Research On The Role And Mechanism Of MicroRNA-146a In Doxorubicin-induced Cardiotoxicity

Objective: The purpose of this study was to investigate the role and mechanism of miR-146 a in low-dose long-term doxorubicin(DOX)-induced chronic myocardial injury by constructing chronic DOX myocardial injury models both in vitro and in vivo,and the relationship between circulating miR-146 a levels and DOX myocardial injury.Methods: Part 1.The human ventricular myocytes cell line(AC16)were stimulated with DOX in different concentration gradients(0-5μM)and time gradients(0-72h).The morphological changes of cells were observed under a microscope.Cell viability was detected by CCK-8 method;the expression of apoptosis and autophagy related proteins were detected by Western Blot,cell apoptosis was detected by TUNEL and flow cytometry;autophagy was detected by GFP-LC3;the expression of miR-146 a were detected by RT-PCR.Then,AC16 s was transfected with miR-146 a mimics and inhibitors before stimulated with DOX(0.5 μM,48 h)to investigate the role of miR-146 a in regulating apoptosis and autophagy in chronic DOX myocardial injury.Finally,Bioinformatics technology were used to predict the target gene TAF9 b of miR-146a;RT-PCR and dual luciferase reporter gene were used to verify;a remedial experiment was established by co-transfect with si TAF9 b and miR-146 a inhibitors in AC16 before DOX(0.5 μM,48h)stimulation.Part2.Six-week-old,male wild-type mice and miR-146 a knockout mice were used and a chronic cardiomyopathy model was established by intraperitoneal injection of DOX(5mg/kg/w,4w).RT-PCR was used to detect the content of miR-146 a in heart tissue at different time points.Heart ultrasound was used to detect mouse heart function.The apoptosis of myocardial cells was detected by TUNEL.The autophagy level of cardiomyocytes was detected by transmission electron microscopy.The expression of target genes and downstream apoptosis and autophagy related molecules were detected by Western Blot.Part 3.Six-week-old,male wild-type mice were used and a chronic cardiomyopathy model was established by intraperitoneal injection of DOX(5mg/kg/w,4w),RT-PCR was used to detect the content of miR-146 a in serum at different time points.Serum samples and clinical data of patients with DOX chemotherapy and non-DOX chemotherapy before and after chemotherapy were collected.Circulating miR-146 a were detected by RT-PCR and its relationship with clinical indicators BNP and troponin were analyzed.Results: Part 1.The cell viability significantly declined detected by CCK-8 and had dropped by about 50% with DOX at 0.5μM for 48h;TUNEL staining,flow cytometry and western blot revealed that DOX significantly increased the rate of apoptosis in AC16 cardiomyocytes;GFP-LC3 and western blot suggested that autophagy flux was inhibited by DOX;miR-146 a was first protectively up-regulated in cardiomyocytes after DOX intervention and subsequently decreased;Under DOX stimulation,overexpression of miR-146 a can inhibit cardiomyocyte apoptosis and promote autophagy,while knocking out miR-146 a further aggravates cardiomyocyte apoptosis and inhibits autophagy;miR-146 a targets TAF9 b and exerts its protective effect on DOX-induced myocardial injury through the TAF9b/P53 pathway.Part 2.After the injection of DOX,the expression of miR-146 a in myocardial tissue increased and was slowly consumed until to lower than normal;Echocardiography showed that compared with wild type mice,cardiac function of miR-146 a knockout mice was significantly reduced after DOX treated;HE staining showed that myocardial fibers were more disordered in miR-146 a knockout mice than wild type mice after DOX treated;TUNEL staining suggested the in situ cardiomyocyte apoptosis of miR-146 a knockout mice was more severe than that of wild type mice after DOX treated;Electron microscopy showed that miR-146 a knockout mice had more obvious mitochondrial swelling and significantly reduced autophagy than wild type mice after DOX treated.Part 3.After the injection of DOX,the expression of miR-146 a in serum increased increased and was slowly consumed until to lower than normal;In patients,the circulating miR-146 a increased in the acute phase of DOX stimulation and was positively correlated with BNP.Conclusion: miR-146 a inhibiting apoptosis promotion and autophagy confusion in DOX-induced cardiotoxicity by targeting TAF9 b and inhibiting the stability of P53.In addition,circulating miR-146 a may be a biomarker in DOX-induced myocardial injury.