Functions Of Differentially Expressed MicroRNAs In Human Heart Failure

BackgroundHeart disease is the greatest noninfectious health hazard ever to confront the human race. It is the leading cause of death in industrialized nations, and accordingly, it is responsible for a huge economic burden to society. It is estimated that 5 million Americans have heart failure (HF), a syndrome with mortality of approximately 50% at 5 years. Now, evidence indicates that the epidemic of HF is extending to the developing world.The road to advances in the treatment of HF is littered with failures, and many new means of diagnosing and tracking disease progression have disappointed. Therefore, there is an urgent need to identify novel mechanisms, markers and therapeutic targets in HF pathogenesis.MicroRNAs were first discovered in 1993 in Caenorhabditis elegans and are known to be critical to early vertebrate development and in an increasing number of basic molecular processes. Over the last few years, miRs have emerged as important mechanisms in human disease, including cardiovascular disease, diabetes and cancer.In recent years, microRNAs (miRs) have caused a true revolution in the cardiovascular research field. MiRs are a class of highly conserved, small noncoding RNAs. They are transcribed from "normal" genes, and their precursor transcripts are enzymatically processed to a mature and active form by Drosha/Dgcr8 and Dicer enzyme complexes. MiRs fine tune the expression of 30% to 50% of the protein-coding genes by binding to partly complementary base pairs in 3’untranslated regions (UTRs) of mRNAs and thereby interfering with translation; targeted mRNAs are either degraded or temporarily silenced. In this way, miRs add another level of complexity to the highly regulated eukaryotic interactome, and their actions (partly) explain why mRNA expression so often does not agree with protein expression levels. So far,>700 human and 500 mouse miR genes have been included in the miRBase (www.microrna.sanger.ac.uk), the primary source of miR data, and computational analyses predict that these numbers will increase.MiRs are of crucial importance to the heart to develop and function properly. In mice, deletion of all of the cardiac miRs by cardiomyocyte Dgcr8 or Dicer knockout is not tolerated, and also the specific deletion of cardiac miRs-1 or-133 leads to (partial) embryonic lethality and heart failure.From 2001 until now, several studies have investigated miR expression in the healthy heart, during pressure overload in mice and rats and in different etiologies of human heart failure. MiRs that consistently come to attention are let-7b and miR-15b,-21,-23a,-27b,-103,-125b,-140*,-195,-199a and-214, which are upregulated in all of the cardiac pathologies studied, as well as miR-30e,-150,-185 and-422b, which are all downregulated during hypertensive heart diseases. Still, the identification of miRs involved in cardiac development and pathology and knowledge on their biological function is exceedingly incomplete, offering an exciting challenge to cardiovascular researchers. Therefore, miRs currently seem to be the most popular kid in the cardiovascular research school. For this reason, we investigated the differentially expressed miRNAs in human heart failure.MethodsUsing miRNAs microarray technology in patients with heart failure and normal myocardium, we observed a set of differentially expressed miRNAs. And these data were verified by real-time PCR. Then, we re-checked the miRNAs expression in three different heart failure models in SD rats:myocardial infarction induced, isoproterenol induced and abdominal aorta constriction induced heart failure.After transfected with certain microRNAs, H9c2 (2-1) myocardial cells were harvested to detect cell proliferation, apoptosis and related signaling pathways. The cell proliferation was tested by SRB and BrdU proliferation assays. The apoptosis was measured by flowcytometry. The activities of related enzyme were determined by ELISA kits and biochemical assays.For microRNA target prediction and validation, we first applied western-blot to screening the targets; then performed luciferase report system to reconfirm them.Finally, we packaged miR-21* and anti-miR-21* into recombinant adeno-associated virus vector 9 (rAAV9), respectively. And we detected the virus titer and transfection efficiency.Results1. Differentially expressed microRNAs screening and verification in myocardial tissue of patients with heart failureUsing Exqion company miRCURY LNATM microRNA Arrays, we found that: compared with normal myocardial tissue,28 miRNAs were upregulated in myocardial tissue from heart failure patients, and three miRNAs were downregulated. Hsa-miR-21*, hsa-miR-361-5p, hsa-miR-519e*, hsa-let-7b and so on were selected for further validation by real-time PCR tests. Further, data from different heart failure models in SD rats reconfirmed these observations.2. Effects of differentially expressed miRNAs in cardiac and endothelial cellsH9c2 (2-1) cells were transfected with miR-21*, miR-361-5p, miR-519e* and let-7b. MiR-21* and miR-519e* treatments significantly promote cell proliferation, prevent apoptosis; and miR-361-5p and let-7b showed the opposite effects. Caspase activity assays then revealed that miR-21* obviously inhibited caspase-3 and caspase-9 activities, suggesting that miR-21* may be involved in oxidative stress. Results from JC-1, SOD, MDA, T-AOC and CAT activity detections further indicated that, miR-21* participate in oxidative stress network.3. Target prediction and identification of miR-21*Running MICROCOSM, TARGETSCAN and PICTAR-VERT programs, we obtained 883 predicted targets of miR-21*. After CpG Island, Diseases, Interaction, Expression location, GO, Promoter, Pathway and Transfactor analysis,50 targets were selected for further verification. Western-blot confirmed 14 of 19 possible targets. Finally, ACADS, MAN1B1, MYBPC3, NOMO1 and WWTR-1 were defined as the targets of miR-21* by luciferase report system.4. The construction of miR-21* expression vector mediated by rAAVrAAV9 packaging system was chosen to express miR-21* and anti-miR-21*. Real-time PCR assay showed that virus titer was sufficient for in vivo research. Transfection efficiency was detected using fluorescence microscope.ConclusionIn this study, cardiac tissues from HF patients and normal hearts were scanned for differentially expressed miRNAs. Using molecular biology, cell biology and bioinformatics technology, we found that:1. hsa-miR-21*, hsa-miR-361-5p, hsa-miR-519e* and hsa-let-7b were significantly highly expressed in cardiac tissue from heart failure patients;2. hsa-miR-21* and hsa-miR-519e* promote cardiac cells proliferation, and prevent apoptosis;3. hsa-miR-361-5p and hsa-let-7b inhibit cardiac cells proliferation, and promote apoptosis;4. hsa-miR-21* participate in oxidative stress in cardiac cells;5. The effects of miR-21* in heart failure were mediated by ACADS, MAN1B1, MYBPC3, NOMO1 and WWRT-1;6. rAAV mediated expression of miR-21* and anti-miR-21* provides a theoretical and practical basis for gene therapy.