The Effect And Mechanism Of Anti-apoptosis Of MicroRNA-21in Rat Myocardium During Early Phase Of Ischemia-repersusion

Objective:Cell apoptosis is the important mechanism in ischemia-reperfusion (I/R) injury. Protective effects on cell apoptosis can avoid from ischemia-reperfusion injury. Evidenced support that the abnormal expression of microRNA-21(miR-21) in I/R animal model, we demonstrated in the present study that the relationship between abnormal expression of miR-21and cell apoptosis in early I/R injury and the mechanism. Trimetazidine (TMZ) is a classic anti-ischemia drug, we plan to determin that miR-21mediated the anti-apoptosis effect of TMZ in early I/R injury.Methods:l.In vivo experiment:Forty-eight SD rats were randomly divided into Sham group, I/R2h group, I/R4h group, I/R6h group. There are12rats in each group. Rats in Sham group were deal with open-chest, rats in other groups suffered ligation of anterior descending coronary atrery for45min and reperfusion for2h,4h,6h respectively. To compare the expression level of miR-21and level of cell apoptosis between I/R groups and Sham group, and between in the diffrent I/R time point groups. Bcl-2/Bax and Caspase-3as classic factors to stand for the level of cell apoptosis. Realtime-PCR was used to assess the expression level of miR-21in ischemic and non-ischemic area; Immunohistochemistry and Western-blot were used to determine the expression of Bcl-2, Bax, Caspase-3and Bcl-2/Bax.2.Thirty rats were divided into five groups randomly:We constructed rAAV9-ZsGreen-pre-miR-21successfully with the titer5.0×1012vg/ml.①Control group(n=6):rats were transfected with rAAV9-ZsGreen by coronary injection;②miR-21group (n=6):rats were transfected rAAV9-ZsGreen-pre-miR-21by coronary injection;③Sham group:rats were dealt with open-chest after transfected with rAAV9-ZsGreen;④I/R group (n=6):rats were treated with I/R after transfected with rAAV9-ZsGreen;⑤I/R+miR-21(n=6):rats were dealt with I/R after transfected rAAV9-ZsGreen-pre-miR-21by coronary injection. Realtime-PCR was used to assess the expression level of miR-21; Immunohistochemistry and Western-blot were used to determine the expression of Bcl-2, Bax, Caspase-3, PTEN, p-AKT and Bcl-2/Bax.3.In vitro experiment:H9C2cell were transfected with different concentration of miR inhibitors and mimics(30nM,50nM and100nM respectively) by liposome LipofectamineTM2000, Realtime-PCR was used to assess the expression level of miR-21,we choose the best concentration. H9C2cell was randomly divided into Vehicle Control (NC),inhibitors negative control (INC), mimics NC (MNC), H/R+INC, H/R+MNC, H/R+inhibitors and H/R+mimics.Cell was treated with hypoxia for4h and reoxygenation for3h. Realtime-PCR was used to assess the expression level of miR-21; Immunohistochemistry and Western-blot were used to determine the expression of Bcl-2, Bax, Caspase-3, PTEN, p-AKT and Bcl-2/Bax. Flow cytometry evaluated apoptosis rate.4. H9C2cell was randomly divided into Control, H/R+Control and H/R+TMZ. There is another2groups, H/R+TMZ+INC and H/R+TMZ+inhibitors. Cell incubated with0μM or10μM TMZ for48hours. Realtime-PCR was used to assess the expression level of miR-21; Immunohistochemistry and Western-blot were used to determine the expression of Bcl-2, Bax, Caspase-3, PTEN, p-AKT and Bcl-2/Bax. Flow cytometry evaluated apoptosis rate.Result:1.miR-21was down-regulated in the ischemic area after I/R compared with the Sham group (P<0.05).but miR-21expression in the non-ischemia area was significantly increased compared with the Sham group (P<0.01). MiR-21expression and the level of Bcl-2/Bax was decreased, the level of Caspase-3was increased in the I/R group than the Sham group at2h,4h and6h after I/R (P<0.05).2.The level of PTEN protein decreased when miR-21over-expressed (P<0.01), and the level of PTEN mRNA was not affected (P>0.05). Compared with I/R group, the expression of miR-21, p-AKT, Bcl-2/Bax was up-regulated in miR-21over-expressed rat suffered from H/R, meanwhile the expression of PTEN, Caspase-3was down-regulated (P<0.05).3. Compared with VC, the protein level of Bcl-2/Bax was up-regulated, which of Caspase-3was down-regulated when cells were dealt with H/R, and cell apoptosis was increased (P<0.05). Cell apoptosis were found to be aggravated after inhibition of miR-21(P<0.05), otherwise it was improved after over-expression of miR-21(P<0.05).4. Rats were treated with TMZ before H/R exhibit increases in Bcl-2/Bax, and decreases Caspase-3protein levels compared H/R rats, and cell apoptosis decreased (P<0.05).When the expression of miR-21was down-regulated after transfected inhibitors, Bcl-2/Bax was decreased, the level of Caspase-3and cell apoptosis rate was increased (P<0.05)Conclusion:1.The expression of miR-21was down-regulated and cell apoptosis was increased in ischemic area at the early phase of I/R, and it was aggravated with the prolonged reperfusion period.2. AAV9was an ideal gene vector who can stably and efficiently expressed in rat myocardium without affecting cardiac function.3. Cell apoptosis can be improved by miR-21over-expressed, otherwise it can be exacerbated when miR-21was inhibited.4.miR-21mediated cell apoptosis induced by I/R via modulation of PTEN/AKT pathway.4. The protective effects of TMZ was partly mediated by miR-21.