The Study Of Myocardial Protective Effect Of RBM10 On Acute Myocardial Infarction In Conditional Gene Knockout Mice And It’s Underlining Mechanisms

Part Ⅰ Construction of RBM10 CKO Mice ModelObjective:Construction of conditional RBM10-knockout mice.Methods:1.RBM10-Lox P mice were induced by CRISPR/Cas9technology.2.RBM10-lox P mice were mated with Cag-Cre/ESR1transgenic mice,which produced Genetic mice of F1 generation.3.F1generation mice were mated with RBM10-Lox P,which produced F2generation mice.4.RBM10fl/fl Cre⁺and RBM10fl/fl Cre^-genotype mice were obtained by the self-mating of F2 generation mice.5.Intraperitoneal injection of tamoxifen induced knockout of RBM10 gene in mice.6.The genotypes of mice were tested by PCR.7.The expression of RBM10protein in myocardial tissue of mouse was detected by Western Blot.Results:1.RBM10 fl/fl Cre⁺and RBM10 fl/fl Cre^-genotype mice were successfully constructed.2.The expression of RBM10 was significantly down-regulated in RBM10^-/-mouse myocardial tissue（P<0.001）.Conclusion:The conditional RBM10-knockout mice were successfully constructed.Part Ⅱ The protective effect of RBM10 on acute myocardial infarction in RBM10 CKO mice and analysis of RBM10 regulated transcriptome during myocardial infarctionObjective:1.Exploring the protective effect of RBM10 by using conditional gene knockout（CKO）mice during acute myocardial infarction in mice;2.The transcriptome changes regulated by RBM10 would be investigated during acute myocardial infarction in mice through the differential genes of myocardial transcriptome which were screened by second generation sequencing technology.Methods:1.The mouse model of acute myocardial infarction was established by ligating the left anterior descending coronary artery.2.The area of acute myocardial infarction would be observed by TTC staining.3.RNA-seq technique was used to detect gene expression in ischemic myocardial tissue of RBM10 wild-type mice and RBM10 conditioned knockout mice,and the myocardial tissue of sham operation group was used as control.The original data would be homogenized by the"TPM".The"DESeq2"package was used to analyze the original read counts data so that the differential genes were obtained.The"pheatmap"package was used to perform cluster analysis of the differential genes.Significantly differential genes were used to perform gene ontology database（GO）enrichment and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis by DAVID.4.The m RNA level of HSPA1B in mouse myocardium was detected by PCR.Results:1.After 6 hours of myocardial infarction,the myocardial infarction area of RBM10^-/-group was significantly larger than that of RBM10^+/+group（P<0.001）.2.Compared with RBM10 non-knockout mice,591 genes were differentially expressed in ischemic myocardial tissue of RBM10 knockout mice,among which 63 genes were significantly up-regulated and 32 genes were significantly down-regulated（P<0.05）.3.GO and KEGG analysis of significantly differentially expressed genes using David online tool showed that target genes regulated by RBM10were involved in multiple myocardial injury-related signaling pathways.4.After 6 hours of myocardial ischemia,the expression level of HSPA1B m RNA was higher than that of RBM10 non-knockout mice（P<0.01）.Conclusion:1.RBM10 had a protective effect on the myocardial tissue of mouse with acute myocardial infarction.2.RBM10 may regulate cytokine-cytokine Receptor interaction,nod-like receptor signaling pathway,Chemokine signaling pathway,and TNF signaling pathway in response to acute myocardial ischemia;3.RBM10 may protect myocardial tissue of mouse with acute myocardial infarction by regulating the expression of Hspa1b.