Depletion of copine-I inhibits cell apoptosis induced by hypoxia/reoxygenation in H9c2 cells

AIM To determine the effects and mechanisms of copine-I(CPNE1) on apoptosis induced by hypoxia/reoxygenation(H/R) in H9c2. METHODS H9c2 myocytes were subjected to H/R(24 h/6 h) and divided into control group(CON), H/R group, negative control(NC)+H/R and CPNE1 siRNA+H/R group. H9c2 myocytes were pretreated with NF-κB inhibitor PDTC(10 μM) for 30 min to determine the potential mechanisms of CPNE1 in cell apoptosis. CPNE1 expression was detected using RT-PCR and Western blot. Activity of LDH was assayed by ELISA and cell apoptosis was measured using flow cytometry after staining with annexin V/PI. Western blot was utilized to detect the expressions of cleaved-caspase 3(c-caspase3), Bax, Bcl-2 and NF-κB and ELISA was used to determine the activity of NF-κB. RESULTS Compared with CON group, mRNA and protein levels of CPNE1 in the H/R group increased. Moreover, H/R resulted in a significant increase in apoptosis rate, activity of LDH, and protein expressions of c-caspase3 and Bax. In addition, the anti-apoptotic protein Bcl-2 was decreased. Depletion of CPNE1 decreased the activity of LDH, apoptosis rate, and expressions of c-caspase3 and Bax but augmented Bcl-2 expression. Silencing of CPNE1 enhanced the activity and expression of NF-κB in the nucleus. Regulation of CPNE1 in cell apoptosis was abolished partly by PDTC. CONCLUSIONS CPNE1 silencing inhibits cell apoptosis induced by H/R in H9c2. The possible mechanism for a myocardial protective role of CPNE siRNA is enhancing the activity of NF-κB.