The Effect To Apoptosis And Molecular Mechanism Of MiR-199a-5p/miR-31a-5p

Cell apoptosis is a type of programmed cell death(PCD),it's a normal phenomenon throughout the life of a cell which is monitored by various genes.In the past time,it is generally believed that cell apoptosis doesn't occur in terminal differentiated cells like myocardial cell,neuron.But it is found that apoptosis occurs in both normal and pathological hearts;Peoples' living standards continue to improving nowadays,at the same time increasing pressure from life and work,unhealthy life style all contribute to heart disease.They seriously endanger people's physical and mental health.Abnormal cell apoptosis induces decreasing of cardiac myocytes number,loss of cardiac tissue,and this pathological process attends and aggravates lots of cardiovascular disease.However,the mechanism of cell apoptosis in the development of cardiovascular disease is not that clear.More and more researchers are committed to the study of apoptosis and cardiovascular disease,but the current role of cardiomyocyte apoptosis,and the regulatory mechanism is still not very comprehensive.Micro RNA(mi RNA)is a recently identified set of noncoding small RNAs.The 3'noncoding region(3'UTR),which normally acts on the target gene,mediates gene silencing in the post-transcriptional level of gene regulation.Current studies have shown that certain specific mi RNAs play a broad role in cardiomyocyte apoptosis-related gene regulation.And in-depth study of this regulatory mechanism is very necessary and worth exploring.On the basis of previous research about the differential expression profile of micro RNAs and transcriptome in the rat model of heart failure after myocardial infarction,in this study,we selected mi R-199a-5p and mi R-31a-5p,which was closely related to the process of heart failure,to further explore the expression of these two mi RNAs in rat cardiomyocytes(H9C2)undergoing angiotensin II(Ang II)inducedcardiomyocyte apoptosis at different concentrations and time;We made mi R-199a-5p/mi R-31a-5p overexpressed and suppression in the myocardial cell,the apoptotic rate of cardiac cells were detected by flow cytometry,Caspase-3 assay kit,Rat Apoptosis RT2 Profiler ? PCR Array;Bioinformatics method was used to predicted target genes for these two mi RNAs primarily,the genes related cell apoptosis,growth,cell cycle related gene were selected and the result were compared with the result of differential expression profile of transcriptome.Then we detected the changes of m RNA,protein by real time PCR and western blot;Furthermore,dual-luciferase report system was constructed to verify the target sequence of mi R-199a-5p and mi R-31a-5p;Next,we explored the function of Junb on cell apoptosis through construction of p BI-CMV3 vector.This study draw following results:1.With different Ang II concentration and duration of stimulation,real time PCR showed that the level of mi R-199a-5p obviously increased with the stimulation time prolonged exposure to 100 n M Ang II or with the concentration elevation when stimulated for 24 h.For mi R-31a-5p,expression level decreased relatively gradually.2.By flow cytometry,Caspase-3 Activity assay kit,mi R-199a-5p/mi R-31a-5p increased/decreased proportion of apoptotic cells undergoing Ang II simulation,and apoptotic rate increased/deceased with mi R-199a-5p and mi R-31a-5p overexpressed respectively.3.Rat Apoptosis RT2 Profiler? PCR Array showed 7 apoptosis-related gene up-regulated significantly through gain of function of mi R-199a-5p;Elevation of mi R-31a-5p intracellular resulted in 20 apoptosis-related genes expression level showed significantly difference.4.Bioinformatic method,real time PCR,western blot results indicated after overexprssion of mi R-199a-5p and mi R-31a-5p,m RNA and protein of Junb and Tp53 decreased,and Junb,Tp53 could be the possible target genes of mi R-199a-5p and mi R-31a-5p respectively.5.Dual-luciferase report system verified mir RNA-m RNA interaction relationship between Junb and mi R-199a-5p,Tp53 and mi R-31a-5p.6.Overexpression of Junb by construction overexpression vector showed Junb played protected role in cell apoptosis.Conclusion:1.With the stimulation of Ang II,expression level of mi R-199a-5p/mi R-31a-5p increased/decreased by the increasing stimulation time and Ang II concentration;2.Overexpression of mi R-199a-5p induced myocardial cell apoptosis and mi R-31a-5p inhibited cell apoptosis3.We found that mi R-199a-5p induced myocardial cell apoptosis directly through down-regulated Junb;mi R-31a-5p targeted Tp53 to reduce cell apoptosis.Over-expression of Junb in vitro have protective effection upon Ang II induced apoptosis.