Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Aug 18, 2016; 8(23): 976-984
Published online Aug 18, 2016. doi: 10.4254/wjh.v8.i23.976
Interplay between microRNA-17-5p, insulin-like growth factor-II through binding protein-3 in hepatocellular carcinoma
Danira Ashraf Habashy, Hend Mohamed El Tayebi, Injie Omar Fawzy, Karim Adel Hosny, Gamal Esmat, Ahmed Ihab Abdelaziz
Danira Ashraf Habashy, Hend Mohamed El Tayebi, Injie Omar Fawzy, Department of Pharmacology and Toxicology, German University in Cairo, Main Entrance Al Tagamoa Al Khames, Cairo 11835, Egypt
Karim Adel Hosny, Department of General Surgery, Faculty of Medicine, Cairo University, Cairo 11562, Egypt
Gamal Esmat, Department of Endemic Medicine and Hepatology, Cairo University, Cairo 11562, Egypt
Ahmed Ihab Abdelaziz, Department of Biology, American University in Cairo, New Cairo City, Cairo 11835, Egypt
Author contributions: Habashy DA planned and executed all the experiments, acquired the data, performed the statistical analysis, interpretation of data, and wrote the manuscript; Habashy DA, El Tayebi HM and Abdelaziz AI contributed in study concept and design; El Tayebi HM contributed in study co-supervision and revision of the manuscript; Fawzy IO assisted in the binding confirmation experiment and revision of the manuscript; Hosny KA and Esmat G provided tissue samples and clinical data; Abdelaziz AI contributed in study supervision, and critical revision of the manuscript for important intellectual content; all authors approved the final version of the article to be published.
Institutional review board statement: The study was approved by the ethical review committees of the German University in Cairo and Cairo University.
Informed consent statement: All liver biopsy specimens from patients and healthy donors were taken after informed consent and ethical permission was obtained for participation in the study.
Conflict-of-interest statement: To the best of our knowledge, no conflict of interest exists.
Data sharing statement: All liver biopsy specimens from patients and healthy donors were taken after informed consent was obtained for participation in the study. Any clinical data stated in the manuscript is anonymous.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Ahmed Ihab Abdelaziz, MD, PhD, Associate Professor of Molecular Medicine, Department of Biology, American University in Cairo, New Cairo City, AUC Avenue, Cairo 11835, Egypt. ahmed.ihab.abdelaziz@gmail.com
Telephone: +20-2-26151000 Fax: +20-2-27957565
Received: March 6, 2016
Peer-review started: March 7, 2016
First decision: May 17, 2016
Revised: June 1, 2016
Accepted: July 11, 2016
Article in press: July 13, 2016
Published online: August 18, 2016
Processing time: 163 Days and 12.3 Hours
Abstract

AIM: To investigate the effect of microRNA on insulin-like growth factor binding protein-3 (IGFBP-3) and hence on insulin-like growth factor-II (IGF-II) bioavailability in hepatocellular carcinoma (HCC).

METHODS: Bioinformatic analysis was performed using microrna.org, DIANA lab and Segal lab softwares. Total RNA was extracted from 23 HCC and 10 healthy liver tissues using mirVana miRNA Isolation Kit. microRNA-17-5p (miR-17-5p) expression was mimicked and antagonized in HuH-7 cell lines using HiPerFect Transfection Reagent, then total RNA was extracted using Biozol reagent then reverse transcribed into cDNA followed by quantification of miR-17-5p and IGFBP-3 expression using TaqMan real-time quantitative PCR. Luciferase reporter assay was performed to validate the binding of miR-17-5p to the 3’UTR of IGFBP-3. Free IGF-II protein was measured in transfected HuH-7 cells using IGF-II ELISA kit.

RESULTS: Bioinformatic analysis revealed IGFBP-3 as a potential target for miR-17-5p. Screening of miR-17-5p and IGFBP-3 revealed a moderate negative correlation in HCC patients, where miR-17-5p was extensively underexpressed in HCC tissues (P = 0.0012), while IGFBP-3 showed significant upregulation in the same set of patients (P = 0.0041) compared to healthy donors. Forcing miR-17-5p expression in HuH-7 cell lines showed a significant downregulation of IGFBP-3 mRNA expression (P = 0.0267) and a significant increase in free IGF-II protein (P = 0.0339) compared to mock untransfected cells using unpaired t-test. Luciferase assay validated IGFBP-3 as a direct target of miR-17-5p; luciferase activity was inhibited by 27.5% in cells co-transfected with miR-17-5p mimics and the construct harboring the wild-type binding region 2 of IGFBP-3 compared to cells transfected with this construct alone (P = 0.0474).

CONCLUSION: These data suggest that regulating IGF-II bioavailability and hence HCC progression can be achieved through targeting IGFBP-3 via manipulating the expression of miRNAs.

Keywords: Insulin-like growth factor binding protein-3; Insulin-like growth factor signaling pathway; MicroRNA; Insulin-like growth factor-II; Hepatocellular carcinoma

Core tip: microRNA-17-5p (miR-17-5p) was extensively underexpressed in hepatocellular carcinoma tissues, while insulin-like growth factor binding protein-3 (IGFBP-3) mRNA showed significant upregulation in the same set of patients. In HuH-7 cell line, miR-17-5p directly targets and downregulates IGFBP-3, consequently elevating the level of free insulin-like growth factor-II (IGF-II). Thus, manipulation of microRNAs can potentially control the activation of the oncogenic IGF axis.