Regulation Of Microrna-34A On The SH2B3 Expression During Cardiac Cells

BackgroundThe function of cardiac is heavily dis-regulated by cardiac fibrosis and the abnormality of extracellular matrix contributed greatly to it. With the ongoing of related study, the primary goal to treat this disease is to regulate the biological activity of cells. It is therefore very important to realize the development and regulation of it. In the presence of extracellular stimuli, the signals could be transmitted into the cell to regulate the function and activity of cardiac cells.As a family member of the adapter protein, SH2B, SH2B3 could mediate the signaling conduction of Janus kinase and receptor tyrosine kinase. Previous studies showed that SH2B3 was related with various disease. In cardiac disease, the fibrosis after myocardial infarction was more obvious in SH2B3 knocking out mice, indicating its potential involvement in cardiac fibrosis. But until now, the regulation of its abnormal expression during cardiac fibrosis remains unknown. miRNAs,which is 22 nucleotides in length and previous regarded as a post-transcriptional regulator, could inhibit translation by base pairing with the 3’untranslated region (3’UTR) of specific messenger RNA transcripts. Functionally,microRNA could exert its activity through directly targeting multiple genes.More and more evidence clearly demonstrated that microRNA played important roles in cardiac disease, especially the fibrosis progression.Recent study showed that miR-34a could regulate the contractile function after myocardiac infarction and silencing miR-34a could alleviate its function. Additionally, Flister et al found that the expression of miR-34a increased significantly in the fibrosis zone of myocardiac infarction. Moreover, miR-34a could promote the cardiac fibrosis through regulating the expression or secretion of fibrosis-related collagen or other components and Smad4 was validated as its direct target.Objective(1) To evaluate the effects of TGF-β1 or hypoxia on the expression of miR-34a and SH2B3;(2).To study the regulation of miR-34a on the expression of SH2B3.MethodsWestern blot and qRT-PCR were applied to detect the expression of SN2B3 at protein and mRNA levels in H9C2 cells after transformation growth factor-β1 and hypoxia stimulation. QRT-PCR was also performed to detect the miR-34a expression after the above treatments. Furthermore, Western blot and qRT-PCR were used to examine the effect of transfection with miR-34a mimic/inhibitor on the SH2B3 expression. Luciferase activity assay was applied to detect the effects of miR-34a on the activity of SH2B33’UTR.ResultsAfter stimulation with transformin growth factor-β1 and hypoxia,SH2B3 expression decreased at both mRNA and protein levels, but the expression of miR-34a increased accordingly. Western blot and qRT-PCR analysis showed that miR-34a could regulate the expression of SH2B3.The luciferase acticity assay confirmed the fact the miR-34a regulated SH2B3 expression through binding to its 3’UTR. The analysis of luciferase activity showed that miR-34a could regulate SH2B3 expression through directly binding to its 3’UTR. In addition, the expression of SH2B3 decreased, but miR-34a increased with the hypoxia exposure.Conclusion1.miR-34a could regulate the expression of SH2B3 in cardiac cells through binding to its 3’UTR.2.The expression of miR-34a and SH2B3 showed the opposite tendency in the presence of TGF-β1 or hypoxia, suggesting the potential involvement of miR-34a/SH2B3 to mediate the effects of TGF-β1 or hypoxia on cardiac cells.