1
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Engin AB, Engin A. MicroRNAs as Epigenetic Regulators of Obesity. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2024; 1460:595-627. [PMID: 39287866 DOI: 10.1007/978-3-031-63657-8_20] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/19/2024]
Abstract
In obesity, the process of adipogenesis largely determines the number of adipocytes in body fat depots. Adipogenesis is regulated by several adipocyte-selective micro-ribonucleic acids (miRNAs) and transcription factors that modulate adipocyte proliferation and differentiation. However, some miRNAs block the expression of master regulators of adipogenesis. Since the specific miRNAs display different expressions during adipogenesis, in mature adipocytes and permanent obesity, their use as biomarkers or therapeutic targets is feasible. Upregulated miRNAs in persistent obesity are downregulated during adipogenesis. Moreover, some of the downregulated miRNAs in obese individuals are upregulated in mature adipocytes. Induction of adipocyte stress and hypertrophy leads to the release of adipocyte-derived exosomes (AdEXs) that contain the cargo molecules, miRNAs. miRNAs are important messengers for intercellular communication involved in metabolic responses and have very specific signatures that direct the metabolic activity of target cells. While each miRNA targets multiple messenger RNAs (mRNAs), which may coordinate or antagonize each other's functions, several miRNAs are dysregulated in other tissues during obesity-related comorbidities. Deletion of the miRNA-processing enzyme DICER in pro-opiomelanocortin-expressing cells results in obesity, which is characterized by hyperphagia, increased adiposity, hyperleptinemia, defective glucose metabolism, and alterations in the pituitary-adrenal axis. In recent years, RNA-based therapeutical approaches have entered clinical trials as novel therapies against overweight and its complications. Development of lipid droplets, macrophage accumulation, macrophage polarization, tumor necrosis factor receptor-associated factor 6 activity, lipolysis, lipotoxicity, and insulin resistance are effectively controlled by miRNAs. Thereby, miRNAs as epigenetic regulators are used to determine the new gene transcripts and therapeutic targets.
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Affiliation(s)
- Ayse Basak Engin
- Faculty of Pharmacy, Department of Toxicology, Gazi University, Hipodrom, Ankara, Turkey.
| | - Atilla Engin
- Faculty of Medicine, Department of General Surgery, Gazi University, Besevler, Ankara, Turkey
- Mustafa Kemal Mah. 2137. Sok. 8/14, 06520, Cankaya, Ankara, Turkey
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2
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Mondal S, Ghosh S. Liposome-Mediated Anti-Viral Drug Delivery Across Blood-Brain Barrier: Can Lipid Droplet Target Be Game Changers? Cell Mol Neurobiol 2023; 44:9. [PMID: 38123863 PMCID: PMC11407177 DOI: 10.1007/s10571-023-01443-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2023] [Accepted: 12/02/2023] [Indexed: 12/23/2023]
Abstract
Lipid droplets (LDs) are subcellular organelles secreted from the endoplasmic reticulum (ER) that play a major role in lipid homeostasis. Recent research elucidates additional roles of LDs in cellular bioenergetics and innate immunity. LDs activate signaling cascades for interferon response and secretion of pro-inflammatory cytokines. Since balanced lipid homeostasis is critical for neuronal health, LDs play a crucial role in neurodegenerative diseases. RNA viruses enhance the secretion of LDs to support various phases of their life cycle in neurons which further leads to neurodegeneration. Targeting the excess LD formation in the brain could give us a new arsenal of antiviral therapeutics against neuroviruses. Liposomes are a suitable drug delivery system that could be used for drug delivery in the brain by crossing the Blood-Brain Barrier. Utilizing this, various pharmacological inhibitors and non-coding RNAs can be delivered that could inhibit the biogenesis of LDs or reduce their sizes, reversing the excess lipid-related imbalance in neurons. Liposome-Mediated Antiviral Drug Delivery Across Blood-Brain Barrier. Developing effective antiviral drug is challenging and it doubles against neuroviruses that needs delivery across the Blood-Brain Barrier (BBB). Lipid Droplets (LDs) are interesting targets for developing antivirals, hence targeting LD formation by drugs delivered using Liposomes can be game changers.
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Affiliation(s)
- Sourav Mondal
- CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Kolkata, West Bengal, 700032, India
| | - Sourish Ghosh
- CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Kolkata, West Bengal, 700032, India.
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3
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Sánchez-Álvarez M, del Pozo MÁ, Bosch M, Pol A. Insights Into the Biogenesis and Emerging Functions of Lipid Droplets From Unbiased Molecular Profiling Approaches. Front Cell Dev Biol 2022; 10:901321. [PMID: 35756995 PMCID: PMC9213792 DOI: 10.3389/fcell.2022.901321] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2022] [Accepted: 05/17/2022] [Indexed: 11/30/2022] Open
Abstract
Lipid droplets (LDs) are spherical, single sheet phospholipid-bound organelles that store neutral lipids in all eukaryotes and some prokaryotes. Initially conceived as relatively inert depots for energy and lipid precursors, these highly dynamic structures play active roles in homeostatic functions beyond metabolism, such as proteostasis and protein turnover, innate immunity and defense. A major share of the knowledge behind this paradigm shift has been enabled by the use of systematic molecular profiling approaches, capable of revealing and describing these non-intuitive systems-level relationships. Here, we discuss these advances and some of the challenges they entail, and highlight standing questions in the field.
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Affiliation(s)
- Miguel Sánchez-Álvarez
- Cell and Developmental Biology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
| | - Miguel Ángel del Pozo
- Cell and Developmental Biology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
| | - Marta Bosch
- Lipid Trafficking and Disease Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
- Department of Biomedical Sciences, Faculty of Medicine, Universitat de Barcelona, Barcelona, Spain
| | - Albert Pol
- Lipid Trafficking and Disease Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
- Department of Biomedical Sciences, Faculty of Medicine, Universitat de Barcelona, Barcelona, Spain
- Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
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4
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Abbey D, Conlon D, Rainville C, Elwyn S, Quiroz-Figueroa K, Billheimer J, Schultz DC, Hand NJ, Cherry S, Rader DJ. Lipid droplet screen in human hepatocytes identifies TRRAP as a regulator of cellular triglyceride metabolism. Clin Transl Sci 2021; 14:1369-1379. [PMID: 34156146 PMCID: PMC8301584 DOI: 10.1111/cts.12988] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2020] [Accepted: 12/08/2020] [Indexed: 12/30/2022] Open
Abstract
Hepatocytes store triglycerides (TGs) in the form of lipid droplets (LDs), which are increased in hepatosteatosis. The regulation of hepatic LDs is poorly understood and new therapies to reduce hepatosteatosis are needed. We performed a siRNA kinase and phosphatase screen in HuH‐7 cells using high‐content automated imaging of LDs. Changes in accumulated lipids were quantified with developed pipeline that measures intensity, area, and number of LDs. Selected “hits,” which reduced lipid accumulation, were further validated with other lipid and expression assays. Among several siRNAs that resulted in significantly reduced LDs, one was targeted to the nuclear adapter protein, transformation/transcription domain‐associated protein (TRRAP). Knockdown of TRRAP reduced triglyceride accumulation in HuH‐7 hepatocytes, in part by reducing C/EBPα‐mediated de novo synthesis of TGs. These findings implicate TRRAP as a novel regulator of hepatic TG metabolism and nominate it as a potential drug target for hepatosteatosis.
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Affiliation(s)
- Deepti Abbey
- Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.,Division of Translational Medicine and Human Genetics, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Donna Conlon
- Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.,Division of Translational Medicine and Human Genetics, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Christopher Rainville
- High Throughput Screening Core, Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Susannah Elwyn
- Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.,Division of Translational Medicine and Human Genetics, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Katherine Quiroz-Figueroa
- Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.,Division of Translational Medicine and Human Genetics, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Jeffrey Billheimer
- Division of Translational Medicine and Human Genetics, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - David C Schultz
- High Throughput Screening Core, Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Nicholas J Hand
- Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Sara Cherry
- Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Daniel J Rader
- Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.,Division of Translational Medicine and Human Genetics, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.,Cardiovascular Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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5
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Huang PS, Chang CC, Wang CS, Lin KH. Functional roles of non-coding RNAs regulated by thyroid hormones in liver cancer. Biomed J 2021; 44:272-284. [PMID: 33077406 PMCID: PMC8358202 DOI: 10.1016/j.bj.2020.08.009] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2020] [Revised: 08/19/2020] [Accepted: 08/24/2020] [Indexed: 02/06/2023] Open
Abstract
Recent reports have shown the important role of the non-coding part of human genome RNA (ncRNA) in cancer formation and progression. Among several kinds of ncRNAs, microRNAs (miRNA) play a pivotal role in cancer biology. Accumulating researches have been focused on the importance of non-coding genes in various diseases. In addition to miRNAs, long non-coding RNAs (lncRNAs) have also been extensively documented. Recently, the study of human liver cancer has gradually shifted to these non-coding RNAs that were originally considered "junk". Notably, dysregulated ncRNAs maybe influence on cell proliferation, angiogenesis, anti-apoptosis, and metastasis. Thyroid hormones play critical roles in human development and abnormalities in thyroid hormone levels are associated with various diseases, such as liver cancer. Thyroid hormone receptors (TR) act as ligand-activated nuclear transcription factors to affect multiple functions through the gene-level regulation in the cells and several studies have revealed that thyroid hormone associated with ncRNAs expression. TR actions are complex and tissue- and time-specific, aberrant expression of the various TR isoforms have different effects and are associated with different types of tumor or stages of development. In this review, we discuss various aspects of the research on the thyroid hormones modulated ncRNAs to affect the functions of human liver cells.
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Affiliation(s)
- Po-Shuan Huang
- Department of Biochemistry, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - Cheng-Chih Chang
- Department of General Surgery, Chang Gung Memorial Hospital at Chia yi, Chia yi, Taiwan
| | - Chia-Siu Wang
- Department of General Surgery, Chang Gung Memorial Hospital at Chia yi, Chia yi, Taiwan
| | - Kwang-Huei Lin
- Department of Biochemistry, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan; Research Center for Chinese Herbal Medicine, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan.
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6
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Peixoto TC, Gaspar de Moura E, Quitete FT, Simino LA, Torsoni AS, Torsoni MA, Manhaes AC, Lisboa PC. Early life nicotine exposure alters mRNA and microRNA expressions related to thyroid function and lipid metabolism in liver and BAT of adult wistar rats. Mol Cell Endocrinol 2021; 523:111141. [PMID: 33359828 DOI: 10.1016/j.mce.2020.111141] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/16/2020] [Revised: 12/17/2020] [Accepted: 12/18/2020] [Indexed: 02/06/2023]
Abstract
In rats, maternal nicotine exposure during lactation induces obesity, thyroid dysfunction, brown adipose tissue (BAT) hypofunction and liver alterations in adult offspring. Both thyroid function and lipid metabolism are influenced by gene silencing mediated by microRNAs (miRNAs). Here we investigated long-term effects of early nicotine exposure on molecular and epigenetic mechanisms closely related to thyroid and lipid metabolism, through the expression of mRNAs and miRNAs in BAT and liver of adult male and female offspring. At postnatal day 2 (PND2), lactating control (CON) or nicotine (NIC) dams were subcutaneously implanted with osmotic minipumps containing, respectively, saline or 6 mg/kg nicotine. Litters were adjusted to 3 males and 3 females. Offspring's euthanasia occurred at PND180. In the BAT, NIC females showed higher Dio2 mRNA expression, while miR-382* expression was not altered in both sexes. In the liver, NIC offspring of both sexes showed lower Dio1 mRNA expression and higher miR-224 expression, while only NIC females had higher miR-383 and miR-21 expressions. NIC offspring of both sexes showed higher mRNA expression of SCD1 in the liver; NIC males had decreased CPT1 expression, whereas NIC females had increased FASN, miR-370 and miR-122 expressions. Regardless of sex, alterations in liver Dio1, miR-224 and SCD1 expressions are involved in the disturbances caused by maternal nicotine exposure during breastfeeding. Interestingly, females had more altered miRs in the liver. Early nicotine exposure induces a sex dimorphism, particularly regarding hepatic lipid metabolism, through miRs expression.
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Affiliation(s)
- Thamara Cherem Peixoto
- Laboratory of Endocrine Physiology, Department of Physiological Sciences, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ, 20551-030, Brazil
| | - Egberto Gaspar de Moura
- Laboratory of Endocrine Physiology, Department of Physiological Sciences, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ, 20551-030, Brazil
| | - Fernanda Torres Quitete
- Laboratory of Endocrine Physiology, Department of Physiological Sciences, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ, 20551-030, Brazil
| | - Laís Angélica Simino
- Laboratory of Metabolic Disorders, School of Applied Sciences, University of Campinas, Limeira, SP, 13484-350, Brazil
| | - Adriana Souza Torsoni
- Laboratory of Metabolic Disorders, School of Applied Sciences, University of Campinas, Limeira, SP, 13484-350, Brazil
| | - Marcio Alberto Torsoni
- Laboratory of Metabolic Disorders, School of Applied Sciences, University of Campinas, Limeira, SP, 13484-350, Brazil
| | - Alex Christian Manhaes
- Laboratory of Neurophysiology, Department of Physiological Sciences, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ, 20551-030, Brazil
| | - Patricia Cristina Lisboa
- Laboratory of Endocrine Physiology, Department of Physiological Sciences, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ, 20551-030, Brazil.
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7
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Saarinen MT, Kärkkäinen O, Hanhineva K, Tiihonen K, Hibberd A, Mäkelä KA, Raza GS, Herzig KH, Anglenius H. Metabolomics analysis of plasma and adipose tissue samples from mice orally administered with polydextrose and correlations with cecal microbiota. Sci Rep 2020; 10:21577. [PMID: 33299048 PMCID: PMC7726573 DOI: 10.1038/s41598-020-78484-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2020] [Accepted: 11/20/2020] [Indexed: 01/07/2023] Open
Abstract
Polydextrose (PDX) is a branched glucose polymer, utilized as a soluble dietary fiber. Recently, PDX was found to have hypolipidemic effects and effects on the gut microbiota. To investigate these findings more closely, a non-targeted metabolomics approach, was exploited to determine metabolic alterations in blood and epididymal adipose tissue samples that were collected from C57BL/6 mice fed with a Western diet, with or without oral administration of PDX. Metabolomic analyses revealed significant differences between PDX- and control mice, which could be due to differences in diet or due to altered microbial metabolism in the gut. Some metabolites were found in both plasma and adipose tissue, such as the bile acid derivative deoxycholic acid and the microbiome-derived tryptophan metabolite indoxyl sulfate, both of which increased by PDX. Additionally, PDX increased the levels of glycine betaine and L-carnitine in plasma samples, which correlated negatively with plasma TG and positively correlated with bacterial genera enriched in PDX mice. The results demonstrated that PDX caused differential metabolite patterns in blood and adipose tissues and that one-carbon metabolism, associated with glycine betaine and L-carnitine, and bile acid and tryptophan metabolism are associated with the hypolipidemic effects observed in mice that were given PDX.
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Affiliation(s)
| | - Olli Kärkkäinen
- Afekta Technologies Ltd., Kuopio, Finland
- School of Pharmacy, University of Eastern Finland, Kuopio, Finland
| | - Kati Hanhineva
- Afekta Technologies Ltd., Kuopio, Finland
- Institute of Public Health and Clinical Nutrition, University of Eastern Finland, Kuopio, Finland
| | - Kirsti Tiihonen
- DuPont Nutrition & Biosciences, Global Health & Nutrition Science, Kantvik, Finland
| | - Ashley Hibberd
- DuPont Nutrition & Biosciences, Genomics & Microbiome Science, St. Louis, MO, USA
| | - Kari Antero Mäkelä
- Institute of Biomedicine, Medical Research Center (MRC), University of Oulu, and University Hospital, Oulu, Finland
| | - Ghulam Shere Raza
- Institute of Biomedicine, Medical Research Center (MRC), University of Oulu, and University Hospital, Oulu, Finland
| | - Karl-Heinz Herzig
- Institute of Biomedicine, Medical Research Center (MRC), University of Oulu, and University Hospital, Oulu, Finland
- Department of Gastroenterology and Metabolism, Poznan University of Medical Sciences, Poznan, Poland
| | - Heli Anglenius
- DuPont Nutrition & Biosciences, Global Health & Nutrition Science, Kantvik, Finland
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8
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Si Z, Guan X, Teng X, Peng X, Wan Z, Li Q, Chen G, Tan J, Li J. Identification of CYP46A1 as a new regulator of lipid metabolism through CRISPR-based whole-genome screening. FASEB J 2020; 34:13776-13791. [PMID: 32816363 DOI: 10.1096/fj.202001067r] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 07/23/2020] [Accepted: 08/03/2020] [Indexed: 12/30/2022]
Abstract
Abnormal lipid droplet (LD) metabolism causes a variety of disorders, especially to nonalcoholic fatty liver disease (NAFLD). But the mechanism of abnormal aggregation of LD is still not fully elucidated. Here, Genome-wide CRISPR-Cas9 knockout (GeCKO) screening was employed to identify candidate genes regulating LD metabolism in L02 cell. We analyzed simultaneously the transcriptomics of liver tissues of NAFLD to find potential genes involved in pathogenesis of NAFLD. After integration these data, we found that the expression of 43 candidate genes from the GeCKO screening was also decreased in tissues of NAFLD patients. Many of these 43 overlapping genes have been reported to play an important role in the formation of LD. Subsequently, we focused on CYP46A1, one of 43 candidate genes and mitochondria-related genes. We confirmed that the protein expression of CYP46A1 is deceased in tissues of NAFLD patients. Downregulation or overexpression of CYP46A1 affected LD accumulation in vitro. Deficiency of CYP46A1 impaired mitochondrial morphology and function, which may be responsible for the accumulation of LD. In summary, this study explored regulatory factors of LD accumulation at the whole-genome level, and demonstrated that CYP46A1 regulated LD formation involving in NAFLD pathogenesis. It provides new clues for studying the molecular mechanisms of diseases related to abnormal lipid metabolism.
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Affiliation(s)
- Zhongzhou Si
- Center for Organ Transplantation, Second Xiangya Hospital, Central South University, Changsha, P.R. China
| | - Xinjie Guan
- Center for Medical Genetics, School of Life Science, Central South University, Changsha, P.R. China
| | | | - Xiaoxia Peng
- Center for Medical Genetics, School of Life Science, Central South University, Changsha, P.R. China
| | - Zhengqin Wan
- Center for Medical Genetics, School of Life Science, Central South University, Changsha, P.R. China
| | - Qiang Li
- Center for Organ Transplantation, Second Xiangya Hospital, Central South University, Changsha, P.R. China
| | - Guangshun Chen
- Center for Organ Transplantation, Second Xiangya Hospital, Central South University, Changsha, P.R. China
| | - Jieqiong Tan
- Center for Medical Genetics, School of Life Science, Central South University, Changsha, P.R. China
| | - Jiequn Li
- Center for Organ Transplantation, Second Xiangya Hospital, Central South University, Changsha, P.R. China
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9
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The role of genetic and epigenetic factors in non alcoholic fatty liver disease (NAFLD) pathogenesis. Meta Gene 2020. [DOI: 10.1016/j.mgene.2020.100647] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
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10
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Reduced miR-181d level in obesity and its role in lipid metabolism via regulation of ANGPTL3. Sci Rep 2019; 9:11866. [PMID: 31413305 PMCID: PMC6694160 DOI: 10.1038/s41598-019-48371-2] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2018] [Accepted: 07/29/2019] [Indexed: 12/14/2022] Open
Abstract
Obesity impacts the endocrine and metabolic functions of the adipose tissue. There is increasing interest in the role of epigenetic factors in obesity and its impact on diabetes and dyslipidemia. One such substance, miR-181, reduces plasma triglyceride levels in mice by targeting isocitrate dehydrogenase 1. In the other hand, the adipocyte differentiation and lipid regulating hormone angiopoietin-like 3 (ANGPTL3) is a known regulator of circulating apolipoproteins through its inhibition of the lipoprotein lipase activity. We aimed to study the miR-181d expression in the blood and adipose tissue in a cohort of obese and non-obese people, assessing its possible role in obesity. We also aimed to confirm whether miR-181d can bind and regulate ANGPTL3. miR-181d expression levels were investigated in 144 participants, 82 who were non-obese (body mass index [BMI] < 30) and 62 who were obese (BMI > 30). miR-181d levels in plasma and adipose tissue were measured by RT-PCR. Hepatocyte cell cultures were assessed by overexpression and 3′-UTR-luciferase assays for miR-181d binding to its target protein and its effect on the protein. The plasma levels of ANGPTL3 were also measured by ELISA. The miR-181d levels were significantly lower in obese than in non-obese individuals. In vitro analysis confirmed miR-181 binding to and repression of the ANGPTL3 transcript. Obesity leads to alterations in miR-181d expression. Its downregulation in obese humans was inversely correlated with ANGPTL3, a protein involved in adipocyte differentiation and lipid metabolism. miR-181d can be used as an inhibitor of ANGPTL3 to reduce the TG plasma level.
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11
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Rodrigues Lopes I, Silva RJ, Caramelo I, Eulalio A, Mano M. Shedding light on microRNA function via microscopy-based screening. Methods 2019; 152:55-64. [DOI: 10.1016/j.ymeth.2018.09.011] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2018] [Revised: 09/13/2018] [Accepted: 09/28/2018] [Indexed: 12/24/2022] Open
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12
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13
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Singh AK, Aryal B, Zhang X, Fan Y, Price NL, Suárez Y, Fernández-Hernando C. Posttranscriptional regulation of lipid metabolism by non-coding RNAs and RNA binding proteins. Semin Cell Dev Biol 2017; 81:129-140. [PMID: 29183708 DOI: 10.1016/j.semcdb.2017.11.026] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2017] [Revised: 11/14/2017] [Accepted: 11/20/2017] [Indexed: 12/14/2022]
Abstract
Alterations in lipoprotein metabolism enhance the risk of cardiometabolic disorders including type-2 diabetes and atherosclerosis, the leading cause of death in Western societies. While the transcriptional regulation of lipid metabolism has been well characterized, recent studies have uncovered the importance of microRNAs (miRNAs), long-non-coding RNAs (lncRNAs) and RNA binding proteins (RBP) in regulating the expression of lipid-related genes at the posttranscriptional level. Work from several groups has identified a number of miRNAs, including miR-33, miR-122 and miR-148a, that play a prominent role in controlling cholesterol homeostasis and lipoprotein metabolism. Importantly, dysregulation of miRNA expression has been associated with dyslipidemia, suggesting that manipulating the expression of these miRNAs could be a useful therapeutic approach to ameliorate cardiovascular disease (CVD). The role of lncRNAs in regulating lipid metabolism has recently emerged and several groups have demonstrated their regulation of lipoprotein metabolism. However, given the high abundance of lncRNAs and the poor-genetic conservation between species, much work will be needed to elucidate the specific role of lncRNAs in controlling lipoprotein metabolism. In this review article, we summarize recent findings in the field and highlight the specific contribution of lncRNAs and RBPs in regulating lipid metabolism.
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Affiliation(s)
- Abhishek K Singh
- Vascular Biology and Therapeutics Program, Integrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine, Department of Pathology, Yale University School of Medicine, 10 Amistad St., New Haven, CT 06510, USA
| | - Binod Aryal
- Vascular Biology and Therapeutics Program, Integrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine, Department of Pathology, Yale University School of Medicine, 10 Amistad St., New Haven, CT 06510, USA
| | - Xinbo Zhang
- Vascular Biology and Therapeutics Program, Integrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine, Department of Pathology, Yale University School of Medicine, 10 Amistad St., New Haven, CT 06510, USA
| | - Yuhua Fan
- Vascular Biology and Therapeutics Program, Integrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine, Department of Pathology, Yale University School of Medicine, 10 Amistad St., New Haven, CT 06510, USA; College of Pharmacy, Harbin Medical University -Daqing, 163000, PR China
| | - Nathan L Price
- Vascular Biology and Therapeutics Program, Integrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine, Department of Pathology, Yale University School of Medicine, 10 Amistad St., New Haven, CT 06510, USA
| | - Yajaira Suárez
- Vascular Biology and Therapeutics Program, Integrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine, Department of Pathology, Yale University School of Medicine, 10 Amistad St., New Haven, CT 06510, USA
| | - Carlos Fernández-Hernando
- Vascular Biology and Therapeutics Program, Integrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine, Department of Pathology, Yale University School of Medicine, 10 Amistad St., New Haven, CT 06510, USA.
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14
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Abstract
Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids for energy or membrane synthesis and act as hubs for metabolic processes. Cells generate LDs de novo, converting cells to emulsions with LDs constituting the dispersed oil phase in the aqueous cytoplasm. Here we review our current view of LD biogenesis. We present a model of LD formation from the ER in distinct steps and highlight the biology of proteins that govern this biophysical process. Areas of incomplete knowledge are identified, as are connections with physiology and diseases linked to alterations in LD biology.
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Affiliation(s)
- Tobias C Walther
- Department of Genetics and Complex Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115; , .,Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.,Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142.,Howard Hughes Medical Institute, Boston, Massachusetts 02115
| | - Jeeyun Chung
- Department of Genetics and Complex Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115; , .,Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
| | - Robert V Farese
- Department of Genetics and Complex Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115; , .,Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.,Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142
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15
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Yu S, Chen E, Sherwood L, Hull M, Woods AK, Tremblay MS. Ex Vivo Cell-Based Screening Platform for Modulators of Hepatosteatosis. ACS Chem Biol 2017; 12:1937-1946. [PMID: 28586195 DOI: 10.1021/acschembio.7b00420] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Nonalcoholic fatty liver disease (NAFLD) is the result of the ectopic accumulation of lipids in hepatic cells and is the early stage of liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma. While some mechanisms of aberrant lipid storage are understood, unbiased phenotypic drug screening holds the potential to identify new therapeutic small molecule mechanisms that reverse lipid accumulation in hepatic cells and prevent disease progression. Immortalized hepatocyte cell lines are often used as in vitro models of hepatocyte function, including in the study of lipid accumulation. However, mechanisms and therapeutic agents studied in these systems suffer from poor translation to primary cells and animal models of disease. Herein, we report an ex vivo high-throughput screening platform using primary mouse hepatocytes with a physiologically relevant lipid-laden phenotype isolated from mice that are administered a choline-methionine deficient diet. This screening platform using primary diseased hepatocytes may help to overcome a major hurdle in liver disease drug discovery and could lead to the development of new therapeutics for hepatosteatosis.
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Affiliation(s)
- Shan Yu
- California Institute for Biomedical Research, La Jolla, California 92037, United States
| | - Emily Chen
- California Institute for Biomedical Research, La Jolla, California 92037, United States
| | - Lance Sherwood
- California Institute for Biomedical Research, La Jolla, California 92037, United States
| | - Mitchell Hull
- California Institute for Biomedical Research, La Jolla, California 92037, United States
| | - Ashley K. Woods
- California Institute for Biomedical Research, La Jolla, California 92037, United States
| | - Matthew S. Tremblay
- California Institute for Biomedical Research, La Jolla, California 92037, United States
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16
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17
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El-Ekiaby NM, Mekky RY, Riad SE, Elhelw DS, El-Sayed M, Esmat G, Abdelaziz AI. miR-148a and miR-30a limit HCV-dependent suppression of the lipid droplet protein, ADRP, in HCV infected cell models. J Med Virol 2017; 89:653-659. [PMID: 27591428 DOI: 10.1002/jmv.24677] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/31/2016] [Indexed: 02/05/2023]
Abstract
Hepatitis C Virus (HCV) promotes lipid droplet (LD) formation and perturbs the expression of the LD associated PAT proteins ADRP and TIP47, to promote its own lifecycle. HCV enhances TIP47 and suppresses ADRP by displacing it from LD surface in infected cell models. We have previously shown that suppression of TIP47 by miR-148a and miR-30a decreased intracellular LDs and HCV RNA. Thus, this study aimed at examining whether this microRNA-mediated suppression of HCV would limit HCV-dependent displacement of ADRP from LDs. ADRP expression was examined in 21 HCV-infected liver biopsies and 9 healthy donor liver tissues as well as in HCV-infected Huh7 cells using qRT-PCR. miR-148a and miR-30a expression was manipulated using specific oligos in JFH-1 infected, oleic acid treated cells, to study their impact on ADRP expression using qRT-PCR, and immunofluorescence microscopy. Intracellular HCV RNA was assessed using qRT-PCR. ADRP is down regulated in patients as well as HCVcc-JFH-I infected cell models. Forcing the expression of both miRNAs induced ADRP on the mRNA and protein levels. This study shows that HCV suppresses hepatic ADRP expression in infected patients and cell lines. Forcing the expression of miR-148a and miR-30a limits the suppressive effect of HCV on ADRP. J. Med. Virol. 89:653-659, 2017. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Nada M El-Ekiaby
- Department of Pharmacology and Toxicology, Molecular Pathology Research Group, German University in Cairo, New Cairo City-Main Entrance Al Tagamoa Al Khames, Cairo, Egypt
- Scientific Affairs Unit, Egyptian Company for Biological Sciences, Giza, Egypt
| | - Radwa Y Mekky
- Department of Pharmacology and Toxicology, Molecular Pathology Research Group, German University in Cairo, New Cairo City-Main Entrance Al Tagamoa Al Khames, Cairo, Egypt
- Faculty of Biotechnology, October University of Modern Sciences and Arts, Giza, Egypt
| | - Sarah E Riad
- Department of Pharmacology and Toxicology, Molecular Pathology Research Group, German University in Cairo, New Cairo City-Main Entrance Al Tagamoa Al Khames, Cairo, Egypt
| | - Dalia S Elhelw
- Department of Pharmacology and Toxicology, Molecular Pathology Research Group, German University in Cairo, New Cairo City-Main Entrance Al Tagamoa Al Khames, Cairo, Egypt
| | | | - Gamal Esmat
- Endemic Medicine and Hepatology, Cairo University, Cairo, Egypt
| | - Ahmed I Abdelaziz
- Department of Pharmacology and Toxicology, Molecular Pathology Research Group, German University in Cairo, New Cairo City-Main Entrance Al Tagamoa Al Khames, Cairo, Egypt
- Biotechnology Graduate Program, American University in Cairo, AUC Avenue, New Cairo, Cairo, Egypt
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18
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Natarajan SK, Rasineni K, Ganesan M, Feng D, McVicker BL, McNiven MA, Osna NA, Mott JL, Casey CA, Kharbanda KK. Structure, Function and Metabolism of Hepatic and Adipose Tissue Lipid Droplets: Implications in Alcoholic Liver Disease. Curr Mol Pharmacol 2017; 10:237-248. [PMID: 26278390 PMCID: PMC4820363 DOI: 10.2174/1874467208666150817111727] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2015] [Revised: 08/07/2015] [Accepted: 08/07/2015] [Indexed: 02/08/2023]
Abstract
For more than 30 years, lipid droplets (LDs) were considered as an inert bag of lipid for storage of energy-rich fat molecules. Following a paradigm shift almost a decade ago, LDs are presently considered an active subcellular organelle especially designed for assembling, storing and subsequently supplying lipids for generating energy and membrane synthesis (and in the case of hepatocytes for VLDL secretion). LDs also play a central role in many other cellular functions such as viral assembly and protein degradation. Here, we have explored the structural and functional changes that occur in hepatic and adipose tissue LDs following chronic ethanol consumption in relation to their role in the pathogenesis of alcoholic liver injury.
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Affiliation(s)
- Sathish Kumar Natarajan
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center
| | - Karuna Rasineni
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Murali Ganesan
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Dan Feng
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Benita L. McVicker
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Mark A. McNiven
- Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota
| | - Natalia A. Osna
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Justin L. Mott
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center
| | - Carol A. Casey
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center
| | - Kusum K. Kharbanda
- Research Service, VA Nebraska-Western Iowa Health Care System (VA NWIHCS), and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center
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19
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Mahdy MM, El-Ekiaby NM, Hashish RM, Salah RA, Hanafi RS, El-Said Azzazy HM, Abdelaziz AI. miR-29a Promotes Lipid Droplet and Triglyceride Formation in HCV Infection by Inducing Expression of SREBP-1c and CAV1. J Clin Transl Hepatol 2016; 4:293-299. [PMID: 28097097 PMCID: PMC5225148 DOI: 10.14218/jcth.2016.00046] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/29/2016] [Revised: 11/21/2016] [Accepted: 12/07/2016] [Indexed: 01/18/2023] Open
Abstract
Aims: To examine the regulation of SREBP-1c and CAV1 by microRNA-29a (miR-29a) in cells infected with hepatitis C virus (HCV) in an attempt to control HCV-induced non-alcoholic fatty liver disease. Methods: In order to examine the manipulation of SREBP-1c and CAV1 by miR-29a, oleic acid (OA)-treated JFH-I-infected Huh-7 cells were used. OA was added 24 h post-transfection and gene expression was investigated by qRT-PCR at 48 h post treatment. The functional impact of the observed alteration in SREBP-1c and CAV1 expression was analyzed by examining lipid droplet (LD) and triglyceride (TG) content at 72 h post-OA treatment using light microscopy and spectrophotometry, respectively. Viral load was quantified by qRT-PCR at 72 h post-transfection. Results: OA treatment induced the expression of miR-29a and SREBP-1c, as compared to untreated cells. Forced miR-29a expression led to a significant up-regulation of SREBP-1c as well as CAV1 compared to mock untransfected cells. Ectopic expression of miR-29a resulted in a marked increase in LDs and their respective TGs, while miR-29a antagomirs decreased both the LD and TG content compared to mock untransfected cells. Moreover, forcing the expression of miR-29a in JFH-1 HCV-infected Huh-7 cells resulted in 53% reduction in viral titers compared to mock untransfected Huh-7 cells. Conclusion: Inducing miR-29a expression significantly induces SREBP-1c and CAV1 expression, thereby increasing LDs as well as their respective TGs. Nonetheless, forcing the expression of miR-29a resulted in reduction of HCV RNA levels in Huh-7 cells.
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Affiliation(s)
| | - Nada Magdy El-Ekiaby
- Department of Pharmacology and Toxicology, German University in Cairo, New Cairo City, Egypt
- School of Medicine, NewGiza University, Cairo, Egypt
| | - Rana Mahmoud Hashish
- Department of Pharmaceutical Biology, German University in Cairo, New Cairo City, Egypt
| | - Radwa Ayman Salah
- Department of Stem Cells and Regenerative Medicine, Zewail City of Science and Technology, Giza, Egypt
| | - Rasha Sayed Hanafi
- Department of Pharmaceutical Chemistry, German University in Cairo, New Cairo City, Egypt
| | | | - Ahmed Ihab Abdelaziz
- Department of Pharmacology and Toxicology, German University in Cairo, New Cairo City, Egypt
- School of Medicine, NewGiza University, Cairo, Egypt
- *Correspondence to: Ahmed Ihab Abdelaziz, Department of Molecular Medicine, School of Medicine, Newgiza University, Cairo 11431, Egypt. Tel: +20-238277847, E-mail:
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20
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Jia Y, Ling M, Zhang L, Jiang S, Sha Y, Zhao R. Downregulation of miR-150 Expression by DNA Hypermethylation Is Associated with High 2-Hydroxy-(4-methylthio)butanoic Acid-Induced Hepatic Cholesterol Accumulation in Nursery Piglets. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2016; 64:7530-7539. [PMID: 27648945 DOI: 10.1021/acs.jafc.6b03615] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
Excess 2-hydroxy-(4-methylthio)butanoic acid (HMB) supplementation induces hyperhomocysteinemia, which contributes to hepatic cholesterol accumulation. However, it is unclear whether and how high levels of HMB break hepatic cholesterol homeostasis in nursery piglets. In this study, HMB oversupplementation suppressed food intake and decreased body weight in nursery piglets. Hyperhomocysteinemia and higher hepatic cholesterol accumulation were observed in HMB groups. Accordingly, HMB significantly increased the protein content of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and glycine N-methyltransferase (GNMT) but decreased that of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1). Significant downregulation of miR-150, miR-181d-5p, and miR-296-3p targeting the 3'-untranslated regions (UTRs) of GNMT and HMGCR was detected in the liver of HMB-treated piglets, and their functional validation was confirmed by dual-luciferase reporter assay. Furthermore, hypermethylation of miR-150 promoter was detected in association with suppressed miR-150 expression in the livers of HMB-treated piglets. This study indicated a new mechanism of hepatic cholesterol unhomeostasis by dietary methyl donor supplementation.
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Affiliation(s)
- Yimin Jia
- Key Laboratory of Animal Physiology & Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing 210095, People's Republic of China
| | - Mingfa Ling
- Key Laboratory of Animal Physiology & Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing 210095, People's Republic of China
| | - Luchu Zhang
- Key Laboratory of Animal Physiology & Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing 210095, People's Republic of China
| | - Shuxia Jiang
- Key Laboratory of Animal Physiology & Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing 210095, People's Republic of China
| | - Yusheng Sha
- China Feed Industry Association, Ministry of Agriculture , Peking 100125, People's Republic of China
| | - Ruqian Zhao
- Key Laboratory of Animal Physiology & Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing 210095, People's Republic of China
- Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, Nanjing Agricultural University , Nanjing 210095, People's Republic of China
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21
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Soronen J, Yki-Järvinen H, Zhou Y, Sädevirta S, Sarin AP, Leivonen M, Sevastianova K, Perttilä J, Laurila PP, Sigruener A, Schmitz G, Olkkonen VM. Novel hepatic microRNAs upregulated in human nonalcoholic fatty liver disease. Physiol Rep 2016; 4:4/1/e12661. [PMID: 26733244 PMCID: PMC4760405 DOI: 10.14814/phy2.12661] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
MicroRNAs (miRNAs) control gene expression by reducing mRNA stability and translation. We aimed to identify alterations in human liver miRNA expression/function in nonalcoholic fatty liver disease (NAFLD). Subjects with the highest (median liver fat 30%, n = 15) and lowest (0%, n = 15) liver fat content were selected from >100 obese patients for miRNA profiling of liver biopsies on microarrays carrying probes for 1438 human miRNAs (a cross‐sectional study). Target mRNAs and pathways were predicted for the miRNAs most significantly upregulated in NAFLD, their cell‐type‐specific expression was investigated by quantitative PCR (qPCR), and the transcriptome of immortalized human hepatocytes (IHH) transfected with the miRNA with the highest number of predicted targets, miR‐576‐5p, was studied. The screen revealed 42 miRNAs up‐ and two downregulated in the NAFLD as compared to non‐NAFLD liver. The miRNAs differing most significantly between the groups, miR‐103a‐2*, miR‐106b, miR‐576‐5p, miRPlus‐I137*, miR‐892a, miR‐1282, miR‐3663‐5p, and miR‐3924, were all upregulated in NAFLD liver. Target pathways predicted for these miRNAs included ones involved in cancer, metabolic regulation, insulin signaling, and inflammation. Consistent transcriptome changes were observed in IHH transfected with miR‐576‐5p, and western analysis revealed a marked reduction of the RAC1 protein belonging to several miR‐576‐5p target pathways. To conclude, we identified 44 miRNAs differentially expressed in NAFLD versus non‐NAFLD liver, 42 of these being novel in the context of NAFLD. The study demonstrates that by applying a novel study set‐up and a broad‐coverage array platform one can reveal a wealth of previously undiscovered miRNA dysregulation in metabolic disease.
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Affiliation(s)
- Jarkko Soronen
- Genomics and Biomarkers Unit, National Institute for Health and Welfare Biomedicum, Helsinki, Finland Minerva Foundation Institute for Medical Research, Helsinki, Finland
| | - Hannele Yki-Järvinen
- Minerva Foundation Institute for Medical Research, Helsinki, Finland Department of Medicine, Division of Diabetes, University of Helsinki, Helsinki, Finland
| | - You Zhou
- Minerva Foundation Institute for Medical Research, Helsinki, Finland
| | - Sanja Sädevirta
- Minerva Foundation Institute for Medical Research, Helsinki, Finland Department of Medicine, Division of Diabetes, University of Helsinki, Helsinki, Finland
| | - Antti-Pekka Sarin
- Genomics and Biomarkers Unit, National Institute for Health and Welfare Biomedicum, Helsinki, Finland Institute for Molecular Medicine Finland FIMM, University of Helsinki, Helsinki, Finland
| | - Marja Leivonen
- Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland
| | - Ksenia Sevastianova
- Minerva Foundation Institute for Medical Research, Helsinki, Finland Department of Medicine, Division of Diabetes, University of Helsinki, Helsinki, Finland
| | - Julia Perttilä
- Minerva Foundation Institute for Medical Research, Helsinki, Finland
| | - Pirkka-Pekka Laurila
- Genomics and Biomarkers Unit, National Institute for Health and Welfare Biomedicum, Helsinki, Finland
| | - Alexander Sigruener
- Institute of Clinical Chemistry and Laboratory Medicine, Regensburg University Medical Center, Regensburg, Germany
| | - Gerd Schmitz
- Institute of Clinical Chemistry and Laboratory Medicine, Regensburg University Medical Center, Regensburg, Germany
| | - Vesa M Olkkonen
- Minerva Foundation Institute for Medical Research, Helsinki, Finland Institute of Biomedicine, Anatomy, University of Helsinki, Helsinki, Finland
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22
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Chen SZ, Ning LF, Xu X, Jiang WY, Xing C, Jia WP, Chen XL, Tang QQ, Huang HY. The miR-181d-regulated metalloproteinase Adamts1 enzymatically impairs adipogenesis via ECM remodeling. Cell Death Differ 2016; 23:1778-1791. [PMID: 27447109 DOI: 10.1038/cdd.2016.66] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2015] [Revised: 05/09/2016] [Accepted: 06/13/2016] [Indexed: 02/08/2023] Open
Abstract
The extracellular matrix (ECM) maintenance is crucial to the structural integrity of adipocytes and whole adipose tissue formation. However, the potential impact of the ECM on adipocyte lineage commitment is unclear. Herein, we demonstrate that forced expression of matrix-associated metalloproteinase Adamts1 (a disintegrin and metalloproteinase with thrombospondin motifs 1), which we show is targeted by microRNA-181d (miR-181d) during BMP4-induced adipocytic lineage commitment, markedly impairs adipocyte commitment. Conversely, siRNA-induced inhibition of Adamts1 promotes adipocyte commitment. Adamst1 metalloprotease activity is required for this inhibition and is determined to function via remodeling ECM components followed by activating FAK-ERK signaling pathway during the commitment process. Furthermore, ablation of Adamts1 in adipose tissue increases adipose tissue mass, reduces insulin sensitivity, and disrupts lipid homeostasis. This finding is consistent with Adamts1 decreased expression in the adipose tissue of obese mice and an inverse correlation of Adamts1 expression with body mass index in humans. Collectively, our results indicate that Adamts1 acts as an ECM 'modifier', with miR-181d-induced downregulation, that regulates adipocyte lineage commitment and obesity.
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Affiliation(s)
- S-Z Chen
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.,Key Laboratory of Metabolism and Molecular Medicine, The Ministry of Education, China
| | - L-F Ning
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.,Key Laboratory of Metabolism and Molecular Medicine, The Ministry of Education, China
| | - X Xu
- Institute of Stem Cell Research and Regenerative Medicine, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China
| | - W-Y Jiang
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.,Key Laboratory of Metabolism and Molecular Medicine, The Ministry of Education, China
| | - C Xing
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.,Key Laboratory of Metabolism and Molecular Medicine, The Ministry of Education, China
| | - W-P Jia
- Department of Endocrinology and Metabolism, Shanghai Clinical Center for Diabetes, Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai 200233, China.,Shanghai Diabetes Institute, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai 200233, China
| | - X-L Chen
- Department of Food Science and Nutrition, University of Minnesota-Twin Cities, Saint Paul, MN 55108, USA
| | - Q-Q Tang
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.,Key Laboratory of Metabolism and Molecular Medicine, The Ministry of Education, China.,Institute of Stem Cell Research and Regenerative Medicine, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China
| | - H-Y Huang
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.,Key Laboratory of Metabolism and Molecular Medicine, The Ministry of Education, China.,Institute of Stem Cell Research and Regenerative Medicine, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China
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23
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Nutrigenetics and Nutrimiromics of the Circadian System: The Time for Human Health. Int J Mol Sci 2016; 17:299. [PMID: 26927084 PMCID: PMC4813163 DOI: 10.3390/ijms17030299] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2016] [Revised: 02/08/2016] [Accepted: 02/16/2016] [Indexed: 12/15/2022] Open
Abstract
Even though the rhythmic oscillations of life have long been known, the precise molecular mechanisms of the biological clock are only recently being explored. Circadian rhythms are found in virtually all organisms and affect our lives. Thus, it is not surprising that the correct running of this clock is essential for cellular functions and health. The circadian system is composed of an intricate network of genes interwined in an intrincated transcriptional/translational feedback loop. The precise oscillation of this clock is controlled by the circadian genes that, in turn, regulate the circadian oscillations of many cellular pathways. Consequently, variations in these genes have been associated with human diseases and metabolic disorders. From a nutrigenetics point of view, some of these variations modify the individual response to the diet and interact with nutrients to modulate such response. This circadian feedback loop is also epigenetically modulated. Among the epigenetic mechanisms that control circadian rhythms, microRNAs are the least studied ones. In this paper, we review the variants of circadian-related genes associated to human disease and nutritional response and discuss the current knowledge about circadian microRNAs. Accumulated evidence on the genetics and epigenetics of the circadian system points to important implications of chronotherapy in the clinical practice, not only in terms of pharmacotherapy, but also for dietary interventions. However, interventional studies (especially nutritional trials) that include chronotherapy are scarce. Given the importance of chronobiology in human health such studies are warranted in the near future.
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24
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El-Ekiaby NM, Mekky RY, El Sobky SA, Elemam NM, El-Sayed M, Esmat G, Abdelaziz AI. Epigenetic harnessing of HCV via modulating the lipid droplet-protein, TIP47, in HCV cell models. FEBS Lett 2015; 589:2266-73. [PMID: 26170028 DOI: 10.1016/j.febslet.2015.06.040] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2015] [Revised: 06/12/2015] [Accepted: 06/25/2015] [Indexed: 11/25/2022]
Abstract
This study aimed at identifying potential microRNAs that modulate hepatic lipid droplets (LD) through targeting the Tail interacting protein of 47 kDa (TIP47) in HCV infection. Bioinformatics analysis revealed that miR-148a and miR-30a potentially target TIP47. Expression profiling showed that both microRNAs were downregulated, while TIP47 was upregulated in liver biopsies of HCV-infected patients. Forcing the expression of both microRNAs in JFH-I infected, oleic acid-treated Huh7 cells, significantly suppressed TIP47 expression and reduced cellular LDs with marked decrease in viral RNA. This study shows that miR-148a and miR-30a, regulate TIP47 expression and LDs in HCV infected cells.
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Affiliation(s)
- Nada M El-Ekiaby
- Molecular Pathology Research Group, Department of Pharmacology and Toxicology, German University in Cairo, Cairo, Egypt
| | - Radwa Y Mekky
- Molecular Pathology Research Group, Department of Pharmacology and Toxicology, German University in Cairo, Cairo, Egypt
| | - Shereen A El Sobky
- Molecular Pathology Research Group, Department of Pharmaceutical Biology, German University in Cairo, Cairo, Egypt
| | - Noha M Elemam
- Molecular Pathology Research Group, Department of Pharmacology and Toxicology, German University in Cairo, Cairo, Egypt
| | | | - Gamal Esmat
- Endemic Medicine and Hepatology, Cairo University, Cairo, Egypt
| | - Ahmed I Abdelaziz
- Molecular Pathology Research Group, Department of Pharmacology and Toxicology, German University in Cairo, Cairo, Egypt.
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25
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Eulalio A, Mano M. MicroRNA Screening and the Quest for Biologically Relevant Targets. ACTA ACUST UNITED AC 2015; 20:1003-17. [DOI: 10.1177/1087057115578837] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2015] [Accepted: 03/03/2015] [Indexed: 12/12/2022]
Abstract
MicroRNAs (miRNAs) are a class of genome-encoded small RNAs that post-transcriptionally regulate gene expression by repressing target transcripts containing partially or fully complementary binding sites. Despite their relatively low number, miRNAs have been shown to directly regulate a large fraction of the transcriptome. In agreement with their pervasive role in the regulation of eukaryotic gene expression, miRNAs have been implicated in virtually all biological processes, including different pathologies. The use of screening technologies to systematically analyze miRNA function in cell-based assays offers a unique opportunity to gain new insights into complex biological and disease-relevant processes. Given the low complexity of the miRNome and the similarities to small interfering RNA (siRNA) screening experimental approaches, phenotypic screening using genome-wide libraries of miRNA mimics or inhibitors is not, per se, technically challenging. The identification of miRNA targets and, more importantly, the characterization of their mechanisms of action through the identification of the key targets underlying observed phenotypes remain the major challenges of this approach. This article provides an overview of cell-based screenings for miRNA function that were performed in different biological contexts. The advantages and limitations of computational and experimental approaches commonly used to identify miRNA targets are also discussed.
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Affiliation(s)
- Ana Eulalio
- Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany
| | - Miguel Mano
- International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
- UC-BIOTECH, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal
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26
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Gerhard GS, DiStefano JK. Micro RNAs in the development of non-alcoholic fatty liver disease. World J Hepatol 2015; 7:226-234. [PMID: 25729477 PMCID: PMC4342604 DOI: 10.4254/wjh.v7.i2.226] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/16/2014] [Revised: 10/16/2014] [Accepted: 11/19/2014] [Indexed: 02/06/2023] Open
Abstract
Nonalcoholic fatty liver disease or nonalcoholic fatty liver disease (NAFLD) refers to a group of disorders that arise from the accrual of fat in hepatocytes. Although various factors have been associated with the development of NAFLD, including genetic predisposition and environmental exposures, little is known about the underlying pathogenesis of the disease. Research efforts are ongoing to identify biological targets and signaling pathways that mediate NAFLD. Emerging evidence has implicated a role for micro RNAs (miRNAs), short single-stranded molecules that regulate gene expression either transcriptionally, through targeting of promoter regions, or post-transcriptionally, by blocking translation or promoting cleavage of specific target mRNAs. Several miRNAs have been associated with NAFLD, although our understanding of the biology underlying their role is still emerging. The goal of this review is to present an overview of the current state of knowledge of miRNAs involved in the development of NAFLD across a range of in vitro and in vivo models, including miRNAs that contribute to pathological mechanisms related to fatty liver in humans. Much less is known about the specific targets of miRNAs in cells, nor the molecular mechanisms involved in the development and progression NAFLD and related outcomes. More recently, the identification and validation of miRNA signatures in serum may facilitate the development of improved methods for diagnosis and clinical monitoring of disease progression.
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Wang XC, Zhan XR, Li XY, Yu JJ, Liu XM. MicroRNA-185 regulates expression of lipid metabolism genes and improves insulin sensitivity in mice with non-alcoholic fatty liver disease. World J Gastroenterol 2014; 20:17914-17923. [PMID: 25548489 PMCID: PMC4273141 DOI: 10.3748/wjg.v20.i47.17914] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/24/2014] [Revised: 08/16/2014] [Accepted: 09/19/2014] [Indexed: 02/06/2023] Open
Abstract
AIM: To assess the regulatory effect of microRNA-185 (miR-185) on lipid metabolism and the insulin signalling pathway in human HepG2 hepatocytes and a high-fat diet mouse model.
METHODS: Quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA levels of lipogenic genes after loss or gain of miR-185. In addition, the amounts of insulin signalling intermediates were determined after transfection of HepG2 cells with pre-miR-185.
RESULTS: MiR-185 levels decreased in a time- and dose-dependent manner in response to palmitic acid in human HepG2 hepatocytes. Transfection of HepG2 cells with miR-185 significantly decreased the mRNA levels of fatty acid synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, sterol-regulatory element binding protein-2, and sterol-regulatory element binding protein-1c, whereas inhibition of miR-185 using an anti-miR-185 oligonucleotide produced the opposite effect in HepG2 cells. In a high-fat diet mouse model, the accumulation of lipids was significantly improved after treatment with miR-185, compared with control animals. Induction of miR-185 enhanced the insulin signalling pathway by up-regulating the insulin-receptor substrate-2.
CONCLUSION: These findings suggest that miR-185 plays an important role in regulating fatty-acid metabolism and cholesterol homeostasis in hepatocytes, as well as in improving insulin sensitivity, both in vitro and in vivo.
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Amacher DE. Progress in the search for circulating biomarkers of nonalcoholic fatty liver disease. Biomarkers 2014; 19:541-52. [PMID: 25189636 DOI: 10.3109/1354750x.2014.958535] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
CONTEXT The definitive standard for the diagnosis of nonalcoholic fatty liver disease (NAFLD) is clinico-pathological correlation, but frequently the only laboratory abnormality is an elevation of serum aminotransferases. OBJECTIVE This has resulted in the search for more specific laboratory biomarkers. METHODS The literature was searched for novel plasma/serum markers of NAFLD. RESULTS Studies reviewed here included histologically-confirmed patients presenting some stage of NAFLD and monitored one or more novel serum/plasma biomarkers. CONCLUSION The most promising application of some of these novel biomarkers for the detection and quantification of NAFLD and particularly NASH appears to be in the combination of several into diagnostic panels.
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Celikbilek M, Baskol M, Taheri S, Deniz K, Dogan S, Zararsiz G, Gursoy S, Guven K, Ozbakır O, Dundar M, Yucesoy M. Circulating microRNAs in patients with non-alcoholic fatty liver disease. World J Hepatol 2014; 6:613-620. [PMID: 25232454 PMCID: PMC4163744 DOI: 10.4254/wjh.v6.i8.613] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/28/2014] [Revised: 03/13/2014] [Accepted: 07/14/2014] [Indexed: 02/06/2023] Open
Abstract
AIM: To identify novel non-invasive biomarkers for non-alcoholic fatty liver disease (NAFLD).
METHODS: Twenty patients with histologically proven NAFLD and 20 controls were included. All NAFLD cases were scored using the NAFLD activity score. The relative expressions of miR-197, miR-146b, miR-10b, miR-181d, miR-34a, miR-122, miR-99a and miR-29a were analyzed using real-time polymerase chain reaction.
RESULTS: Serum levels of miR-181d, miR-99a, miR-197 and miR-146b were significantly lower in biopsy-proven NAFLD patients than in the healthy controls. Serum levels of miR-197 and miR-10b were inversely correlated with degree of inflammation and miR-181d and miR-99a were inversely correlated with serum gamma glutamyl transferase levels in non-alcoholic steatohepatitis patients.
CONCLUSION: NAFLD is associated with altered serum miRNA expression pattern. This study provides clues for defining the non-invasive diagnosis of NAFLD.
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Nie YQ, Cao J, Zhou YJ, Liang X, Du YL, Wan YJY, Li YY. The effect of miRNA-122 in regulating fat deposition in a cell line model. J Cell Biochem 2014; 115:839-846. [PMID: 24288170 PMCID: PMC3991241 DOI: 10.1002/jcb.24725] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2013] [Accepted: 11/19/2013] [Indexed: 12/17/2022]
Abstract
Accumulating evidence supports the role of miR-122 in fatty liver disease. We investigated miR-122 expression in a steatotic hepatocyte model, the effect of miR-122 over-expression and inhibition in the pathogenesis. Human hepatic cell line L02 was induced with oleic acid to establish the steatotic hepatocyte model. Intracellular lipid content was observed with laser scanning confocal microscope (LSCM), and triglyceride content was determined with kits. Total RNA was extracted and reversely transcribed into cDNA. miR-122 expression was measured using qRT-PCR. Subsequently, miR-122 mimic and miR-122 inhibitor were transfected into steatotic hepatocytes to observe their effect on intracellular lipid content. The lipid fluorescence intensity and triglyceride content within the steatotic hepatocytes were significantly higher than those in normal control (860.01 ± 26.52 vs. 257.77 ± 29.69 and 3.47 ± 0.12 vs. 1.85 ± 0.02 at 24 h) (P < 0.01). miR-122 expression in steatotic hepatocytes was down-regulated compared with that in control (2-ΔCt value: 0.0286 ± 0.0078 vs. 0.0075 ± 0.0012) (P ≪ 0.01). After transfection, miR-122 expression (2-ΔCt value) in the miR-122 mimic group increased 2.96-fold compared with that in control, and its lipid fluorescence intensity was significantly lower than that in control (790.92 ± 46.72 vs. 1,022.16 ± 49.66) (P < 0.01). Nevertheless, miR-122 expression decreased 3.45-fold in the miR-122 inhibitor group compared with that in control, and its fluorescence intensity was significantly higher than that in control (1,386.49 ± 40.34 vs 1,022.16 ± 49.66)(P ≪ 0.01). We concluded that miR-122 was down-regulated in steatotic hepatocytes model. The pathogenesis of hepatocyte steatosis was enhanced by miR-122 mimic and reduced with miR-122 inhibitor.
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Affiliation(s)
- Yu-Qiang Nie
- Department of Gastroenterology and Hepatology, Guangzhou Institute of Digestive Disease, Guangzhou first People’s Hospital, Guangzhou Medical University, Guangzhou 510180, China
| | - Jie Cao
- Department of Gastroenterology and Hepatology, Guangzhou Institute of Digestive Disease, Guangzhou first People’s Hospital, Guangzhou Medical University, Guangzhou 510180, China
| | - Yong-Jian Zhou
- Department of Gastroenterology and Hepatology, Guangzhou Institute of Digestive Disease, Guangzhou first People’s Hospital, Guangzhou Medical University, Guangzhou 510180, China
| | - Xia Liang
- Department of Gastroenterology and Hepatology, Guangzhou Institute of Digestive Disease, Guangzhou first People’s Hospital, Guangzhou Medical University, Guangzhou 510180, China
| | - Yan-Lei Du
- Department of Gastroenterology and Hepatology, Guangzhou Institute of Digestive Disease, Guangzhou first People’s Hospital, Guangzhou Medical University, Guangzhou 510180, China
| | - Yu-Jui Yvonne Wan
- Department of Medical Pathology and Laboratory Medicine, University of California, Davis Health Systems, USA
| | - Yu-Yuan Li
- Department of Gastroenterology and Hepatology, Guangzhou Institute of Digestive Disease, Guangzhou first People’s Hospital, Guangzhou Medical University, Guangzhou 510180, China
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Singh S, Carpenter AE, Genovesio A. Increasing the Content of High-Content Screening: An Overview. ACTA ACUST UNITED AC 2014; 19:640-50. [PMID: 24710339 PMCID: PMC4230961 DOI: 10.1177/1087057114528537] [Citation(s) in RCA: 134] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2013] [Accepted: 12/31/2013] [Indexed: 01/17/2023]
Abstract
Target-based high-throughput screening (HTS) has recently been critiqued for its relatively poor yield compared to phenotypic screening approaches. One type of phenotypic screening, image-based high-content screening (HCS), has been seen as particularly promising. In this article, we assess whether HCS is as high content as it can be. We analyze HCS publications and find that although the number of HCS experiments published each year continues to grow steadily, the information content lags behind. We find that a majority of high-content screens published so far (60−80%) made use of only one or two image-based features measured from each sample and disregarded the distribution of those features among each cell population. We discuss several potential explanations, focusing on the hypothesis that data analysis traditions are to blame. This includes practical problems related to managing large and multidimensional HCS data sets as well as the adoption of assay quality statistics from HTS to HCS. Both may have led to the simplification or systematic rejection of assays carrying complex and valuable phenotypic information. We predict that advanced data analysis methods that enable full multiparametric data to be harvested for entire cell populations will enable HCS to finally reach its potential.
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Affiliation(s)
- Shantanu Singh
- Imaging Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Anne E Carpenter
- Imaging Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Auguste Genovesio
- Imaging Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA École Normale Supérieure, 45, Rue d'Ulm, 75005 Paris
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Maternal high-fat diet consumption modulates hepatic lipid metabolism and microRNA-122 (miR-122) and microRNA-370 (miR-370) expression in offspring. Br J Nutr 2014; 111:2112-22. [DOI: 10.1017/s0007114514000579] [Citation(s) in RCA: 103] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Maternal consumption of a high-fat diet (HFD) during pregnancy and lactation is closely related to hepatic lipid accumulation, insulin resistance and increased serum cytokine levels in offspring and into their adulthood. MicroRNA (miRNA) have been implicated in cholesterol biosynthesis and fatty acid metabolism. We evaluated the modulation of hepatic fatty acid synthesis (de novo), β-oxidation pathways, and miRNA-122 (miR-122) and miRNA-370 (miR-370) expression in recently weaned offspring (day 28) of mouse dams fed a HFD (HFD-O) or a standard chow (SC-O) during pregnancy and lactation. Compared with SC-O mice, HFD-O mice weighed more, had a larger adipose tissue mass and were more intolerant to glucose and insulin (P< 0·05). HFD-O mice also presented more levels of serum cholesterol, TAG, NEFA and hepatic IκB kinase and c-Jun N-terminal kinase phosphorylation compared with SC-O mice (P< 0·05). Protein levels of fatty acid synthase, acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase were similar in HFD-O and SC-O mice, whereas expression levels of SCD1 mRNA and protein were more abundant in HFD-O mice than in SC-O mice (P< 0·05). Interestingly, mRNA expression levels of the β-oxidation-related genes ACADVL and CPT1 were decreased in HFD-O mice (P< 0·05). Furthermore, the expression of miR-122 was reduced but that of miR-370 was increased in HFD-O mice compared with that in SC-O mice (P< 0·05). Changes in hepatic lipid metabolism were accompanied by increased mRNA content of AGPAT1 and TAG deposition in HFD-O mice (P< 0·05). Taken together, the present results strongly suggest that maternal consumption of a HFD affects the early lipid metabolism of offspring by modulating the expression of hepatic β-oxidation-related genes and miRNA that can contribute to metabolic disturbances in adult life.
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Ye CG, Sun SL, Bai R, Zhu WX, Chen CP, Xie P, Zhao H, Tu WJ, Gao DY, Liu LM. Differentially expressed microRNAs in plasma of pretreated patients with/without anti-tuberculosis drugs-induced hepatotoxicity. Shijie Huaren Xiaohua Zazhi 2014; 22:415-422. [DOI: 10.11569/wcjd.v22.i3.415] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate differential expression profile of microRNAs (miRNAs) in plasma of pretreated patients with/without anti-tuberculosis drug-induced hepatotoxicity (ATDH).
METHODS: Plasma samples were collected from patients with/without ATDH before anti-tuberculosis treatment and subjected to miRNA microarray analysis. Twenty-five miRNAs were tested using high-flux real-time quantitative PCR. The target genes of miRNAs were predicted using the Internet software, and the GO functional classification of target proteins was analyzed using the PANTHER tool.
RESULTS: Compared with patients without ATDH, there were 7 miRNAs differentially expressed in patients with ATDH before anti-tuberculosis drug therapy, 4 of which were up-regulated, including miR-4284, miR-3620, miR-652-5p and miR-4800-5p, and 3 down-regulated, including miR-338-3p, miR-424-5p and miR-194-5p.
CONCLUSION: There are differentially expressed miRNAs in the circulation of patients with ATDH before anti-tuberculosis drug therapy, and the up-regulated miRNAs (esp. miR-4284) may be new biological markers for screening ATDH susceptible population.
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McDaniel K, Herrera L, Zhou T, Francis H, Han Y, Levine P, Lin E, Glaser S, Alpini G, Meng F. The functional role of microRNAs in alcoholic liver injury. J Cell Mol Med 2014; 18:197-207. [PMID: 24400890 PMCID: PMC3930407 DOI: 10.1111/jcmm.12223] [Citation(s) in RCA: 97] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2013] [Accepted: 11/28/2013] [Indexed: 12/16/2022] Open
Abstract
The function of microRNAs (miRNAs) during alcoholic liver disease (ALD) has recently become of great interest in biological research. Studies have shown that ALD associated miRNAs play a crucial role in the regulation of liver-inflammatory agents such as tumour necrosis factor-alpha (TNF-α), one of the key inflammatory agents responsible for liver fibrosis (liver scarring) and the critical contributor of alcoholic liver disease. Lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, is responsible for TNF-α release by Kupffer cells. miRNAs are the critical mediators of LPS signalling in Kupffer cells, hepatocytes and hepatic stellate cells. Certain miRNAs, in particular miR-155 and miR-21, show a positive correlation in up-regulation of LPS signalling when they are exposed to ethanol. ALD is related to enhanced gut permeability that allows the levels of LPS to increase, leads to increased secretion of TNF-α by the Kupffer cells and subsequently promotes alcoholic liver injury through specific miRNAs. Meanwhile, two of the most frequently dysregulated miRNAs in steatohepatitis, miR-122 and miR-34a are the critical mediators in ethanol/LPS activated survival signalling during ALD. In this review, we summarize recent findings regarding the experimental and clinical aspects of functions of specific microRNAs, focusing mainly on inflammation and cell survival after ethanol/LPS treatment, and advances on the role of circulating miRNAs in human alcoholic disorders.
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Affiliation(s)
- Kelly McDaniel
- Research, Central Texas Veterans Health Care System, Temple, TX, USA; Department of Medicine, Scott & White Digestive Disease Research Center, Texas A&M University Health Science Center and Scott & White Healthcare, Temple, TX, USA; Academic Operations, Scott & White Hospital, Temple, TX, USA
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35
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Abstract
Today's world population is currently faced with a new type of non-transmissible pandemic: obesity. This lifestyle-related condition is driving the emergence of the diabetes pandemic through the development of low-level chronic inflammation. In recent years, a novel class of non-coding RNA, microRNA (miRNA), have emerged as being important regulators of numerous biological functions. Among these functions are basic maintenance of cell signalling and tissue architecture. Disruption of miRNA levels can contribute not only to the development of the chronic inflammation observed in obese diabetics, but also the development of both pancreatic β-cell dysfunction and loss, along with insulin resistance in metabolic tissues. These primary events set the scene for dysfunction of other tissues, including the retina, kidney, peripheral nerves, heart and the vasculature as a whole. Here, miRNAs again play a deterministic role in the development of a range of diseases collectively termed diabetic complications. Disturbances in miRNA levels appear to be reflected in the serum of patients and this may prove to be diagnostic in patients prior to clinical manifestation of disease, thus improving management of diabetes and its associated complications. Not only are miRNAs displaying promise as an early biomarker for disease, but a number of these miRNAs are displaying therapeutic potential with several in pre-clinical development. The present review aims to highlight our current understanding of miRNAs and their interaction with inflammatory signalling in the development and progression of diabetes and its complications. Utilization of miRNAs as biomarkers and therapeutic targets will also be considered.
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36
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Yap CS, Sinha RA, Ota S, Katsuki M, Yen PM. Thyroid hormone negatively regulates CDX2 and SOAT2 mRNA expression via induction of miRNA-181d in hepatic cells. Biochem Biophys Res Commun 2013; 440:635-639. [PMID: 24103759 DOI: 10.1016/j.bbrc.2013.09.116] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2013] [Accepted: 09/24/2013] [Indexed: 12/12/2022]
Abstract
Thyroid hormones (THs) regulate transcription of many metabolic genes in the liver through its nuclear receptors (TRs). Although the molecular mechanisms for positive regulation of hepatic genes by TH are well understood, much less is known about TH-mediated negative regulation. Recently, several nuclear hormone receptors were shown to downregulate gene expression via miRNAs. To further examine the potential role of miRNAs in TH-mediated negative regulation, we used a miRNA microarray to identify miRNAs that were directly regulated by TH in a human hepatic cell line. In our screen, we discovered that miRNA-181d is a novel hepatic miRNA that was regulated by TH in hepatic cell culture and in vivo. Furthermore, we identified and characterized two novel TH-regulated target genes that were downstream of miR-181d signaling: caudal type homeobox 2 (CDX2) and sterol O-acyltransferase 2 (SOAT2 or ACAT2). CDX2, a known positive regulator of hepatocyte differentiation, was regulated by miR-181d and directly activated SOAT2 gene expression. Since SOAT2 is an enzyme that generates cholesteryl esters that are packaged into lipoproteins, our results suggest miR-181d plays a significant role in the negative regulation of key metabolic genes by TH in the liver.
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Affiliation(s)
- Chui Sun Yap
- Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, 8, College Road, Singapore 169857, Singapore
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37
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Abstract
Inflammation has been considered as one of the hallmarks of cancer, and chronic hepatitis is a major cause of liver cancer. This review will focus on the pathogenic role of inflammation in hepatocarcinogenesis and will discuss recent advances in understanding the chronic hepatitis-liver cancer link based on hot spots in liver cancer research, including cellular interaction, cytokines, microRNA and stem cells. All of these mechanisms should be taken into consideration because they are crucial for the development of more efficacious therapeutic strategies for preventing and treating human chronic hepatitis and hepatocellular carcinoma.
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Vickers KC, Sethupathy P, Baran-Gale J, Remaley AT. Complexity of microRNA function and the role of isomiRs in lipid homeostasis. J Lipid Res 2013; 54:1182-91. [PMID: 23505317 DOI: 10.1194/jlr.r034801] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
MicroRNAs (miRNAs) are key posttranscriptional regulators of biological pathways that govern lipid metabolic phenotypes. Recent advances in high-throughput small RNA sequencing technology have revealed the complex and dynamic repertoire of miRNAs. Specifically, it has been demonstrated that a single genomic locus can give rise to multiple, functionally distinct miRNA isoforms (isomiR). There are several mechanisms by which isomiRs can be generated, including processing heterogeneity and posttranscriptional modifications, such as RNA editing, exonuclease-mediated nucleotide trimming, and/or nontemplated nucleotide addition (NTA). NTAs are dominant at the 3'-end of a miRNA, are most commonly uridylation or adenlyation events, and are catalyzed by one or more of several nucleotidyl transferase enzymes. 3' NTAs can affect miRNA stability and/or activity and are physiologically regulated, whereas modifications to the 5'-ends of miRNAs likely alter miRNA targeting activity. Recent evidence also suggests that the biogenesis of specific miRNAs, or small RNAs that act as miRNAs, can occur through unconventional mechanisms that circumvent key canonical miRNA processing steps. The unveiling of miRNA diversity has significantly added to our view of the complexity of miRNA function. In this review we present the current understanding of the biological relevance of isomiRs and their potential role in regulating lipid metabolism.
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Affiliation(s)
- Kasey C Vickers
- Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA.
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Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis. PLoS One 2013; 8:e55511. [PMID: 23405163 PMCID: PMC3566132 DOI: 10.1371/journal.pone.0055511] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2012] [Accepted: 01/02/2013] [Indexed: 12/03/2022] Open
Abstract
Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3′,5′-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.
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Li YY. Genetic and epigenetic variants influencing the development of nonalcoholic fatty liver disease. World J Gastroenterol 2012; 18:6546-6551. [PMID: 23236228 PMCID: PMC3516206 DOI: 10.3748/wjg.v18.i45.6546] [Citation(s) in RCA: 58] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2012] [Revised: 07/24/2012] [Accepted: 08/14/2012] [Indexed: 02/06/2023] Open
Abstract
Nonalcoholic fatty liver disease (NAFLD) is common worldwide. The importance of genetic and epigenetic changes in etiology and pathogenesis of NAFLD has been increasingly recognized. However, the exact mechanism is largely unknown. A large number of single nucleotide polymorphisms (SNPs) related to NAFLD has been documented by candidate gene studies (CGSs). Among these genes, peroxisome proliferatoractivated receptor-γ, adiponectin, leptin and tumor necrosis factor-α were frequently reported. Since the introduction of genome-wide association studies (GWASs), there have been significant advances in our understanding of genomic variations of NAFLD. Patatin-like phospholipase domain containing family member A3 (PNPLA3, SNP rs738409, encoding I148M), also termed adiponutrin, has caught most attention. The evidence that PNPLA3 is associated with increased hepatic fat levels and hepatic inflammation has been validated by a series of studies. Epigenetic modification refers to phenotypic changes caused by an adaptive mechanism unrelated to alteration of primary DNA sequences. Epigenetic regulation mainly includes microRNAs (miRs), DNA methylation, histone modifications and ubiquitination, among which miRs are studied most extensively. miRs are small natural single stranded RNA molecules regulating mRNA degradation or translation inhibition, subsequently altering protein expression of target genes. The miR-122, a highly abundant miR accounting for nearly 70% of all miRs in the liver, is significantly under-expressed in NAFLD subjects. Inhibition of miR-122 with an antisense oligonucleotide results in decreased mRNA expression of lipogenic genes and improvement of liver steatosis. The investigation into epigenetic involvement in NAFLD pathogenesis is just at the beginning and needs to be refined. This review summarizes the roles of genetics and epigenetics in the development of NAFLD. The progress made in this field may provide novel diagnostic biomarkers and therapeutic targets for NAFLD management.
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Bladé C, Baselga-Escudero L, Salvadó MJ, Arola-Arnal A. miRNAs, polyphenols, and chronic disease. Mol Nutr Food Res 2012; 57:58-70. [PMID: 23165995 DOI: 10.1002/mnfr.201200454] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2012] [Revised: 09/03/2012] [Accepted: 09/13/2012] [Indexed: 12/18/2022]
Abstract
MicroRNAs (miRNAs) are small noncoding RNAs, approximately 18-25 nucleotides in length, that modulate gene expression at the posttranscriptional level. Thousands of miRNAs have been described, and it is thought that they regulate some aspects of more than 60% of all human cell transcripts. Several polyphenols have been shown to modulate miRNAs related to metabolic homeostasis and chronic diseases. Polyphenolic modulation of miRNAs is very attractive as a strategy to target numerous cell processes and potentially reduce the risk of chronic disease. Evidence is building that polyphenols can target specific miRNAs, such as miR-122, but more studies are necessary to discover and validate additional miRNA targets.
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Affiliation(s)
- Cinta Bladé
- Department of Biochemistry and Biotechnology, Universitat Rovira i Virgili, 43007 Tarragona, Spain.
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42
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Abstract
Post-transcriptional gene regulation by microRNAs (miRNAs) and RNA-binding proteins (RBPs) is central to many biological functions. Aberrant gene expression patterns underlie many metabolic diseases that represent major public health concerns and formidable therapeutic challenges. Several studies have established a number of post-transcriptional regulators implicated in metabolic diseases such as diabetes and obesity. In addition, emerging knowledge of metabolically active and insulin-sensitive organs, such as the pancreas, liver, muscle and adipose compartment, is rapidly expanding the panel of potential therapeutic targets for the treatment of metabolic diseases. Here, we review our current understanding of miRNAs and RBPs that affect glucose and lipid homeostasis, and their roles in normal physiology and metabolic disorders, especially type 2 diabetes and obesity.
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Affiliation(s)
- Wook Kim
- Laboratory of Clinical Investigation, National Institute on Aging-Intramural Research Program, NIH, Baltimore, MD, USA
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43
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Rimon N, Schuldiner M. Getting the whole picture: combining throughput with content in microscopy. J Cell Sci 2012; 124:3743-51. [PMID: 22124141 DOI: 10.1242/jcs.087486] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
The increasing availability and performance of automated scientific equipment in the past decades have brought about a revolution in the biological sciences. The ease with which data can now be generated has led to a new culture of high-throughput science, in which new types of biological questions can be asked and tackled in a systematic and unbiased manner. High-throughput microscopy, also often referred to as high-content screening (HCS), allows acquisition of systematic data at the single-cell level. Moreover, it allows the visualization of an enormous array of cellular features and provides tools to quantify a large number of parameters for each cell. These features make HCS a powerful method to create data that is rich and biologically meaningful without compromising systematic capabilities. In this Commentary, we will discuss recent work, which has used HCS, to demonstrate the diversity of applications and technological solutions that are evolving in this field. Such advances are placing HCS methodologies at the frontier of high-throughput science and enable scientists to combine throughput with content to address a variety of cell biological questions.
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Affiliation(s)
- Nitzan Rimon
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel 76100
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44
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Ikeda Y, Tanji E, Makino N, Kawata S, Furukawa T. MicroRNAs associated with mitogen-activated protein kinase in human pancreatic cancer. Mol Cancer Res 2011; 10:259-69. [PMID: 22188669 DOI: 10.1158/1541-7786.mcr-11-0035] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Aberrant expression of microRNAs (miRNA) is associated with phenotypes of various cancers, including pancreatic cancer. However, the mechanism of the aberrant expression is largely unknown. Activation of the mitogen-activated protein kinase (MAPK) signaling pathway plays a crucial role in gene expression related to the malignant phenotype of pancreatic cancer. Hence, we studied the role of MAPK in the aberrant expression of miRNAs in pancreatic cancer cells. The alterations in expression of 183 miRNAs induced by activation or inactivation of MAPK were assayed in cultured pancreatic cancer cells and HEK293 cells by means of the quantitative real-time PCR method. We found that four miRNAs, namely, miR-7-3, miR-34a, miR-181d, and miR-193b, were preferentially associated with MAPK activity. Among these miRNAs, miR-7-3 was upregulated by active MAPK, whereas the others were downregulated. Promoter assays indicated that the promoter activities of the host genes of miR-7-3 and miR-34a were both downregulated by alteration in MAPK activity. Exogenous overexpression of the MAPK-associated miRNAs had the effect of inhibition of the proliferation of cultured pancreatic cancer cells; miR-193b was found to exhibit the most remarkable inhibition. A search for target genes of miR-193b led to identification of CCND1, NT5E, PLAU, STARD7, STMN1, and YWHAZ as the targets. Translational suppression of these genes by miR-193b was confirmed by reporter assay. These results indicate that activation of MAPK may play a significant role in aberrant expression of miRNAs and their associated phenotypes in pancreatic cancer.
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Affiliation(s)
- Yushi Ikeda
- International Research and Educational Institute for Integrated Medical Sciences, Tokyo Women's Medical University, Tokyo, Japan
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Ali AS, Ali S, Ahmad A, Bao B, Philip PA, Sarkar FH. Expression of microRNAs: potential molecular link between obesity, diabetes and cancer. Obes Rev 2011; 12:1050-62. [PMID: 21767342 DOI: 10.1111/j.1467-789x.2011.00906.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Clinicians are routinely challenged in their management of cancer patients because of the complexities of obesity and diabetes that are often found as comorbid conditions. Although attention has been given to optimizing treatment planning for these patients, less attention has been given to manage their obesity and diabetes. This suggests that newer, comprehensive approaches must be developed for the treatment of cancer patients as a 'whole' rather than as a single disease. While the specific pathologies of each are unique, years of research have indicated intimate molecular links between these chronic diseases. The contribution of sedentary lifestyles and poor dietary habits is recognized; however, the precise molecular links are still not well-explored. In addition, emerging evidence suggests the important role of microRNAs (miRNAs) in the development and progression of several diseases, yet their roles in linking obesity, diabetes and cancer are only now beginning to be recognized. It is hoped that miRNAs will serve as novel biomarkers and molecular targets for cancer therapy in patients with comorbid conditions. In this review, we discuss the current understanding of the pathobiology of obesity, diabetes and cancer, and document molecular roles of miRNAs linking cancer with obesity and diabetes.
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Affiliation(s)
- A S Ali
- Department of Pathology, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, USA
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Ranall MV, Gabrielli BG, Gonda TJ. High-content imaging of neutral lipid droplets with 1,6-diphenylhexatriene. Biotechniques 2011; 51:35-6, 38-42. [PMID: 21781051 DOI: 10.2144/000113702] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2010] [Accepted: 05/16/2011] [Indexed: 11/23/2022] Open
Abstract
Neutral lipid droplets (LDs) are dynamic lipid storage organelles found in all eukaryotic cells from yeast to mammals and higher plants. LDs are important to many physiological processes that include basic cellular maintenance, metabolism, and diverse medical pathologies. LD accumulation has been studied extensively by a range of methods, but particularly by microscopy with several fluorescent dyes extensively used for qualitative and quantitative imaging. Here, we compared established LD stains Nile Red and BODIPY 493/503 to the 4', 6-diamidino-2-phenylindole (DAPI)-range dye 1,6-diphenyl-1,3,5-hexatriene (DPH; excitation/emission λmax=350 nm/420 nm) using high-content image analysis. HeLa cells treated with oleic acid or vehicle were used to compare staining patterns between DPH and Nile Red as well as DPH and the LD protein adipophilin. DPH, Nile Red, and BODIPY 493/503 were compared as assay reagents in oleic acid dose-response experiments. Treatment of MCF-7 cells with sodium butyrate was used as a second cellular system for high-content analysis of LD formation. In this experimental context, we demonstrate the compatibility of DPH with GFP, a technical limitation of Nile Red and BODIPY 493/503 dyes. These data show that DPH has comparable sensitivity and specificity to that of Nile Red. Z'-factor analysis of dose-response experiments indicated that DPH and BODIPY 493/503 are well suited for quantitative analysis of LDs for high-throughput screening (HTS) applications.
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Affiliation(s)
- Max V Ranall
- University of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane, QLD, Australia
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Serva A, Claas C, Starkuviene V. A Potential of microRNAs for High-Content Screening. J Nucleic Acids 2011; 2011:870903. [PMID: 21922044 PMCID: PMC3172976 DOI: 10.4061/2011/870903] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2010] [Revised: 05/15/2011] [Accepted: 06/03/2011] [Indexed: 12/19/2022] Open
Abstract
In the last years miRNAs have increasingly been recognised as potent posttranscriptional regulators of gene expression. Possibly, miRNAs exert their action on virtually any biological process by simultaneous regulation of numerous genes. The importance of miRNA-based regulation in health and disease has inspired research to investigate diverse aspects of miRNA origin, biogenesis, and function. Despite the recent rapid accumulation of experimental data, and the emergence of functional models, the complexity of miRNA-based regulation is still far from being well understood. In particular, we lack comprehensive knowledge as to which cellular processes are regulated by which miRNAs, and, furthermore, how temporal and spatial interactions of miRNAs to their targets occur. Results from large-scale functional analyses have immense potential to address these questions. In this review, we discuss the latest progress in application of high-content and high-throughput functional analysis for the systematic elucidation of the biological roles of miRNAs.
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Affiliation(s)
- Andrius Serva
- BioQuant, University of Heidelberg, 69120 Heidelberg, Germany
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Fernandez-Valverde SL, Taft RJ, Mattick JS. MicroRNAs in β-cell biology, insulin resistance, diabetes and its complications. Diabetes 2011; 60:1825-31. [PMID: 21709277 PMCID: PMC3121441 DOI: 10.2337/db11-0171] [Citation(s) in RCA: 163] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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Abstract
PURPOSE OF REVIEW To summarize currently available information about the mechanisms involved in liver fat accumulation. RECENT FINDINGS The contribution of functional genomics approaches, such as those represented by high-throughput analysis and genetically modified mice, may envision a complex network involving fatty acid, triglyceride and phospholipid metabolisms and lipid droplet dynamics. Likewise, it may pose an exquisite regulation exerted through insulin, glucocorticoids, thyroid hormones, transcription factors and microRNAs, orchestrated with sexual differences and able to respond to environmental factors such as nutritional or viral influences among others. SUMMARY The information gathered will facilitate further research to complete gaps of interacting pieces among regulators and new contributing agents emerging from high-throughput analyses. With this new paradigm, new biomarkers able to discriminate the progression of hepatic steatosis into human steatohepatitis will eventually emerge, and hopefully new therapeutic approaches will be developed.
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Affiliation(s)
- José M Lou-Bonafonte
- Departamento de Farmacología y Fisiología, Facultad de Ciencias de Salud y del Deporte, Instituto de Investigación Sanitaria de Aragón, Universidad de Zaragoza, Zaragoza, Spain
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McDonough PM, Ingermanson RS, Loy PA, Koon ED, Whittaker R, Laris CA, Hilton JM, Nicoll JB, Buehrer BM, Price JH. Quantification of hormone sensitive lipase phosphorylation and colocalization with lipid droplets in murine 3T3L1 and human subcutaneous adipocytes via automated digital microscopy and high-content analysis. Assay Drug Dev Technol 2011; 9:262-80. [PMID: 21186937 PMCID: PMC3102254 DOI: 10.1089/adt.2010.0302] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets. HSL:lipid droplet colocalization was quantified through use of Pearson's correlation, Mander's M1 Colocalization, and the Tanimoto coefficient. For murine 3T3L1 adipocytes, isoproterenol, Lys-γ3-melanocyte stimulating hormone, and forskolin elicited the appearance and colocalization of pHSLser660, whereas atrial natriuretic peptide (ANP) did not. For human subcutaneous adipocytes, isoproterenol, forskolin, and ANP activated HSL phosphorylation/colocalization, but Lys-γ3-melanocyte stimulating hormone had little or no effect. Since ANP activates guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase, HSL serine 660 is likely a substrate for cGMP-dependent protein kinase in human adipocytes. For both adipocyte model systems, adipocytes with the greatest lipid content displayed the greatest lipolytic responses. The results for pHSLser660 were consistent with release of glycerol by the cells, a well-established assay of lipolysis, and the HCA methods yielded Z' values >0.50. The results illustrate several key differences between human and murine adipocytes and demonstrate advantages of utilizing HCA techniques to study lipolysis in cultured adipocytes.
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