Analysis Of Characteristic Molecular Changes In Mouse Model Of Cardiac Hypertrophy By Multi-omics Method

Objective Myocardial hypertrophy is the most common structural abnormality of the myocardium and is closely associated with heart failure.However,the molecules related to the phenotype of cardiac hypertrophy are still not completely clear.Single-cell RNA sequencing and miRNA sequencing techniques were used to explore the molecular changes in the pathogenesis of cardiac hypertrophy induced by pressure overload.Methods A mouse model of cardiac hypertrophy was established by transverse aortic constriction(TAC).Multiple omics studies were performed on 23033 cells from the cardiac hypertrophy group and the control group,including the integration and analysis of single-cell RNA sequencing and miRNA sequencing datasets.The sequencing results were verified by immunofluorescence staining.Myocardial fibroblasts from primary neonatal mice were extracted and treated with angiotensin Ⅱ to construct fibroblasts under the pathological conditions of myocardial hypertrophy and heart failure.The upstream miRNA of differentially expressed transcription factors was intervened by miRNA inhibitor,and the transfection effect was verified by fluorescence quantitative polymerase chain reaction and Western Blot.The possible function of target gene on cardiac hypertrophy was verified by Cell Counting Kit-8.Results Single-cell sequencing was used to analyze the changes in the number of five main cell types in the myocardial tissues of the cardiac hypertrophy group and the control group.Compared with the control group,the number of B cells and T cells in myocardial hypertrophy group was significantly different,and the increasing trend of B cells was statistically significant.In order to verify the sequencing results,Coro1a and CD74 was used to label B cells and T cells respectively.The results of miRNA sequencing showed that mmu-miR-3473a and mmu-miR-1983 were two important up-regulated miRNAs.It was found that the differentially expressed genes were mainly the downstream target molecules of these two miRNAs.We further found that Rora and Ddr2,which are specifically expressed in fibroblasts,are down-regulated in cardiac hypertrophic fibroblasts.Their potential upstream miRNA is up-regulated,so we speculate that miRNA has a negative regulation mechanism for the two downstream target molecules.In vitro,miRNA inhibitor was used to interfere with mmu-miR-3473a.And we found that the expression of Rora was up-regulated and the proliferation of fibroblasts in cardiac hypertrophic cell model was restricted after knocking down miR-3473a.So we speculated that limiting the function of miR-3473a would reverse the proliferation of fibroblasts in cardiac hypertrophy and reduce the occurrence of myocardial fibrosis and heart failure.Conclusion Through single-cell RNA and miRNA sequencing techniques,miR-3473a was found to be differentially expressed in the cardiac hypertrophy group and the control group.Inhibition of the function of miR-3473a could promote the expression of downstream target genes Rora,and thus might play the role of weakening the pathological phenotype of cardiac hypertrophy.