The Study On Mechanism Of CircHNRNPH1 In Myocardial Fibrosis After Myocardial Infarction

Objective:circular RNA High-throughput sequencing of rat myocardial infarction models,we know that CircHNRNPH1 is highly expressed in ischemic heart disease,but its formation mechanism and mechanism are still lack of systematic research.In order to elucidate the function of circHNRNPH1 in ischemic heart disease,our group mainly clarified the bioinformatics of circHNRNPH1;studied the function,regulation and mechanism of circHNRNPH1 at the cellular level;and verified circHNRNPH1 in ischemic heart disease at the anima level Effects on cardiac function and degree of myocardial fibrosisMethods:High-throughput sequencing of circular RNA and its differential expression in myocardial infarction and normal tissues of Norwegian rat hearts were performed to select the differential RNA circHNRNPH1.The outer loop primer and the inner loop primer were designed according to the circHNRNPH1 predicted sequence,and the authenticity of circHNRNPH1 was confirmed by RNAase R experiment,and the products were amplified by PCR amplification and sequenced respectively to determine the circHNRNPH1 sequence.According to the circHNRNPH1 sequence,it was genetically mapped and analyzed to characterize the introns upstream and downstream of its exons.The mfold software was used to predict the stable secondary structure of circHNRNPH1.The expression of circHNRNPH1 in cardiomyocytes and cardiac fibroblasts was detected by qRT-PCR.The localization of circHNRNPHl in cardiac fibroblasts was detected using a circHNRNPH1-specific probe.qRT-PCR and western blot were used to detect the role of ADAR1 in the regulation of TGF β regulation of circHNRNPHl formation Targeted miRNAs for circHNRNPHl were predicted using regRNA 2.0 RIP,FISH experiments,dual luciferase assays,and western blot experiments confirmed the interaction of circHNRNPHl with target miRNAs and their effects on downstream target genes.Scratch test and transwell experiment confirmed the effect of circHNRNPHl on myocardial fibroblast migration,TUNEL assay and cell flow assay to detect the effect of circHNRNPHl on myocardial fibrocyte apoptosis.An animal model of myocardial infarction in rats was established.The rAAV-sh-circHNRNPHl virus was injected in situ to inhibit the expression of circHNRNPHl in the heart.The cardiac function of rats after circHNRNPHl was down-regulated by cardiac Doppler ultrasound,and immunohistochemistry was performed by Masson staining.Tissue protein western blot was used to detect the effect of circHNRNPHl on myocardial fibrosis in ischemic heart diseaseResults:RNAase R and DNA agarose gel experiments confirmed the true presence of circHNRNPHl and determined the sequence of circHNRNPHl by sanger sequencing.qRT-PCR experiments confirmed that circHNRNPHl is highly expressed in cardiac fibroblasts,and circHNRNPH1 and miR216-5p colocalize in the cytoplasm of cardiac fibroblasts.circHNRNPH1 directly interacts with miR216-5p,promotes SMAD7 expression,promotes TβRI degradation,inhibits aSMA and collagen I protein expression,and reduces fibrosis;scratch assay,transwell assay confirms that circHNRNPHl inhibits myocardial fibroblast migration;TUNEL assay and cell flow The experimental results confirmed that circHNRNPHl can promote myocardial fibroblast apoptosis through SMAD7 protein.In vivo experiments confirmed that knocking down circHNRNPHl significantly promoted the expression of aSMA and collagen I mRNA and protein levels,and promoted TβRI protein expression and decreased SMAD7 protein expression.Masson staining showed inhibition of circHNRNPHl expression,which significantly enhanced collagen fibers in myocardial infarction.Expression;immunohistochemistry results showed that inhibition of circHNRNPHl expression can significantly promote the expression of aSMA and collagen I;cardiac Doppler color Doppler ultrasound detection of cardiac function,the results show that inhibition of circHNRNPHl expression further aggravates cardiac dysfunction,leading to cardiac ejection fraction(EF),the left ventricular short axis shortening rate(FS)decreased to varying degrees,left ventricular systolic and diastolic left ventricular diameter and left ventricular volume increased to varying degrees.Conclusion:In ischemic heart disease,the expression of circHNRNPHl is increased,which plays a major role in cardiac fibroblasts.ADAR1 stabilizes the expression of circHNRNPHl induced by TGFβ;CircHNRNPH1 acts as a sponge of miR216-5p,increases the expression of SMAD7 protein,promotes the degradation of TβRI,and promotes the apoptosis of myocardial fibroblasts through SMAD7,thereby inhibiting the progression of cardiac fibrosis.In vivo experiments have further demonstrated that knocking down circHNRNPHl can significantly promote myocardial fibrosis and aggravate cardiac dysfunction.