Effects And Mechanism Of Dock1 On Hypoxia/Reoxygenation-induced Apoptosis And Proliferation Of H9C2 Cardiomyocytes

Objective : To explore the effect of guanine nucleotide exchange factor Dock1 on hypoxia/reoxygenation(H/R)-induced apoptosis and proliferation of H9C2 cardiomyocytes and its underlying mechanisms.Methods : H9C2 cardiomyocytes were respectively infected with negative control lentivirus(NC shRNA) and target of Dock1 silence lentivirus(Dock1 shRNA), Screened and established of stable cell lines by using Puromycin, and the Dock1 silence cell lines were randomly transfected with pCXN2-Flag plasmid and hDock1-pCXN2-Flag plasmid.The above three groups of intervened-cells were prepared in two copies,and then randomly treated with H/R. Thus the experiments were randomly divided into six groups, namely NC shRNA group; Dock1 shRNA group;Dock1 shRNA+hDock1 group; NC shRNA+H/R group; Dock1 shRNA+H/R group; Dock1 shRNA+hDock1 +H/R group. The expression level of Dock1 mRNA was detected by PT-RCR. The protein levels of Dock1, AKT,p-AKT,p-ERK1/2, Bcl-2, Bax and Flag were detected by Western blotting.The cell proliferation rate was observed by MTT assay. The cellapoptosis rate was examined with flow cytometry.Results:More than 80% of H9C2 cardiomyocytes were infected with lentivirus. Compared with NC shRNA group, the expression of Dock1 mRNA, Dock1 protein, p-AKT protein, p-ERK1/2protein, Bcl-2 protein and the cell proliferation rate were remarkably decreased, the Bax protein and cell apoptosis rate were significantly increased in the Dock1 shRNA group(P<0.05). Compared with Dock1 shRNA group, the expression of Rat Dock1 mRNA was had no statistical significance, while expressed Human Dock1 mRNA, and the expression of Dock1 protein, p-AKT protein, p-ERK1/2protein,Bcl-2 protein and the cell proliferation rate were remarkably increased, the Bax protein and cell apoptosis rate were significantly decreased in the Dock1 shRNA+hDock1 group(P<0.05).Compared with normal condition incubated groups, the expression of Dock1 mRNA, Dock1 protein, p-AKT protein,p-ERK1/2protein, Bcl-2 protein and the cell proliferation rate were remarkably decreased, the Bax protein and cell apoptosis rate were significantly increased in the H/R treated group(P<0.05). Compared the NC shRNA group with the Dock1 shRNA+hDock1 +H/R group, There was no statistical significance in the detection index.Conclusion : Successfully established the hypoxia / reoxygenation(ischemia / reperfusion) injury model on H9C2 cardiomyocytes in vitro.Dock1 silence can aggravate H/R-induced apoptosis, On the basis ofDock1 gene silence,overexpression of Dock1 gene can completely reverse the effects of H/R-induced injury on H9C2 cardiomyocytes. Thus, Dock1 gene can inhibit apoptosis and promote proliferation in H9C2 cardiomyocytes.This effect is positively correlated to the expression level of Dock1 gene.The mechanism might be mediated by p-AKT, p-ERK1/2,and Bcl2 family.