Bioinformatic Analyses And Cytological Verification Of STAT3 Expression Difference In Hypertrophic Cardiomyocytes

Background: As the response of the heart to various pathophysiological stresses,cardiac hypertrophy has compensatory significance in the early stage.However,continuous myocardial hypertrophic growth will provoke poor cardiac remodeling and cardiac dysfunction,eventually leading to cardiovascular adverse events including heart failure,arrhythmia and death.Moderate level of autophagy is vital for cell survival and growth,and also affects the development of hypertrophic cardiomyocytes.Meanwhile,inflammatory signal transduction and immune cells play an indispensable pathophysiological role in myocardial hypertrophy.However,upstream regulators of genes related to autophagy and immune regulation remain to be elucidated.Objective: Hub genes associated with autophagy and immune infiltration that were differentially expressed in hypertrophic cardiomyocytes were identified by bioinformatic analyses,and the differential expression was further verified in cytological experiments.Methods:(1)Bioinformatic analyses: The transcriptome array expression profiling dataset GSE36961 and the high-throughput sequencing expression profiling dataset GSE160997 were selected from Gene Expression Omnibus(GEO)database,both of which included hypertrophic cardiomyopathy(HCM)and healthy control heart tissue samples.Differentially expressed genes(DEGs)between HCM and control heart tissues were obtained by differential expression analysis of the above datasets using R software,and common DEGs of the two datasets were gained by intersection.The common DEGs were analyzed by gene ontology(GO)and Kyoto encyclopedia genes and genomes(KEGG),and gene set enrichment analysis(GSEA)was performed in the two datasets.The protein-protein interaction(PPI)network of common DEGs was constructed by STRING online website and Cytoscape software,followed by 10 hub genes screening,and the key autophagy related genes were acquired by intersection with the autophagy related gene set integrated from Human Autophagy Database(HADb)and MSig DB database.Immune infiltration analysis was performed on the combined datasets using Sanger Box online website based on x Cell database,where immune and stromal cell types between the HCM group and the healthy control group were compared,and Pearson correlation coefficients among immune cell subtypes as well as 10 key genes and immune cell subtypes were calculated.(2)Cell experimental verification: H9c2 cardiomyocytes were stimulated with isoproterenol(ISO)and phenylephrine(PE)to induce cardiac hypertrophy,and the expression differences of hub genes connected with autophagy and immune infiltration in hypertrophic cardiomyocytes were validated.ISO(10μmol/ L,that is,10μM)and PE(100μM)were applied to H9c2 cardiomyocytes for 48 h respectively.Quantitative PCR and immunofluorescence assay were used to detect H9c2 cardiomyocyte hypertrophy models.H9c2 cardiomyocytes were treated with ISO(0,5,10,20μM)and PE(0,50,100,200μM)at different concentrations for 24 h and 48 h.The expressions of key genes and the autophagy marker molecule Beclin1 were detected by Western blot.The corresponding gene expressions in cardiomyocytes treated by ISO(10μM)and PE(100μM)for 48 h were detected by quantitative PCR.(3)Statistical analysis of cell experimental results: Graph Pad Prism 7 software was used for plotting and statistical analysis of quantitative PCR results and cell surface area results.Parameter test of student T test was applied to detect whether there was statistical difference between the experimental group and the control group.P < 0.05 was considered statistically significant.Results:(1)Bioinformatic analyses: 971 and 2527 DEGs were obtained from GSE36961 and GSE160997,respectively.The 93 common up-regulated DEGs were mainly enriched in biological processes including "circulatory system development","muscle structure development" and "actin filament based process",as well as Janus kinase(JAK)-signal transducer and activator of transcription(STAT)and AGE-RAGE signaling pathways.The 191 common down-regulated DEGs were enriched in immunerelated biological processes and inflammatory signaling pathways.GSEA results showed that in GSE36961,"JAK＿STAT＿SIGNALING＿PATHWAY"(FDR=0.0034,p=0,NES=-1.8908)and "HYPERTROPHIC＿CARDIOMYOPATHY＿HCM"(FDR=0.1174,p =0.0281,NES=-1.3593)were significantly down-regulated in the control group,while "REGULATION＿OF＿AUTOPHAGY" was significantly upregulated in the HCM group(FDR=0.1203,p =0.0285,NES=1.4859).In GSE160997,"JAK＿STAT＿SIGNALING＿PATHWAY"(FDR=0.0920,p =0.0635,NES=-1.3921),"HYPERTROPHIC＿CARDIOMYOPATHY＿HCM"(FDR=0.0948,p =0.0759,NES=-1.3746)and "REGULATION＿OF＿AUTOPHAGY"(FDR=0.1012,p =0.0557,NES=-1.4386)were down-regulated in the control group.PPI network analysis identified the top 10 key genes(TYROBP,STAT3,ITGB2,ITGAM,CSF1 R,IL6,CD163,FCER1 G,HCK and PIK3R1).Through intersecting them with 714 autophagy related genes integrated from HADb and MSig DB databases,STAT3 was identified as a critical autophagy related gene.The results of immune infiltration analysis suggested that there was a significant positive correlation between macrophages and monocytes(M1 type macrophages and macrophages: Pearson correlation coefficient =0.87,M1 type macrophages and monocytes: Pearson correlation coefficient =0.82,macrophages and monocytes: Pearson correlation coefficient =0.79),and CD8+ T cells were significantly negatively correlated with M1 type macrophages,monocytes and macrophages(Pearson correlation coefficients were-0.36,-0.32,-0.30,respectively).IL6 was positively correlated with all immune cells,and the other key genes were negatively correlated with most of the immune cells.(2)Cell experimental verification: H9c2 cardiomyocytes were treated with ISO(10μM)and PE(100μM)for 48 h,respectively.Quantitative PCR and immunofluorescence assay demonstrated that the models of cardiomyocyte hypertrophy were successfully established.In the experimental group,H9c2 cardiomyocytes were stimulated by ISO and PE respectively,and the protein expression levels of STAT3 and Beclin1 in different concentrations of drugs were upregulated compared with the control group by Western blot analysis after 24 h and 48 h treatment.The results of quantitative PCR showed that the gene expresion levels of STAT3(P =0.0117)and Beclin1(P =0.0481)in ISO group(10μM,48h)as well as the ones of STAT3(P =0.0068)and Beclin1(P =0.0002)in PE group(100μM,48h)were increased in different degrees compared with the control group.Conclusion:(1)Through bioinformatic analyses,it was found that STAT3 was closely related to the autophagy and immune infiltration in cardiomyocytes,and may have a regulatory effect on the autophagy and immune infiltration in hypertrophic cardiomyocytes.(2)At the cellular level,the expression levels of STAT3 and autophagy related gene Beclin1 were significantly up-regulated in cardiomyocytes with a certain degree of hypertrophy.(3)The results of this study provide a theoretical basis for clarifying the mechanism of regulating autophagy and immune infiltration in cardiomyocytes,as well as clues for further exploring the pathophysiological mechanisms and potential intervention targets of cardiac hypertrophy.