H3K9ac Modification Was Involved In Doxorubicin Induced Apoptosis By Regulating Pik3ca Transcription In H9c2 Cells

Objective: In doxorubicin induced H9c2 cells,we evaluated the effect of H3K9 ac modification on Pik3ca transcription levels and the mechanism of Pik3ca by inducing apoptosis in rats.Methods: To establish doxorubicin induced H9c2 cells model: the experiment was divided into control group(added the same amount of high sugar medium for 8h),doxorubicin group(1uM doxorubicin cultured H9c2 cells for 8h),the Cell Counting Kit8(CCK8)and Annexin V-FITC/PI experiments detected H9c2 cells apoptosis and activity,amount of protein of Bcl2,BAX,Cleaved-caspase3 were examined by Western blot.The levels of histone H3K9 acetylation in Pik3ca transcription initiation region was tested with ChIP-Seq.The level of Pik3ca transcription initiation region fragment enriched by H3K9 ac antibody was detected by ChIP-QPCR.Protein and RNA levels of Pik3ca was detected by fluorescence quantitative PCR technique and Western blot.Transcription efficiency of Pik3ca siRNA was detected by immunofluorescence and Western blot.Cell activity was inspected by Cell Counting Kits(CCK8),and cardiac myocyte apoptosis rate was detected by Annexin V-FITC/PI.Protein expression levels of PI3K,P-AKT,AKT,Bcl2,BAX and Cleaved-caspase3 in H9c2 cells were detected by Western blot.Results: H9c2 cells were cultured with 1u M doxorubicin for 8h.CCK8 and Annexin V-FITV/PI showed obvious apoptosis,decreased expression of Bcl2 protein and increased expression of BAX and Cleaved-caspased3 protein.ChIP-Seq sequencing analysis showed that the level of histone H3K9 acetylation in the Pik3ca promoter region was significantly increased in the Dox group in comparison with the Con group.Meanwhile,ChIP-q PCR further proved that in the doxorubicin group,the fragment in the Pik3ca promoter region enriched by H3K9 ac antibody was significantly higher than that in the control group.PCR and western blot results displayed that in comparison with the Con group,the m RNA and protein expression of Pik3ca were elevated in the Dox group,and the protein expressions of PI3K and P-AKT were also elevated.The efficiency of Pik3ca siRNA transfection was 70% by immunofluorescence and western blot.After Pik3ca silenced,CCK8 and Annexin V-FITC/PI showed a significant decrease in H9c2 cells apoptosis.In western blot results,the protein expression levels of PI3K,P-AKT and Bcl2 were lifted,the protein expression level of AKT was not changed,and protein expression levels of BAX,Cleaved-caspase3 were also declined.Conclusion: In conclusion,in doxorubicin-induced H9c2 cells,Histone H3K9 acetylation in the promoter region of Pik3ca significantly increased and promoted transcription of Pik3ca,and Pik3ca promoted doxorubicin-induced apoptosis of H9c2 cells by activating the PI3K/ Akt signaling pathway,providing a new theoretical basis for the study of doxorubicin-induced cardiomyopathy.