Tanshinone IIA Ameliorates Progression of CAD Through Regulating Cardiac H9c2 Cells Proliferation and Apoptosis by miR-133a-3p/EGFR Axis

Figures & data

Figure 1 miR-133a-3p targets EGFR 3ʹ-UTR. There were binding sites for miR-133a-3p on EGFR 3ʹ-UTR (A). miR-133a-3p mimics significantly decreased the luciferase activity when co-transfected with pGL3-EGFR-WT (B). **p<0.01.

Figure 2 miR-133a-3p was decreased and EGFR was increased in H₂O₂-induced H9c2 cells. Compared with control group, miR-133a-3p level was significantly lower in H₂O₂ group, which was reversed by Tan IIA (A). Compared with control group, EGFR level was significantly increased by H₂O₂, which was reduced by Tan IIA in H9c2 cells (B). ***p<0.001.

Figure 3 The successful transfection of miR-133a-3p inhibitor and siRNA-EGFR. Compared with control group, miR-133a-3p level was significantly inhibited by miR-133a-3p inhibitor but not miR-NC inhibitor (A). Compared with control group, EGFR level was significantly reduced by siRNA-EGFR-1and siRNA-EGFR-2 but not siRNA-NC (B). ***p<0.001.

Figure 4 miR-133a-3p inhibitor negatively regulated and siRNA-EGFR positively regulated the expression of EGFR. Compared with miR-NC inhibitor group, a significant increase of mRNA level of EGFR was found in miR-133a-3p inhibitor group, which was reversed by siRNA-EGFR (A). Compared with miR-NC inhibitor group, protein level of EGFR was upregulated by inhibition of miR-133a-3p, which was reversed by siRNA-EGFR (B). **p<0.01, *p<0.05.

Figure 5 Tan IIA reversed H₂O₂-induced H9c2 cell proliferation reduction by miR-133a-3p/EGFR axis. Results of CCK-8 (A) analysis showed Tan IIA reversed H₂O₂-induced H9c2 cell proliferation reduction, which was partially attenuated by miR-133a-3p inhibitor but not miR-133a-3p inhibitor+siRNA-EGFR (A). *p<0.05 and **p<0.01. Results of EdU staining showed that Tan IIA reversed cell proliferation decreased in H₂O₂-induced H9c2 cells, which was partially rescued by miR-133a-3p inhibitor but not in miR-133a-3p inhibitor+siRNA-EGFR group (B). Quantified results of B (C). *p<0.05 and **p<0.01. Western blot analysis indicated that the expressions of p21 and p53 showed the opposite trends (D). Quantified results of D (E). **p<0.01 and ***p<0.001.

Figure 6 Flow cytometry results showed Tan IIA protected H₂O₂-induced H9c2 cells from apoptosis by miR-133a-3p/EGFR axis. Results of FCM analysis showed Tan IIA protected H₂O₂-induced H9c2 cells from apoptosis, which was partially attenuated by miR-133a-3p inhibitor but not miR-133a-3p inhibitor+siRNA-EGFR. *p<0.05, **p<0.01.

Figure 7 Tunnel staining results showed Tan IIA protected H₂O₂-induced H9c2 cells from apoptosis by miR-133a-3p/EGFR axis. Results of TUNNEL analysis showed Tan IIA protected H₂O₂-induced H9c2 cells from apoptosis, which was partially attenuated by miR-133a-3p inhibitor but not miR-133a-3p inhibitor+siRNA-EGFR. **p<0.01, ***p<0.001.

Figure 8 Tan IIA reversed H₂O₂-induced changes in the expressions of apoptosis and cell cycle-related proteins of H9c2 cells by miR-133a-3p/EGFR axis. The expression profile of cleaved caspase-3 and Bax showed the similar trends to cell apoptosis; while Bcl-2 showed the opposite trends. The expression profile of CyclinD1 and CDK4 exhibited the similar trends with that of cell cycle. **p<0.01, ***p<0.001.

Figure 9 Tan IIA reversed H₂O₂-induced G0/G1 phase reduction of H9c2 cells by miR-133a-3p/EGFR axis. Tan IIA reversed H₂O₂-induced G0/G1 phase reduction of H9c2 cells, which was partially attenuated by miR-133 inhibitor but not miR-133a-3p inhibitor +siRNA-EGFR. *p<0.05.