Effect of silencing troponin I3 gene expression on biological property of rat embryonic H9C2 cardiomyocytes / 中华实用儿科临床杂志

ABSTRACT

Objective@#To investigate the effect of silencing troponin I3 (Tnni3) gene expression on biological property of rat embryonic H9C2 cardiomyocytes.@*Methods@#The rat embryonic H9C2 cardiomyocytes were cultured and divided into 2 groups control group transfected with negative control small interfering RNA (NC-siRNA group) and experimental group transfected with Tnni3 small interfering RNA (Tnni3-siRNA group). At 48 h, 72 h after transfection, the cells were collected, and real time quantitative polymerase chain reaction (qPCR)was used to detect the mRNA expressions of Tnni3 and Caspase-3, and Western blot was used to detect the protein expressions of Tnni3, Cyclin A1 and Cyclin B1.Annexin V-fluorescein isothiocyanate(FITC) apoptosis detection kit was used to analyze cell apoptosis.Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) solution and cell cycle was detected by flow cytometry.@*Results@#At 48 h post-transfection with Tnni3-siRNA, H9C2 cells exhibited a significant decrease in Tnni3 mRNA (0.27±0.05 vs. 1.00±0.00) and protein (0.18±0.03 vs. 1.00±0.00) compared with those transfected with NC-siRNA, and the differences were statistically significant (t=25.26, 47.40, all P<0.01). Apoptotic cells were observed in the NC-siRNA group and the Tnni3-siRNA group.At 72 h post-transfection, the percentage of apoptotic cells significantly increased in H9C2 cells transfected with Tnni3-siRNA [(11.30±1.85)% vs. (0.33±0.15)%] compared with those transfected with NC-siRNA, an increased expression of Caspase-3 mRNA was also observed in Tnni3-siRNA-transfected H9C2 cells (1.39±0.13 vs. 1.00±0.00), and the differences were statistically significant (t=10.24, 5.19, all P<0.01). Compared with NC-siRNA-transfected H9C2 cells, a time-dependent reduction in cell proliferation was observed in Tnni3-siRNA-transfected H9C2 cells (48 h 0.32±0.06 vs. 0.46±0.03; 72 h 0.31±0.01 vs. 0.63±0.04; 96 h 0.36±0.01 vs 0.75±0.04), and the differences were statistically significant (t=3.62, 13.45, 16.39, all P<0.01). At 72 h post-transfection with Tnni3-siRNA, the percentage of G1 phase, S phase and G2 phase cells was (71.25±3.82)%, (18.28±2.78)% and (9.94±1.09)%, respectively.There was a significant increase in the proportion of G2 phase cells [(9.94±1.09)% vs. (4.54±0.99)%] in H9C2 cells transfected with Tnni3-siRNA compared with those transfected with NC-siRNA, an increased expression of Cyclin A1 protein (1.89±0.09 vs.1.00±0.00) and a decreased expression of Cyclin B1 protein (0.47±0.06 vs.1.00±0.00) were observed in Tnni3-siRNA-transfected H9C2 cells, respectively, and the differences were statistically significant (t=6.35, 17.12, 15.32, all P<0.01).@*Conclusions@#Silencing Tnni3 gene expression in rat embryonic H9C2 cardiomyocytes can induce cell apoptosis, suppress cell proliferation, and led to G2 cell cycle arrest.