Overexpression Of CircRNA SNRK Targets MiR-103-3p To Promote Cardiac Repair Through GSK3β/β-catenin Pathway In Rats With Myocardial Infarction

Objective: Circular RNAs(circ RNAs)play an important role in development of cardiovascular diseases.However,it remains unclear whether circ RNA can protect cardiac function after myocardial infarction(MI).Complete transcriptome sequencing of ischemic myocardium is helpful to discover the key genes and signaling pathway in the development of myocardial infarction.Therefore,the purpose of this study was to identify the key circ RNA and signaling pathway in the pathological process of myocardial infarction,and to explore the mechanism of their regulatory role in the heart.Material and method: 1.In Sprague-Dawley(SD)rats,the left anterior descending branch(LAD)was ligated to induce myocardial infarction.The infarct border zone of myocardium was collected for complete transcriptome sequencing at 3 days post MI,and differentially expressed circ RNAs were revealed.Meanwhile,RT-PCR was performed to verify some circ RNAs with significant differences.2.In vitro experiments,circSNRK overexpression plasmid was constructed and transfected into primary cardiomyocytes.Cell apoptosis was detected by flow cytometry,TUNEL staining and WB,cell viability was detected by CCK8 assay,and cardiomyocytes proliferation was detected by Ed U,Ki67 staining and flow cytometry.3.The interaction between circSNRK and mi R-103-3p,as well as the interaction between mi R-103-3p and its target gene SNRK were verified by bioinformatics techniques,q RT-PCR techniques,double luciferase reporter gene assay and WB assay.4.Primary cardiomyocytes were transfected with mi R-103-3p mimics and inhibitor to detect the effects of mi R-103-3p on cell apoptosis and proliferation.5.By constructing SNRK overexpression plasmid,the interaction of circSNRK,mi R-103-3p and SNRK,and their effects on apoptosis and proliferation of cardiomyocytes were determined.6.Adeno associated virus 9(AAV9)mediated myocardium overexpression of circSNRK in vivo.We used Echocardiography to detect cardiac function.HE,Masson and TTC staining were used to observe cardiac structure and fibrosis.Apoptosis was detected by TUNEL staining and WB staining.Angiogenesis was evaluated by CD31 and α-SMA staining.7.GSK3β/β-catenin signaling pathway was detected by WB and Co-Immunoprecipitation(Co-IP).Results: 1.66 differentially expressed circ RNAs were screened by high throughput sequencing.The result showed that 19 circ RNAs are up-regulated and 47 circ RNAs are down-regulated.q RT-PCR results showed that the expression of circSNRK was significantly down-regulated in myocardium of MI and in hypoxic and serum deprivation(H/SD)cardiomyocytes.2.Overexpression of circSNRK in cardiomyocytes significantly reduced apoptosis and promoted proliferation of cardiomyocytes.3.q RT-PCR,double luciferase reporter gene assay and WB assay confirmed that circSNRK binds to mi R-103-3p directly,SNRK is the direct target gene of mi R-103-3p,and circSNRK can significantly inhibit the binding effect between mi R-103-3p and SNRK.4.Overexpression of mi R-103-3p in vitro promoted apoptosis and inhibited proliferation of cardiomyocytes,whereas inhibition of mi R-103-3p had the opposite results.5.Mi R-103-3p mimics impaired the effect of circSNRK overexpression on apoptosis and proliferation,which was reversed by SNRK up-regulation.6.In vivo,overexpression of circSNRK can significantly reduce myocardial apoptosis,promote angiogenesis,alleviate fibrosis,reduce pathological remodeling,and improve cardiac function.7.SNRK regulates the phosphorylation of GSK3β,thereby regulating the apoptosis and proliferation of cardiomyocytes.Conclusion: Circ SNRK is a key molecule in the occurrence and development of MI.Overexpression of circSNRK can reduce cardiomyocytes apoptosis,promotes proliferation and functional recovery after MI by mi R-103-3p/ SNRK/ GSK3β/β-catenin axis,which is expected to provide a promising therapeutic target for MI.