The Investigation Of CircNFIX’s Function In Cardiomyocyte Apoptosis

Objective Myocardial infarction is a cardiovascular disease with high mortality and poor prognosis.The decrease of cardiomyocyte apoptosis is of great significance to improve the survival rate of patients with myocardial infarction.Characterized by stable structure,tissue and developmental stage-specific expression,circRNAs play vital roles in development and process of multiple diseases.However,the roles of circRNAs in cardiomyocyte apoptosis remain unclear.The purpose of this study was to explore the roles of circRNAs in cardiomyocyte apoptosis and to further clarify the mechanism of myocardial infarction.Methods CircRNAs associated with cardiomyocyte apoptosis were screened in the database and sequencing literature,and circRNAs with significant change in H9c2 cell apoptosis induced by H2O2(100 μM)were detected.Divergent primers and convergent primers were used to perform PCR,agarose gel electrophoresis was applied to confirm the molecular weight of PCR product,and the sequence of reverse splicing site was detected by sanger sequencing.The interspecies conservation was analyzed by Phylop.RNase R assay was performed to confirm the resistance of circNFIX to RNase R digestion,and Actinomycin D assay was applied to detect the stability of circNFIX.The apoptosis of cardiomyocytes was induced by hydrogen peroxide(100 μM)or hypoxia,and the expression of circNFIX was measured in different stimulation times.A mouse model of myocardial infarction was constructed,Evan’s blue staining analysis and Western blot were used to verify the effect of myocardial ischemia,the expression of circNFIX were detected at different ischemic times.Small interfering RNA(si-circRNA)was transfected and its inhibitory effect on circRNA was confirmed,then the effect of si-circNFIX on hydrogen peroxide(100 μM)induced apoptosis of H9c2 cells was detected.Furthermore,the effect of overexpression plasmids(oe-circ RNA)was transfected and its effect to circRNA was verified,then the effect of overexpression vector oe-circNFIX on the apoptosis of H9c2 induced by hydrogen peroxide(50 μM)was detected.TUNEL staining was performed to detect the effect of si-circNFIX or oe-circNFIX on the apoptosis rate of H9c2 cells.Western blot assay was used to determine the effects of si-circNFIX or oe-circNFIX to the protein level of bax and bcl-2 in H9c2 cells.Results The circNFIX screened in this study showed the most significant change in H9c2 cell apoptosis.Agarose gel electrophoresis suggested that divergent PCR product was single,specific,consistent with molecular weight band,and sanger sequencing showed that its base sequence was consistent with circBase.CircNFIX is conservative in humans,mice and rats.RNase R assay demonstrated that circNFIX was resistant to the digestion of RNase R.The actinomycin D assay demonstrated that circNFIX was more stable than linear NFIX and had a longer half-time.In hydrogen peroxide(100 μM)or hypoxia induced H9c2 cell apoptosis,the expression of circNFIX was downregulated in a time-dependent manner.Evans blue staining analysis revealed that ischemic area of the left ventricle was increased significantly after ischemic surgery.Western blot assay demonstrated that the ratio of bax and bcl-2 was increased in myocardial ischemia group.These results indicated that myocardial infarction area and apoptotic levels were increased in the mouse myocardial infarction model.Moreover,the expression of circNFIX was decreased in a time-dependent manner.Si-circNFIX inhibited the expression of circNFIX significantly,and oe-circNFIX has a contrary effect.TUNEL staining indicated that knockdown of circNFIX inhibited the increase of TUNEL-positive cell rate induced by hydrogen peroxide(100 μM),and overexpression of circNFIX promoted the augmentation of TUNEL-positive cell rate induced by low concentration of hydrogen peroxide(50 μM).Western blot analysis demonstrated that the reduction of circNFIX inhibited the augmentation of bax/bcl-2 ratio induced by hydrogen peroxide(100 μM),and the overexpression of circNFIX promoted the augmentation of bax/bcl-2 induced by low concentration of hydrogen peroxide(50 μM).Conclusion In this study,circNFIX was found to be abundant in heart with advanced conservation and stability.The expression of circNFIX was decreased during apoptosis of cardiomyocytes and tissue.Silencing circNFIX inhibited H9c2 cells apoptosis induced by hydrogen peroxide,and overexpression of circNFIX promoted the susceptibility of cardiomyocyte to apoptosis.CircNFIX can promote apoptosis in the process of myocardial infarction.