RAAV9 Mediates SiRNA-targeted Intervention Of P65 On Cardiomyocyte Hypertrophy And Fibrosis-related Factors

Objective: Inhibition of p65 gene expression in rat ventricular myocytes H9c2 cells by RNA interference mediated by type 9 adeno-associated virus(AAV9),further study of the effect of targeted inhibition of NF-κB signaling pathway p65 on cardiomyocyte hypertrophy and fibrosis.Methods:rAAV9-eGFP and rAAV9-eGFP-NF-κB p65-siRNA were transfected into H9c2 cells according to different multiplicities of infection(MOI).The expression of enhanced green fluorescent protein(eGFP)positive at 24h-168 h after transfection was observed under fluorescence inverted microscope and the optimal multiplicity and time were determined.The transfection efficiency was detected by flow cytometry.The effect of virus on the viability of H9c2 cells was detected by CCK-8method.AngII was added to the cells as a stimulating factor,and the expression of p65 at mRNA and protein levels was analyzed by qRT-PCR and Western blotting.Flow cytometry was used to analyze the apoptosis of the control group and each experimental group.ELISA for detection of atrial natriuretic peptide(ANP),type B natriuretic peptide(BNP),type I collagen(Col I),type III collagen(Col III),and transforming growth factor-β(TGF-β)at the protein level expression.qRT-PCR detection of ANP,BNP,Col I,Col III,TGF-β expression at mRNA level.Results: After 48 h of virus transfection,eGFP began to express,and the expression intensity increased with time,and reached the highest value at 120 h.At this time,the transfection rate was detected by flow cytometry(66.96±1.12)%.Compared with the blank group,the activity of H9c2 cells did not change significantly after CCK-8 detection of transfected virus.NF-κB p65 was activated in H9c2 cells at rest,and the activity of NF-κB p65 was significantly increased after stimulation with AngII,while transfection of rAAV9-eGFP-NF-κB p65-siRNA was effective in inhibiting the activity of NF-κB p65.Flow cytometry showed that the degree of apoptosis in the AngII-stimulated group was significantly higher than that in the blank group,whilethe apoptosis in the rAAV9-eGFP-NF-κB p65-siRNA group was significantly inhibited.ELISA and qRT-PCR showed that transfection of rAAV9-eGFP-NF-κB p65-siRNA could effectively inhibit the expression of ANP,BNP,Col I,Col III and TGF-β,while the expression of ANP,BNP,Col I,Col III and TGF-β was significantly higher in AngII stimulation group than of the blank control group.Compared with the blank control group,the expression levels of BNP,Col I,Col III and TGF-β were significantly decreased in rAAV9-eGFP-NF-κB p65-siRNA+ AngII group,but the expression of ANP was not significantly decreased.Conclusion: rAAV9-mediated RNA interference can effectively transfect and inhibit the expression of NF-κB p65 gene in H9c2 cells,and inhibit the activity of H9c2 cells,which can reduce the apoptosis induced by angiotensin II.And can effectively inhibit the expression of ANP,BNP,Col I,Col III,TGF-β.