| Background:Rheumatic heart disease(RHD)is a disease that seriously damages the health of humans,and it may be fatal in the later stages of the disease.According to relevant reports,the number of RHD patients in China is up to 2.5 million.The disease is described as an autoimmune sequelae of acute rheumatic fever(ARF),which is caused by infection with Streptococcus pyogenes or Group A streptococci(GAS).RHD can cause or worsen heart failure and is a risk factor for ischemic stroke caused by thromboembolism.Many proteins involved in the autoimmune response and control of the inflammatory process,which resulted in the occurrence of RHD heart valve injury and the formation of calcified valves,but their molecular pathways are not fully understood.Objectives:1.To identify differential genes and key molecular pathways of rheumatic heart disease(RHD)based on bioinformatics methods.2.To explore the expression of MMP-12,STAT4 and p-STAT4 in rheumatic heart disease calcified valves through experiments.3.To investigate whether MMP-12 and STAT4 act on rheumatic heart disease through JAK/STAT pathway.Methods:In this experiment,GEO2R online software was used to analyze the microarray data provided by the original provider,and to find the differentially expressed gene DEG.The R language is used as the basic data to read,filter and standardize the processing,which allowing users to compare different sample groups in the GEO database and screen for differentially expressed genes under experimental conditions.Correlation analysis was conducted by Wilcoxon rank sum test and one-way variance statistics.The differential gene screening criteria:|logFC|≥1,P<0.05,At the same time,we use the software package of R software which named pheatmap、ggplot2 to obtain the hierarchical clustering heat map and volcano map of differentially expressed genes.Differential expression analysis aims to identify genes that are differentially expressed between different samples,and conduct further in-depth functional mining of the differentially expressed genes,and find that STAT4,MMP-12 and other genes are differentially expressed.The expression level was divided into STAT4 low expression group and high expression group,and the differentially expressed genes were analyzed.Differential gene screening conditions:a.Fold change(FC)is greater than or equal to 1;b.The corrected P value is less than 0.05.We use the clusterProfiler software package in R to annotate and visualize the pathways enriched by GO entries.GO annotation was performed using the DAVID database(https://david.ncifcrf.gov/),and the DEGs up-and down-regulated were used for GO enrichment analysis.Take P value<0.05 as the threshold.There are a large number of gene annotation terms in the GO database,which allows the use of consistent terms for genome annotation.GO enrichment analysis includes molecular function(MF),cellular composition(CC),and biological process(BP)to determine which GO annotation genes are over-or under-expressed in a given set of genes.20 patients with rheumatic aortic valve calcified or rheumatic combined valvular aortic calcified who underwent aortic valve replacement or double valve replacement in cardiothoracic surgery of the First Affiliated Hospital of Guangxi Medical University from November 2017 to November 2019 were selected(test group).At the same time,12 patients(No aortic valve disease)with type A aortic dissection underwent Bentall or Bentall+Suns operation(control group)were selected in this hospital.Both groups of patients were incised from the root of the aorta,and the aortic valve was excised after cardioplegia by perfusing cardioplegia.Western Blot method and immunohistochemistry were used to detect the expression of p-STAT4,STAT4 and MMP-12 in valve calcification at the protein level.Real time PCR was used to verify the expression of STAT4 specific sequence messenger RNA(STAT4mRNA)at the molecular level.To investigate whether the STAT pathway factor STAT4 and stromal cell factor MMP-12 participate in RHD aortic valve inflammation and calcification through the JAK/STAT pathway.Results:In the Malacards database,255 up-regulated genes related to RHD calcification were obtained,and 436 down-regulated genes were uploaded to the STRING online tool.As a result,there were 636 nodes and 5857 edges,and each node represented a protein(gene).Each edge represents an interactive relationship.After being combined with the identified DEG,96 RHD valve calcification-related genes were obtained.Make a PPI network after screening through STRING online tool.Based on the overlap of genes calculated by three algorithms,22 core genes are obtained:IGLL5,FCER1A,ATP6V0D2,ACP5,CA2、SOST、MMP-13、MMP-7、GREM1、SFRP1、FABP5、FABP4、ADIPOQ、TNFSF11、TNNC1、MYOZ2、ACP5、DSG2、PPL、EFNA1、NGEF、SEMA3G,The differential genes of STAT4 and MMP-12 are non-core genes in the PPI network.There was no significant difference in age,weight,cardiac function classification,and cardiothoracic ratio between the two groups divided by collecting patient information(P>0.05).The two groups of cases are comparable.Western Blot calcification group MMP-12 protein expression was higher than the control group,the difference was statistically significant.(P<0.05).The expression level of STAT4 protein in the calcification group was not significantly different from that in the normal group(P>0.05).The expression level of p-STAT4 protein in the calcification group was higher than that in the control group(P<0.05).The expression of STAT4mRNA in the calcification group was not significantly different from that in the control group by qRT-PCR(P>0.05).The expression of MMP-12mRNA in the calcification group was higher than that in the control group,and the difference was statistically significant(P<0.001).In the calcification group(experimental group),the positive rate of p-STAT4 protein expression in the aortic valve tissue was 83.33%,and the areal density was 0.12497266.The positive rate of p-STAT4 protein expression in the normal group(control group)was 20%,and the areal density was 0.07796184.The positive expression rate and areal density calcification group were significantly higher than the normal group,and the difference was statistically significant(P<0.05).Conclusion:1.MMP-12 and STAT4 combined with JAK/STAT signaling pathway regulates the progression of RHD aortic valve calcification,and their expression levels are positively correlated with chronic RHD inflammatory response,STAT4 regulates RHD aortic valve calcification after forming p-STAT4 through phosphorylation.2.MMP-12 and STAT4 differential genes are up-regulated genes,and their expression affects RHD aortic valve calcification,but the differential genes of the two are non-core genes in multiple pathways. |
PDF Full Text Request