MiR-153-3p Targets βⅡ Spectrin To Regulate Formaldehyde-induced Cardiomyocyte Apoptosis

Objective:To study the role and regulatory mechanism of miR-153-3p in formaldehyde-induced cardiomyocyte apoptosis,and to explore its potential as a therapeutic target for congenital heart disease(CHD).Methods:(1)According to previous studies,βⅡ spectrin is highly conserved and essential in the development of embryonic heart,intraperitoneal injection of formaldehyde can cause abnormal embryonic heart development and cardiomyocyte apoptosis.Real-time quantitative PCR analysis(RealTime Quantitative PCR,RT-qPCR)and Western blotting(Western Blot,WB)can detect the expression of βⅡ spectrin in H9C2 cells induced by formaldehyde.(2)RT-qPCR and WB were used to detect the expression of Caspase7,Cleaved caspase7,Bax and Bcl-2 in H9C2 cells induced by formaldehyde.(3)TdT-mediated dUTP Nick-End Labeling(Tunel)method and flow cytometry were used to detect formaldehyde-induced apoptosis of H9C2 cells.(4)Using bioinformatics methods to predict the upstream binding miRNA of rat βⅡ spectrin and its binding ability.Using RT-qPCR experiment to further screen and identify the results.RT-qPCR was used to detect the expression of βⅡ spectrin after knockdown or overexpression of miR-153-3p.The luciferase reporter gene experiment and RNA pull-down detect the binding of miR-153-3p to the downstream protein βⅡ spectrin.RT-qPCR was used to detect the expression of miR-153-3p in H9C2 induced by formaldehyde.(5)Design and synthesize the small interfering RNA of miR-153-3p,and verify its knockdown efficiency in H9C2.(6)After knocking down miR-153-3p with small interfering RNA in H9C2,the apoptosis of cells was detected by Tunel and flow cytometry.RT-qPCR and WB were used to detect the expression of Caspase7,Cleaved caspase7,Bax and Bcl-2 in H9C2.CCK-8 and Tunel were used to detect the effect of knockdown of miR-153-3p in H9C2 and then formaldehyde induction on proliferation and apoptosis.Tunel and flow cytometry were used to detect the biological function of H9C2 after miR-153-3p knockdown and βⅡ spectrin knockdown together.(7)Designed miR-153-3p overexpression and verify its expression efficiency in H9C2 by RT-qPCR.CCK-8,Tunel and WB experiments were used to detect the biological function of H9C2 after miR-153-3p overexpression.Tunel and flow cytometry were used to detect the biological function of H9C2 after miR-153-3p and βⅡ spectrin co-expression.(8)Tunel and flow cytometry were used to detect the effect of miR-153-3p overexpression on H9C2 apoptosis induced by formaldehyde,CCK8 was used to detect cell proliferation,WB was used to detect the expression of H9C2 apoptotic gene and βⅡ spectrin.(9)To construct a model of formaldehyde-induced fetal rat cardiomyocyte apoptosis,immunohistochemistry(IHC)and fluorescence in situ hybridization(FISH)experiments were used to detect the expression of βⅡ spectrin and miR-153-3p.At the same time,RT-qPCR and WB were used to detect the expression of βⅡ spectrin and apoptosis genes Caspase7,Cleaved caspase7,Bax and Bcl-2.Masson staining was used to detects cardiac fibrosis.Results:(1)βⅡ spectrin was expressed in high abundance in H9C2.Formaldehyde-stimulated H9C2 apoptosis was significantly increased,and βⅡ spectrin was down-regulated in formaldehyde-stimulated H9C2(P<0.05).(2)The prediction results of the Targetscan website show that miR-153-3p is most likely upstream of βⅡspectrin;the experimental analysis of RNA pulldown and luciferase reporter gene proves the interaction of miR-153-3p and βⅡ spectrin;(3)We transfected H9C2 cells with the miR-153-3p mimic or inhibitor with high transfection efficiency(P<0.05);Apoptosis was significantly reduced after knockdown of miR-153-3p in H9C2;while overexpression of miR-153-3p can significantly promote apoptosis;overexpression of miR-153-3p and H9C2 cells induced by formaldehyde can significantly promote apoptosis(P<0.05).(4)The results of Tunel and flow cytometry experiments showed that compared with the NC group,overexpression of miR-153-3p and H9C2 cells induced by formaldehyde increased the apoptosis(P<0.05);(5)The results of flow cytometric showed that compared with the overexpression of miR-153-3p group,the overexpression of miR-153-3p+si-βⅡ spectrin group had significantly increased apoptosis in H9C2(P<0.05);(7)Compared with the healthy group,induced by formaldehyde increased expression of miR-153-3p,Bax and Cleaved caspase7 in the myocardial tissue of embryonic rat.In contrast,the expression of βⅡ spectrin,Bcl-2 and Caspase7 were decreased(P<0.05).Conclusion:We found for the first time that miRNA-miR-153-3p has a certain regulatory role in the process of formaldehyde-induced cardiomyocyte apoptosis.miR-153-3p regulates the expression of Bax,Cleaved caspase7,Bcl-2 and Caspase7 by directly binding to βⅡ spectrin.Thereby promoting the apoptosis of embryonic rat cardiomyocytes.This study enriches the functional regulation and mechanism of cardiomyocytes,explores its clinical significance,and provides potential therapeutic targets for the diagnosis and treatment of congenital heart disease.