Antisense circular RNA is a special type of circular RNA that is derived from the antisense complementary strand of parental mRNA. However, the function of antisense circRNA in hepatocellular carcinoma (HCC) is still unclear. Here, we reported that CSF3R-AS was upregulated in HCC and correlated with a poor prognosis. CSF3R-AS promoted the proliferation, angiogenesis, and metastasis of HCC, and inhibited apoptosis. Mechanistically, CSF3R-AS has a 180-base complementary pairing sequence with its parental mRNA CSF3R, which can directly bind to CSF3R and recruit RBMS3 to stabilize its parental mRNA, and finally activate JAK2/STAT3 signaling pathway. Interestingly, STAT3 can act as a transcription factor of CSF3R-AS, which means that there is a CSF3R-AS/CSF3R/JAK2/STAT3 positive feedback loop in HCC. Finally, the CSF3R-AS/CSF3R/JAK2/STAT3 positive feedback loop was also activated in HCC sorafenib-resistant cells, and blocking this loop was expected to improve the sensitivity of HCC to sorafenib. These findings suggested that the CSF3R-AS/CSF3R/JAK2/STAT3 positive feedback loop could promote HCC progression and sorafenib resistance. Blocking this loop is expected to provide new research directions and therapy targets for HCC.

Regnase-1 is an ribonuclease that plays essential roles in restricting inflammation through degrading messenger RNAs (mRNAs) involved in immune reactions via the recognition of stem-loop (SL) structures in the 3' untranslated regions (3'UTRs). Dysregulated expression of Regnase-1 is associated with the pathogenesis of inflammatory and autoimmune diseases in mice and humans. Here, we developed a therapeutic strategy to suppress inflammatory responses by blocking Regnase-1 self-regulation, which was mediated by the simultaneous use of two antisense phosphorodiamidate morpholino oligonucleotides (MOs) to alter the binding of Regnase-1 toward the SL structures in its 3'UTR. Regnase-1-targeting MOs not only enhanced Regnase-1 expression by stabilizing mRNAs but also effectively reduced the expression of multiple proinflammatory transcripts that were controlled by Regnase-1 in macrophages. Intratracheal administration of Regnase-1-targeting MOs ameliorated acute respiratory distress syndrome and chronic fibrosis through suppression of inflammatory cascades. In addition, intracranial treatment with Regnase-1-targeting MOs attenuated the development of experimental autoimmune encephalomyelitis by promoting the expansion of homeostatic microglia and regulatory T cell populations. Regnase-1 expression was inversely correlated with disease severity in patients with multiple sclerosis, and MOs targeting human Regnase-1 SL structures were effective in mitigating cytokine production in human immune cells. Collectively, MO-mediated disruption of the Regnase-1 self-regulation pathway is a potential therapeutic strategy to enhance Regnase-1 abundance, which, in turn, provides therapeutic benefits for treating inflammatory diseases by suppressing inflammation.

The initial identification of long non-coding RNA myocardial infarction associated transcript (MIAT) as a genetic risk factor of myocardial infarction has made this lncRNA (designated as lncR-MIAT here) a focus of intensive studies worldwide. Emerging evidence supports that lncR-MIAT is susceptible in its expression to multiple deleterious factors like angiotensin II, isoproterenol, hypoxia, and infection and is anomaly overexpressed in serum, plasma, blood cells and myocardial tissues under a variety of cardiovascular conditions including myocardial infarction, cardiac hypertrophy, diabetic cardiomyopathy, dilated cardiomyopathy, sepsis cardiomyopathy, atrial fibrillation and microvascular dysfunction. Experimental results consistently demonstrated that upregulation of lncR-MIAT plays active roles in the pathological processes of the cardiovascular system and knockdown of this lncRNA effectively ameliorates the adverse conditions. The available data revealed that lncR-MIAT acts through multiple mechanisms such as competitive endogenous RNA, natural antisense RNA and RNA/protein interactions. Moreover, the functional domains of lncR-MIAT accounting for certain specific cellular functions of the full-length transcript have been identified and characterized. These insights will not only tremendously advance our understanding of lncRNA biology and pathophysiology, but also offer good opportunities for more innovative and precise design of agents that have the potential to be developed into new drugs for better therapy of cardiovascular diseases (CVDs) in the future. Herein, we provide an overview of lncR-MIAT, focusing on its roles in cardiovascular diseases, underline the unique cellular/molecular mechanisms for its actions, and speculate the perspectives about the translational studies on the potential diagnostic and therapeutic applications of lncR-MIAT.

Drug candidates derived from oligonucleotides (ON) are receiving increased attention that is supported by the clinical approval of several ON drugs. Such therapeutic ON are designed to alter the expression levels of specific disease-related proteins, e.g., by displaying antigene, antisense, and RNA interference mechanisms. However, the high polarity of the polyanionic ON and their relatively rapid nuclease-mediated cleavage represent two major pharmacokinetic hurdles for their application in vivo. This has led to a range of non-natural modifications of ON structures that are routinely applied in the design of therapeutic ON. The polyanionic architecture of ON often hampers their penetration of target cells or tissues, and ON usually show no inherent specificity for certain cell types. These limitations can be overcome by conjugation of ON with molecular entities mediating cellular 'targeting', i.e., enhanced accumulation at and/or penetration of a specific cell type. In this context, the use of small molecules as targeting units appears particularly attractive and promising. This review provides an overview of advances in the emerging field of cellular targeting of ON via their conjugation with small-molecule targeting structures.

Ciliopathies are a group of hereditary disorders that result from structural or functional abnormalities of cilia. Recent intense research efforts have uncovered the genetic bases of ciliopathies, and our understanding of the assembly and functions of cilia has been improved significantly. Although mechanism-specific therapies for ciliopathies have not yet received regulatory approval, the use of innovative therapeutic modalities such as oligonucleotide therapy, gene replacement therapy, and gene editing in addition to symptomatic treatments are expected to provide valid treatment options in the near future. Moreover, candidate chemical compounds for developing small molecule drugs to treat ciliopathies have been identified. This review introduces the key features of cilia and ciliopathies, and summarizes the advances as well as the challenges that remain with the development of therapies for treating ciliopathies.

Pulsed electron double resonance (PELDOR) experiments on oligonucleotides provide a distance ruler that allows the measurement of nanometer distances accurately. The technique requires attachment of nitroxide spin labels to the nucleotides, which may possibly perturb its conformation. To study to what extent nitroxide spin labels may affect RNA structure, all-atom molecular dynamics simulations in explicit solvent are performed for six double-labeled RNA duplexes. A new parametrization of the force field for the nitroxide spin label is developed, which leads to intramolecular distances that are in good agreement with experimental results. Comparison of the results for spin-labeled and unlabeled RNA reveals that the conformational effect of the spin label depends significantly on whether the spin label is attached to the major or the minor groove of RNA. While major-groove spin labeling may to some extent affect the conformation of nearby base pairs, minor-groove spin labeling has the advantage of mostly preserving the RNA conformation.

Studies on the regulatory RNA MicF in Enterobacteriaceae reveal a pivotal role in gene regulation. Multiple target gene mRNAs were identified and, importantly, MicF RNA regulates the expression of the global regulatory gene lrp (Holmqvist et al., 2012; Corcoran et al., 2012). Thus MicF RNA is a central factor in a regulatory network that regulates bacterial cell physiology.

Recent progress in the analyses of the mouse transcriptome leads to unexpected discoveries. The mouse genomic sequences read by RNA polymerase II may be six times more than previously expected for human chromosomes. The transcript-abundant regions (named "transcription forests") occupy more than half of the genomic sequence and are divided by transcript-scarce regions (transcription deserts). Many of the coding mRNAs may have partially overlapping antisense RNAs. There are transcripts bridging several adjacent genes that were previously regarded as distinct ones. The transcription start sites appearing as cap analysis of gene expression (CAGE) tags are mapped on the mouse genomic sequences. Distributions of CAGE tags show that the shapes of mammalian gene promoters can be classified into four major categories. These shapes were conserved between mouse and human. Most of the gene has exonic transcription start sites, especially in the 3' untranslated region (3' UTR) sequences. The term "RNA continent" has been invented to express this unexpectedly complex and prodigious mouse transcriptome. More than a half of the RNA polymerase II transcripts are regarded as noncoding RNAs (ncRNAs). The great variety of ncRNAs in mammalian transcriptome implies that there are many functional ncRNAs in the cells. Especially, the evolutionarily conserved microRNAs play critical roles in mammalian development and other biological functions. Moreover, many other ncRNAs have also been shown to have biological significant functions, mainly in the regulation of gene expression. The functional survey of the RNA continent has just started. We will describe the state of the art of the RNA continent and its impact on the modern molecular biology, especially on the cancer research.

To explore the possibility of repression of chloromycetin (Cm) acyl transferase by using external guided sequence (EGS) in order to converse the clinical E coli isolates from Cm- resistant to Cm- sensitive.

EGS directed against chloromycetin acetyl transferase gene (cat) was cloned to vector pEGFP-C1 which contains the kanamycin (Km) resistance gene. The recombinant plasmid pEGFP-C1+EGScat1+cat2 was constructed and the blank vector without EGS fragment was used as control plasmids. By using the CaCl(2) transformation method, the recombinant plasmids were introduced into the clinically isolated Cm resistant but Km sensitive E coli strains. Transformants were screened on LB agar plates containing Km. Extraction of plasmids and PCR were applied to identify the positive clones. The growth curve of EGS transformed bacteria cultured in broth with Cm resistance was determined by using spectrophotometer at A(600). Drug sensitivity was tested in solid culture containing Cm by using KB method.

Transformation studies were carried out on 16 clinically isolated Cm-resistant (250 microg/mL of Cm) E coli strains by using pEGFP-C1-EGScat1cat2 recombinant plasmid. Transformants were screened on LB-agar plates containing Km after the transformation using EGS. Of the 16 tested strains, 4 strains were transformed successfully. Transformants with EGS plasmid showed growth inhibition when grown in liquid broth culture containing 200 microg/mL of Cm. In drug sensitivity test, these strains were sensitive to Cm on LB-agar plates containing 200 microg/mL of Cm. Extraction of plasmids and PCR amplification showed the existence of EGS plasmids in these four transformed strains. These results indicated that the Cat of the four clinical isolates had been suppressed and the four strains were converted to Cm sensitive ones.

The EGS directed against Cat is able to inhibit the expression of Cat, and hence convert Cm-resistant bacteria to Cm-sensitive ones. Thus, the EGS has the capability of converting the phenotype of clinical drug-resistant isolates strains to drug-sensitive ones.

In prokaryotes, a number of endogenous antisense RNAs have been detected and found to exert various biological functions. In eukaryotes antisense RNAs have been found; however, a lack of experimental methodologies that permit the identification of overlapping transcripts in cells presents a barrier to a more systematic identification of antisense RNA. Here we have developed an experimental strategy that allows systematic identification of endogenous mRNAs with long complementary regions to other transcripts. The method was applied to human normal mammary epithelial and breast cancer cells. Experimental validation of the presence of the sense and antisense transcripts by various techniques (e.g., northern blots, RT-PCR) supports the specificity of the method. When the antisense RNAs were specifically targeted, their corresponding mRNA levels significantly altered, a result consistent with a regulatory role for the identified antisense RNAs.

Rodent cells, widely used for the industrial production of recombinant human glycoproteins, possess CMP-N-acetylneuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase; EC 1.14.13.45) which is the key enzyme in the formation of the sialic acid, N-glycolylneuraminic acid (Neu5Gc). This enzyme is not expressed in an active form in man and evidence suggests that the presence of Neu5Gc in recombinant therapeutic glycoproteins may elicit an immune response. The aim of this work was, therefore, to reduce CMP-Neu5Ac hydroxylase activity in a Chinese Hamster Ovary (CHO) cell line, and thus the Neu5Gc content of the resulting glycoconjugates, using a rational antisense RNA approach. For this purpose, the cDNA of the hamster hydroxylase was partially cloned and sequenced. Based on the sequence of the mouse and hamster cDNAs, optimal antisense RNA fragments were selected from preliminary in vitro translation tests. Compared to the parental cell line, the new strain (CHO-AsUH2), which was transfected with a 199-bp antisense fragment derived from the mouse CMP-Neu5Ac hydroxylase cDNA, showed an 80% reduction in hydroxylase activity. An analysis of the sialic acids present in the cells' own glycoconjugates revealed a decrease in the percentage of Neu5Gc residues from 4% in the parental cells to less than 1% in the CHO-AsUH2 cell line.

micF RNA, a small regulatory RNA found in bacteria, post-transcriptionally regulates expression of outer membrane protein F (OmpF) by interaction with the ompF mRNA 5'UTR. Phylogenetic data can be useful for RNA/RNA duplex structure analyses and aid in elucidation of mechanism of regulation. However micF and associated genes, ompF and ompC are difficult to annotate because of either similarities or divergences in nucleotide sequence. We report by using sequences that represent "gene signatures" as probes, e.g., mRNA 5'UTR sequences, closely related genes can be accurately located in genomic sequences.

Alignment and search methods using NCBI BLAST programs have been used to identify micF, ompF and ompC in Yersinia pestis and Yersinia enterocolitica. By alignment with DNA sequences from other bacterial species, 5' start sites of genes and upstream transcriptional regulatory sites in promoter regions were predicted. Annotated genes from Yersinia species provide phylogenetic information on the micF regulatory system. High sequence conservation in binding sites of transcriptional regulatory factors are found in the promoter region upstream of micF and conservation in blocks of sequences as well as marked sequence variation is seen in segments of the micF RNA gene. Unexpected large differences in rates of evolution were found between the interacting RNA transcripts, micF RNA and the 5' UTR of the ompF mRNA. micF RNA/ompF mRNA 5' UTR duplex structures were modeled by the mfold program. Functional domains such as RNA/RNA interacting sites appear to display a minimum of evolutionary drift in sequence with the exception of a significant change in Y. enterocolitica micF RNA.

Newly annotated Yersinia micF and ompF genes and the resultant RNA/RNA duplex structures add strong phylogenetic support for a generalized duplex model. The alignment and search approach using 5' UTR signatures may be a model to help define other genes and their start sites when annotated genes are available in well-defined reference organisms.

An increasing number of eukaryotic genes are being found to have naturally occurring antisense transcripts. Here we study the extent of antisense transcription in the human genome by analyzing the public databases of expressed sequences using a set of computational tools designed to identify sense-antisense transcriptional units on opposite DNA strands of the same genomic locus. The resulting data set of 2,667 sense-antisense pairs was evaluated by microarrays containing strand-specific oligonucleotide probes derived from the region of overlap. Verification of specific cases by northern blot analysis with strand-specific riboprobes proved transcription from both DNA strands. We conclude that > or =60% of this data set, or approximately 1,600 predicted sense-antisense transcriptional units, are transcribed from both DNA strands. This indicates that the occurrence of antisense transcription, usually regarded as infrequent, is a very common phenomenon in the human genome. Therefore, antisense modulation of gene expression in human cells may be a common regulatory mechanism.

The micF gene is a stress response gene found in Escherichia coli and related bacteria that post-transcriptionally controls expression of the outer membrane porin gene ompF. The micF gene encodes a non-translated 93 nt antisense RNA that binds its target ompF mRNA and regulates ompF expression by inhibiting translation and inducing degradation of the message. In addition, other factors, such as the RNA chaperone protein StpA also play a role in this regulatory system. Expression of micF is controlled by both environmental and internal stress factors. Four transcriptional regulators are known to bind the micF promoter region and activate micF expression. The crystal structure of one these transcriptional activators, Rob, complexed with the micF promoter has been reported. Here, we review new developments in the micF regulatory network.

Because the expression level of hTERT, a catalytic subunit of human telomerase, is a rate-limiting determinant of telomerase activity, hTERT mRNA would be an excellent target of hammerhead ribozymes for the regulation of telomerase activity. We studied the efficiency of several hammerhead ribozymes targeting hTERT mRNA by transient and stable transfection procedures. To screen the potency of the ribozymes, transient ribozyme transfection and telomerase determination were performed. The ribozyme targeting 13 nucleotides downstream from the 5'-end of hTERT mRNA (13-ribozyme) exhibited the strongest telomerase-inhibitory activity, and the ribozyme to target 59 nucleotides upstream from the poly(A) tail showed clear activity. A stable transfection study confirmed that the 13-ribozyme suppressed telomerase. These observations suggest that the 13-ribozyme can regulate telomerase activity and may possess potential for cancer therapy.

Although lipid-based DNA delivery systems are being assessed in gene therapy clinical trials, many investigators in this field are concerned about the inefficiency of lipid-based gene transfer technology, a criticism directed at all formulations used to enhance transfer of plasmid expression vectors. It is important to recognize that many approaches have been taken to improve transfection efficiency, however because of the complex nature of the formulation technology being developed, it has been extremely difficult to define specific carrier attributes that enhance transfection. We believe that these optimization processes are flawed for two reasons. First, a very defined change in formulation components affects the physical and chemical characteristics of the carrier in many ways. As a consequence, it has not been possible to define structure/activity relationships. Second, the primary endpoint used to assess plasmid delivery has been transgene expression, an activity that is under the control of cellular processes that have nothing to do with delivery. Gene expression following administration of a plasmid expression vector involves a number of critical steps: (i) DNA protection, (ii) binding to a specific cell population, (iii) DNA transfer across the cell membrane, (iv) release of DNA into the cytoplasm, (v) transport through the cell and across the nuclear membrane as well as (vi) transcription and translation of the gene. The objective of this review is to describe lipid-based DNA carrier systems and the attributes believed to be important in regulating the transfection activity of these formulations. Although membrane destabilization activity of the lipid-based carriers plays an important role, we suggest here that a critical element required for efficient transfection is dissociation of lipids bound to the plasmid expression vector following internalization.

OxyS is a small untranslated RNA which is induced in response to oxidative stress in Escherichia coli. This novel RNA acts as a global regulator to activate or repress the expression of as many as 40 genes, including the fhlA-encoded transcriptional activator and the rpoS-encoded sigma(s) subunit of RNA polymerase. Deletion analysis of OxyS showed that different domains of the small RNA are required for the regulation of fhlA and rpoS. We examined the mechanism of OxyS repression of fhlA and found that the OxyS RNA inhibits fhlA translation by pairing with a short sequence overlapping the Shine-Dalgarno sequence, thereby blocking ribosome binding/translation.

Antisense molecules and ribozymes capture the imagination with their promise of rational drug design and exquisite specificity. However, they are far more difficult to produce than was originally anticipated, and their ability to eliminate the function of a single gene has never been proven. Furthermore, a wide variety of unexpected non-antisense effects have come to light. Although some of these side effects will almost certainly have clinical value, they make it hard to produce drugs that act primarily through true antisense mechanisms and complicate the use of antisense compounds as research reagents. To minimize unwanted non-antisense effects, investigators are searching for antisense compounds and ribozymes whose target sites are particularly vulnerable to attack. This is a challenging quest.