Relationship Between Hspa1a Gene Expression And Cardiac Hypertrophy

Research background:Cardiac hypertrophy refers to the increase of the volume of cardiomyocytes,which mainly occurs in the case of long-term pressure overload.The total amount of myocardial cells increases and the contractility strengthens,so that the heart can maintain normal blood circulation and considerable reserve power.But this compensatory function will gradually develop into decompensation,resulting in myocardial ischemia,myocardial contractility decline,and eventually develop into heart failure.Cardiac hypertrophy can be divided into pathological hypertrophy and physiological hypertrophy.Pathological hypertrophy is a complex process,which is related to capillary sparsity,fibrosis,increased production of pro-inflammatory cytokines,poor epigenetic modification and cell dysfunction.New evidence suggests a link between Cardiac hypertrophy,immune response and activated inflammation.In the past decade,researchers have made great progress in the formation mechanism of cardiac hypertrophy,and found many modification factors and signal molecules in cardiac hypertrophy.Gene expression regulation of cardiac hypertrophy is considered to be a very important research direction in this field.Therefore,we made animal models of cardiac hypertrophy to find genes closely related to the regulation of cardiac hypertrophy,and to explore the role and mechanism of its abnormal expression.In this study,we studied the relationship between hspa1 a and cardiac hypertrophy and its mechanism from the perspective ofRNA binding protein and its function.The research in the field of RBP is the frontier hotspot ofRNA research.The experimental results will be beneficial to explore the regulation of gene expression and its modifying factors and signal molecules in cardiac hypertrophy,It is of great clinical significance to re understand the mechanism of cardiac hypertrophy and to think about the therapeutic targets of related diseases.Research objective:To explore the correlation between abnormal expression of heat shock protein gene(hspa1a)and cardiac hypertrophy by making animal models of cardiac hypertrophy.Research content:1.Establishment of a mouse model of cardiac hypertrophy by coarctation of aortic arch(TAC),it can be compared with sham group.In this study,8-week-old healthy male(C57BL / 6J)mice(weighing23-33g)from China were used.The mice were divided into four groups:TAC + dantrolene,TAC + DMSO(control reagents),sham + dantrolene and sham + DMSO(control reagents).Each group was given drugs(dantrolene,30 mg / kg,daily,4 weeks in total.)or control reagents on the second day after operation,and the weight of mice was measured after 4 weeks.After the mice were killed,the heart and tibia were taken to measure the dry weight of the heart and the length of the tibia,then averaged the results.The ratio of heart weight to tibia length and the ratio of heart weight to body weight were calculated.HE staining and Masson staining were performed on the left ventricular tissue of mouse heart,and the fibrotic area of myocardial tissue was quantified by Image J software.Mice with successful cardiac hypertrophy models(The ratio of heart weight to tibial length(HW / TL)(P < 0.001)and heart weight to body weight(HW / BW)(Fig.1)showed that the hearts in all models were significantly hypertrophic.)were selected for further studies to explore the gene regulatory mechanisms of cardiac hypertrophy.2.Determination of Hspa1 a Four groups of mouse ventricular tissue samples were established for high-throughput sequencing.Using illunima hiseq-x-ten sequencing platform,transcriptome data were obtained by paired-end sequencing.Differential expression genes(DEGs)were obtained by differential expression analysis of four groups of transcriptome data.The expression of hspa1 a which isRNA binding protein gene was significantly different between TAC + dantrolene group and sham + dantrolene group,and between TAC + DMSO group and sham + DMSO group.The expression of mki67 and gm5619 was significantly different between TAC +dantrolene group and TAC + DMSO group.These results suggest that dantrolene may delay cardiac hypertrophy and ventricular remodeling and improve heart failure by regulating the expression ofRNA binding protein genes mki67 and gm5619.3.Overexpression of Hspa1 a in mouse heart HL-1 cells In order to clarify the correlation and mechanism between hspa1 a and cardiac hypertrophy,in this project,we overexpressed(OE)Hspa1a in mouse cardiac HL-1 cells and identified 121 differentially expressed genes(DEGs)among Hspa1a-overexpressed(Hspa1a-OE)HL-1 and monitoring cells using edge R,including 69 significantly upregulated genes and 52 downregulated genes.Heatmap study of the DEG expression patterns inRNA-seq specimens illustrated a great agreement of the Hspa1a-mediated transcription in both series of results.GO and KEGG pathway annotation database was used to analyze all 121Hspa1a-regulated DEGs in HL-1 cells.In terms of the biological process GO analysis,the up-regulated genes in Hspa1a-OE HL-1 cells were mainly enriched in G protein-coupled receptor signaling pathway,signal transduction,transportation,and other biological processes,which is less relevant to cardiac hypertrophy.In hsps1a-oe-hl-1 cells,a large number of non codingRNAs were upregulated.The first two ncRNAs(Rn7sk,RMRP)with the highest expression level were associated with cardiac hypertrophy.Down regulated genes are mainly related to inflammation and immune response,including CXCL1,CCL2,CCL7,CXCL5,Fas and C3,which are basically related to cardiac hypertrophy.In addition,Lcn2,the gene with the highest expression among all down regulated genes,is an immune mediator and plays a key role in the ontogeny of heart failure and cardiac hypertrophy.4.Hspa1 a regulates the expression of ncRNAs in HL-1 cells and Validation of Hspa1a-regulated expression of ncRNAs and immune response-related genes in HL-1 cells In order to confirm the different expression of ncRNAs and hspa1 a regulatory genes in HL-1 cells and the correlation between cardiac hypertrophy and immune response,GO and KEGG pathway annotation database analysis showed that the down-regulated genes were mainly related to inflammatory and immune response,including CXCL1,CCL2,CCL7,CXCL5,Fas and C3.These genes were basically related to cardiac hypertrophy.In addition,Lcn2,the gene with the highest expression among all down-regulated genes,is also an immune mediator and plays a key role in the ontogeny of heart failure and cardiac hypertrophy.The expression of seven immune response related genes was verified by RT qPCR experiment.5.Using software tools to analyze the alternative splicing events regulated by Hspa1 a in HL-1 cells ABLas software was used to further analyze the alternative splicing events regulated by Hspa1 a in HL-1 cells.We detected known alternative splicing events(ASEs)in the model gene we entitled in the new ASEs,and reference genome.Through exerting a rigorous cutoff of p-value?0.05,modified AS ratio?0.2,we determined 366 credible regulated alternative splicing events(RASEs),mainly including alternative 5' splice-site(A5SS)events,exon skipping(ES)events,alternative 3' splice site(A3SS)events,and intron retention(IR)events.By significant regulation of genes at alternative splicing level by hspa1 a,we found that among these RASGs related to the overexpression of Hspa1 a in HL-1 cells,Asxl2 and Runx1 are associated with cardiac hypertrophy.6.Validation of Hspa1a-regulated alternative splicing events in HL-1 cells.In order to explore the significant regulation of hspa1 a gene at the level of alternative splicing,we found that Asxl2 and Runx1 were associated with cardiac hypertrophy,and further found that the alternative splicing events of Asxl2 and Runx1 were consistent with the results verified by RT qPCR.Research results:1.C57 BL / 6J mouse model of pathological cardiac hypertrophy was successfully established by transverse aortic arch constriction(TAC).Differential expression genes(DEGs)were obtained by differential expression analysis of four groups of transcriptome data.2.Hspa1 a regulates the transcriptome of HL-1 cells,69 genes are significantly up regulated and 52 genes are down regulated.3.Hspa1 a selectively regulates the expression of ncRNAs related to cardiac hypertrophy.Rn7 sk and RMRP are related to cardiac hypertrophy.The expression of Rn7 sk and RMRP was validated by RT-qPCR experiment.The results are consistent.4.Hspa1 a negatively regulates the expression of genes related to inflammation and immune response in cardiac hypertrophy,including CXCL1,CCL2,CCL7,CXCL5,Fas and C3.The expression of 7 immune response-related genes was verified by RT-qPCR experiment.The results are consistent.5.ABLas software was used to analyze alternative splicing regulated by hspa1 a.We found that Asxl2 and Runx1 were associated with cardiac hypertrophy.Hspa1 a negatively regulates the alternative splicing of ASXL2 and positively regulates the alternative splicing of RUNX1.The alternative splicing events of Asxl2 and Runx1 were consistent with the results of RT-qPCR verification.Research conclusion:1.The expression of Hspa1 a was significantly up regulated in TAC mice,which indicated that Hspa1 a was closely related to cardiac hypertrophy.2.Hspa1 a regulates the transcriptome of HL-1 cells,69 genes are significantly up regulated and 52 genes are down regulated.3.Hspa1 a selectively positively regulates the expression of ncRNAs(RN7SK and RMRP)related to cardiac hypertrophy.4.Hspa1 a negatively regulates the expression of inflammation and immune response related genes(CXCL1,CCL2,CCL7,CXCL5,Fas,C3 and Lcn2).5.Hspa1 a can play a role in cardiac hypertrophy by regulating the alternative splicing of Asxl2 and Runx1.Hspa1 a negatively regulates the alternative splicing of ASXL2 and positively regulates the alternative splicing of RUNX1.