Biological Effect And Mechanism Of Synergistic Interaction Between Tnni3 Gene And Tbx20 Gene On Rat H9C2 Cardiomyocyte Apoptosis

Objective:Cardiomyocyte proliferation and apoptosis are essential cellular basis in maintaining cardiac homeostasis and the disequilibrium between proliferation and apoptosis will lead to cardiac defects.Therefore,it is useful to clarify the biological effect of candidate cardiac pathogenic genes on proliferation and apoptosis,which will help understand the potential pathogenic mechanism and provide new clues for treatment,prevention and intervention.Tbx20 gene is the important candidate for cardiac diseases.Previous studies in our group confirmed that silencing Tbx20 gene expression could induce cell apoptosis,suppress cell proliferation and lead to G2 cell cycle arrest.Meanwhile,Tbx20 gene negatively regulated the expression of its downstream target Lta and participated in cardiomyocyte apoptosis.Interaction between Tbx20 and cTnI was also identified by co-immunoprecipitation,but little was known about the biological effect of Tbx20-cTnI interaction.cTnI encoded by Tnni3 gene is the inhibitory subunit of the troponin complex and plays a central role in the regulation of cardiac contraction and relaxation.Loss-of-function mutations in Tnni3 gene have been identified in patients with cardiac diseases.These pathogenic mutations often result in decreased expression of the Tnni3gene,but little is known about the mechanism how expression reduction in the Tnni3gene participates in the pathogenesis of cardiac diseases.In the present study,RNA interference?RNAi?was carried out to investigate the biological effect of silencing Tnni3 gene expression on rat H9C2 cardiomyocytes,including cell apoptosis,cell proliferation and cell cycle.Furthermore,co-transfection and luciferase assays were performed to elucidate the synergistic collaboration between Tnni3 gene and Tbx20 gene and reveal its effect on Tbx20 gene function such as participating cell apoptosis and regulating Lta expression,which will provide a theoretical basis for the etiology and treatment of cardiac diseases.Methods:The rat H9C2 cardiomyocytes were cultured and divided into 4 groups:control group transfected with negative control small interfering RNA?NC-siRNA?,Tnni3-siRNA group,Tbx20-siRNA group and experimental group co-transfected with Tnni3-siRNA and Tbx20-siRNA.At 48h and 72h after transfection,the cells were collected,followed by real time quantitative polymerase chain reaction and Western Blot to detect the mRNA and protein expression of several genes including Tnni3,Tbx20,Caspase3,PCNA,CyclinA1,CyclinB1 and Lta.Annexin V-FITC/PI apoptosis detection kit and DeadEnd^TM Fluorometric TUNEL System were used to explore early and late cell apoptosis,respectively.Cell proliferation was measured by cell counting kit-8?CCK-8?solution and cell cycle was detected by flow cytometry with PI staining.Dual-Luciferase Reporter System was used to measure the luciferase activity of Lta promoter.Results:1.At 48h post-transfection with Tnni3-siRNA,H9C2 cells exhibited a significant decrease in Tnni3 mRNA and protein compared with those transfected with NC-siRNA?P<0.01?,suggesting that Tnni3-siRNA could effectively inhibit the expression of Tnni3 gene.2.At 72h post-transfection,early apoptotic cells,late apoptotic bodies and the percentage of apoptotic cells were significantly increased in H9C2 cells transfected with Tnni3-siRNA compared with those transfected with NC-siRNA?P<0.01?.An increased expression of Caspase3 mRNA and protein were also observed in Tnni3-siRNA-transfected H9C2 cells?P<0.01?,suggesting that silencing Tnni3 gene expression could induce cell apoptosis.3.Compared with NC-siRNA-transfected H9C2 cells,a time-dependent reduction in cell proliferation was observed in Tnni3-siRNA-transfected H9C2 cells.At 72h post-transfection with Tnni3-siRNA,H9C2 cells exhibited a significant decrease in PCNA mRNA and protein?P<0.01?,suggesting that silencing Tnni3 gene expression could suppress cell proliferation.4.At 72h post-transfection with Tnni3-siRNA,the percentage of G1 phase,S phase and G2 phase cells were?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 in H9C2 cells transfected with Tnni3-siRNA compared with those transfected with NC-siRNA?P<0.01?.An increased expression of CyclinA1 mRNA and protein were observed in Tnni3-siRNA-transfected H9C2 cells?P<0.05?.In contrast,a decreased expression of CyclinB1 mRNA and protein were observed in Tnni3-siRNA-transfected H9C2 cells?P<0.05?,suggesting that silencing Tnni3 gene expression could lead to G2 cell cycle arrest.5.At 72h post-transfection,early apoptotic cells,late apoptotic bodies and the percentage of apoptotic cells were significantly increased in H9C2 cells co-transfected with Tnni3-siRNA and Tbx20-siRNA compared with those transfected with Tnni3-siRNA or Tbx20-siRNA alone?P<0.01?.An increased expression of Caspase3 protein was also observed in H9C2 cells co-transfected with Tnni3-siRNA and Tbx20-siRNA?P<0.05?,suggesting that Tnni3 gene and Tbx20 gene acted synergistically to induce cell apoptosis.6.Compared with Tnni3-siRNA-transfected H9C2 cells or Tbx20-siRNA-transfected H9C2 cells,an increased expression of Lta mRNA was observed at 72h post-transfection in H9C2 cells co-transfected with Tnni3-siRNA and Tbx20-siRNA?P<0.01?.Meanwhile,wild-type Lta promoter exhibited an increased luciferase activity?P<0.01?while mutant-type Lta promoter displayed no changes in luciferase activity?P>0.05?,suggesting that Tnni3 gene and Tbx20 gene could act synergistically to regulate Lta expression.Conclusions:1.Silencing Tnni3 gene expression in rat H9C2 cardiomyocytes can induce cell apoptosis,suppress cell proliferation and lead to G2 cell cycle arrest.2.Tnni3 gene and Tbx20 gene act synergistically to regulate the expression of Lta gene and participate in cell apoptosis.