The Protective Effect To Apoptosis And Molecular Mechanism Of MiR-214in Cardiomyocytes

Background:With the changes in modern lifestyle, the accelerated pace of life and the growing aging population, the increased incidence and prevalence of heart disease has become increasingly prominent. Myocardial apoptosis is not an independent heart disease, but a pathological process involved in the occurrence and development of a variety of heart disease, such as ischemic heart disease, arrhythmia, congenital heart disease, cardiac overload, viral infections, myocardial remodeling, heart failure and others. However, we are still lack of adequate understanding of myocardial apoptosis. Many factors that causes apoptosis, interactions between these factors, and the downstream signaling interaction between them are still unknown. As a class of non-coding RNA, microRNAs are single-stranded RNA molecules, with a length of about18-24nucleotides, encoded by endogenous genes. Current research has shown that this type of small molecules play an important role in the regulation of gene expression. For the research purposes of myocardial apoptosis, to clarify how microRNA regulates apoptosis in certain ways by complex process is necessary, and worth further exploration. In this study, we hope to know how a certain microRNA plays a role in myocardial apoptosis.Methods and results:With different concentrations and duration of stimulation of hydrogen peroxide respectively in primary rat cardiomyocytes, real time PCR was used to detect the changes of miR-214expression.We made miR-214overexpressed in myocardial cells and then exposured to the appropriate concentration of hydrogen peroxide treatment, compared with the control group, the proportion of apoptotic cells and the related protein expression were detected by flow cytometry and western blot method respectively. miR-214target genes were predicted by online software. And then we detected the changes of mRNA and protein expression level by real time PCR and western blot. Further more, we used dual luciferase report system to verify the targeting sequence of miR-214. With the application of miR-214transgenic mouse model, we used real time PCR, Western blot and immunohistochemical methods to verify the miR-214target genes and analyze the specific molecular mechanism of apoptosis regulation.The expression level of miR-214showed a gradual decline with the increasing concentrations of hydrogen peroxide and the prolonged duration of oxidative stress of100μM hydrogen peroxide respectively. We made in vitro transfection of miR-214mimic to myocardial cells to enable the overexpression of miR-214, and then exposure100μM hydrogen peroxide treatment with the negative control. By flow cytometry and western blot, we found that the myocardial cells with miR-214overexpression have a lower rate of apoptosis. Furthermore, after overexpression of miR-214, we found the decreasing mRNA and protein levels of bim, bax and casp7. Dual reporter luciferase system analysis indicates that they may be direct targets of miR-214. Finally, by using the miR-214transgenic mice, we validated the lower expression of pten and puma, compared to the wild type.Conclusion:With the stimulation of hydrogen peroxide to myocardial cells, the expression of miR-214showed a gradual decline by the increasing time or increasing concentration of hydrogen peroxide. Overexpression of miR-214in vitro enhances the ability of anti-apoptotic in myocardial cells, and the anti-apoptosis ability by overexpression of miR-214may be achieved through synergies of these multiple downstream targets.