MicroRNA - Volume 4, Issue 2, 2015

Micro-RNAs (miRNAs) are responsible for important and evolutionary-conserved regulatory functions in several cellular processes such as apoptosis, signalling, differentiation and proliferation. There is a growing interest in understanding more clearly the mechanisms regulating activation and suppression of miRNAs expression in benefit of health prevention advancement. It is now acknowledged that physical activity represents one of the most effective preventive agents in chronicdegenerative diseases. Indeed, a regular exercise exerts a great influence on several parameters and biological pathways, both at genomic and post-genomic levels. Recent works have highlighted the effects of structured physical activity on miRNAs modulation. Modulation of MiRNAs, regulated by exercise in human skeletal muscle, depends on type, duration and intensity of an exercise performed. The aim of this review is to provide a comprehensive overview of scientific evidence concerning the effects of physical activity on miRNAs and its relevance for chronic-degenerative diseases prevention.

Several indirect evidences are available to indicate that abnormalities in cell cycle may contribute to pathogenesis of Huntington’s disease (HD). Here, we show that the cell cycle progression in STsdhQ111/HdhQ111cells, a cell model of HD, is delayed in S and G2-M phases compared to control STHdhQ7/HdhQ7cells. Expression of 17 genes, like PCNA and CHEK1, was increased in STHdhQ111/HdhQ111cells. Increased expressions of PCNA, CHEK1 and CCNA2, and an enhanced phosphorylation of Rb1 were observed in primary cortical neurons expressing mutant N-terminal huntingtin (HTT), R6/2 mice and STHdhQ111/HdhQ111 cells. This increase in the expressions of PCNA, CHEK1 and CCNA2 was found to be the result of decreased expressions of miR-432, miR-146a, and (miR-19a and miR-146a), respectively. Enhanced apoptosis was observed at late S phase and G2-M phase in STHdhQ111/HdhQ111cells. Exogenous expressions of these miRNAs in STHdhQ111/HdhQ111 cells rescued the abnormalities in cell cycle and apoptosis. We also observed that inhibitors of cell cycle could decrease cell death in a cell model of HD. Based on these results obtained in cell and animal model of HD, we propose that inhibition of cell cycle either by miRNA expressions or by using inhibitors could be a potential approach for the treatment of HD.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression posttranscriptionally by interfering with translation of their target mRNAs. Typically, miRNAs bind to the 3’ UTRs of mRNAs to induce repression or degradation. Neurotrophins are growth factors in brain required for neuronal survival, synapse formation, and plasticity mechanisms. Neurotrophins are not only regulated by miRNAs, but they in turn regulate miRNA expression. Accumulating data indicate there is a regulatory negative feedback loop between one ubiquitous neurotrophin, brain-derived neurotrophic factor (BDNF), and miRNAs. That is, while BDNF treatment stimulates neuronal miRNA expression, miRNAs generally function to inhibit expression of BDNF. This negative feedback loop is maintained in a state of equilibrium in normal cells. However, in Alzheimer’s Disease (AD), a progressive neurodegenerative disorder resulting in memory loss and eventually dementia that is characterized by reduced levels of BDNF in brain, the balance between BDNF and miRNA is shifted toward inhibitory control by miRNAs. Here, we will briefly review the evidence for a positive action of BDNF on miRNA expression and a negative action of miRNAs on BDNF. We propose that the reduction in BDNF that occurs in the AD brain is the result of two independent mechanisms: 1) a failure in the proteolytic conversion of BDNF precursor protein to its functional mature form, and 2) inhibition of BDNF gene expression by miRNAs. The role of miRNAs in BDNF regulation should be considered when developing BDNF-based therapeutic treatments for AD.

Eukaryotic ribonucleases (RNase) H1 and H2 are endonucleases that cleave RNA in a double- stranded RNA-DNA molecule. RNase H2 can also cleave a single ribonucleotide embedded in DNA duplex. While the activity of RNase H1 and H2 has been extensively characterized in vitro, still much is unclear about the specific targets of these enzymes in vivo. We recently demonstrated that yeast cells can repair a double-strand break (DSB) in DNA by homologous recombination (HR) using antisense (non-coding) RNA, either directly, or indirectly after converting RNA into cDNA. In wildtype RNase H1 and/or H2 cells, repair by cDNA dominates, whereas in the absence of RNase H1 and H2 functions cDNA and, in particular, direct transcript-RNA repair mechanisms are markedly stimulated. Here we found that null alleles of any of the three RNase H2 subunits stimulate DSB repair by cDNA significantly more than a null allele of RNase H1. These results show that RNase H2 is the preferred RNase H enzyme to target cDNA in yeast. Targeting of cDNA by RNase H2 does not require RNase H2 interaction with the DNA clamp proliferating cell nuclear antigen (PCNA). Moreover, yeast RNase H2 orthologous mutants of two common RNase H2 defects associated with Aicardi Goutières syndrome (AGS) in humans, displayed elevated cDNA-driven repair of a DSB when combined with each other or with RNase H1 null mutation. Our findings support the hypothesis that defective RNase H2 alleles have higher level of cDNA derived from either coding or non-coding RNA in the form of RNA-cDNA hybrids.

T-cell activation affects microRNA (miRNA) expression in T-cell subsets. However, little is known about the kinetics of miRNA regulation and possible differences between CD4 and CD8 T cells. In this study we set out to analyze the kinetics of activation-induced expression regulation of twelve pre-selected miRNAs. The dynamics of the expression of these miRNAs was studied in sorted CD4 and CD8 CD45RO- T cells of healthy individuals stimulated with αCD3/αCD28 antibodies. Analysis of miRNA levels at day 3, 5, 7 and 10 showed significant activation-induced changes in expression levels of all twelve miRNAs. Expression levels of nine miRNAs, including miR-21, miR-146a and miR-155, were induced following activation, whereas expression of three miRNAs, including miR-31, were decreased following activation. The expression changes of miR-18a and miR-155 was relatively early, at day 3, whereas expression of miR-451, miR-21 and miR-146a was evident at day 5, 7 and 10, respectively. Four miRNAs showed a differential regulation between CD4 and CD8 T cells. Induction of miR-18a and miR-21 was more pronounced and occurred earlier in CD4 T cells compared to CD8 T cells. Downregulation of miR-223 and miR-451 was also more pronounced in CD4 T cells compared to CD8 T cells. In conclusion, we show a complex pattern of miRNA expression regulation upon T-cell activation with early and late as well as CD4 and CD8 T-cell specific changes. These differences might be the result of differences in kinetics and efficiency of CD4 and CD8 T cells in response to antigen priming.

Hepatitis B virus (HBV) infection can lead to various disease states including asymptomatic, acute hepatitis, chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC), which remain a major health problem worldwide. Previous studies demonstrated that microRNA (miRNAs) plays an important role in viral replication. This study aimed to predict and evaluate human miRNAs targeting multiple genotypes of HBV. Candidate human miRNAs were analyzed by data obtained from miRBase and RNAhybrid. Then miRNAs were selected based on hybridization patterns and minimum free energy (MFE). The silencing effect of miRNA was evaluated by real-time PCR, the luciferase reporter assay and the ELISA assay. Five human miRNAs including miR- 142-5p, miR-384, miR-500b, miR-4731-5p and miR-5193 were found to target several HBV genotypes. Interestingly, miR-5193 was found to be the most potent miRNA that could target against all HBV transcripts in almost all HBV genotypes with a highly stable hybridization pattern (5' canonical with MFE lower than -35 kcal/mol). Moreover, miR-5193 caused significant silencing in luciferase activity (53% reduction), luciferase transcript (60% reduction) and HBV surface antigen (HBsAg) production (20-40% reduction depending on genotypes). Therefore, miR-5193 might be useful and have a vital role for inhibition of HBV replication in the future.

Brain glial tumors, and particularly glioblastomas, are tumors with a very poor prognosis. Currently, the parameters that control aggressiveness, migration, or chemoresistance are not well known. In this tumor context, microRNAs are thought to be essential actors of tumorigenesis as they are able to control the expression of numerous genes. microRNAs are not only active in controlling tumor cell pathways, they are also secreted by cells, inside microvesicles called exosomes, and may play specific roles outside the tumor cells in the tumor microenvironment. We analyzed the microRNA content of exosomes produced in vitro by normal glial cells (astrocytes) and tumor glial cells (U87 MG) using Affymetrix microarrays. It appears that the exosome microRNA profiles are qualitatively quite similar. Nevertheless, their quantitative profiles are different and may be potentially taken as an opportunity to carry out assays of diagnostic interest. We submitted the cultured cells to several stresses such as oxygen deprivation or treatments with chemical drugs (GW4869 or 5-Aza-2’- deoxycitidine) to assess the impact of the cellular microRNA profile modifications on the exosome microRNA profiles. We found that modifications of the cellular microRNA content are not strictly mirrored in exosomes. On the basis of these results, we propose that the way microRNAs are released in exosomes is probably the result of a combination of different excretion mechanisms or constraints that concur in a controlled regulation of the exosome microRNA secretion.