The Study Of MiR-10 And MiR-542 Target Proteins In The Intervention Of Aerobic Exercise On Early Heart Failure Rats

Objectives: rno-mi R-10a-5p and rno-mi R-542-5p take part in the intervention of early heart failure by aerobic exercise,which were identified by our laboratory initially.They were down-regulated in the myocardium of chronic heart failure rats by stress load.However,they were up-regulated by the aerobic exercise.Meanwhile it accompanied with mitochondrial function enhanced and the heart failure process delayed.The purpose of this study is to determine the target proteins of mi R-10a-5p or mi R-542-5p and to explore the mechanism of intervening early heart failure by aerobic exercise and enrich the research of micro RNAs' regulation on postranscriptional level.Methods: 1.RIP and screened of target genes: H9C2 cells were overexpressed mi R-10a-5p or mi R-542-5p respectively.m RNAs pool acquired by RNA-Protein coimmunoprecipitation to enrich m RNAs in the RNA-induced silencing complex(RISC)was tested by gene chip.By the results of gene chip,we analyzed the variety and cluster of m RNA.The candidate genes were computed and predicted by mi RBase?Target Scan?RNAhybrid database after processing data preliminarily.We understood the main physiological functions of candidate genes in order to further research.2.Designed primer: the primer was send to synthesis in Sangon biotech company which designed by Oligo Primer Analysis Software 7.SYBR Green RT-PCR was used to detect whether can work and appear dimer.Agarose gel electrophoresis was to verify the product of PCR.3.SYBR Green RT-PCR: The expression of candidate genes were detected in vivo and in vitro respectively.H9C2 cells overexpressed mi R-10a-5p or mi R-542-5p respectively and tissues from sham-operated control group(SC),sham-operated plus training group(ST),abdominal aorta constriction control group(AC)and abdominal aorta constriction plus training group(AT)were extracted the total RNA and detected by the method of RT-PCR.Results: 1.The analysis of GO showed that many genes are related with the morphology of heart and mitochondria,the occurrence of blood vessels,cell apoptosis and inflammation.It is in accord with our initial idea.2.By screening and verifying,we suggested that the target gene of mi R-10a-5p may be Fat4 and the target gene of mi R-542-5p may be Tfrc and Vav2.The results of RT-q PCR displaied that in the H9C2 cell expressed mi R-10a-5p,atypical cadherin Fat4 was significantly increased compared with expressed empty vehicle.And the expression of Vav2 was changed the same with Fat4 in the H9C2 cell expressed mi R-542-5p.The validation was also carried out in model animal tissues.Compared with group SC,the expression of transferrin receptor Tfrc in group AC was increased significantly.But it returned to normal level after aerobic exercise.Compared with group SC,the expression of Vav2 in group AC was decreased significantly.The cardiac function and the expression of Vav2 were recovered to some extent after aerobic exercise.Compared with group SC,the expression of Fat4 in group AC was decreased significantly.But it had no obvious changes after aerobic exercise.Compared with group SC,the expression of COX ? in group AC was decreased significantly.The expression of COX ? in group AC increase to some extent by aerobic exercise for eight weeks.Conclusion: By the analysis of gene chip and RT-PCR in vivo and in vitro,we found that the target gene of mi R-10a-5p may be Fat4 and the target gene of mi R-542-5p may be Tfrc and Vav2 in the intervention of early heart failure by aerobic exercise.In addition,we have theoretical basis as support.Transferrin receptor Tfrc is associated with the oxidative phosphorylation of mitochondria and autophagy.Vav2 is related to the cardiovascular homeostasis and Fat4 can regulate cell proliferation.