Effect And Mechanism Of POSTN And Its Exon 17 Alternative Splicing On Acute Myocardial Infarction

Objective: Acute myocardial infarction(AMI)is the main cause of death after cancer at present,which is characterized by high morbidity and mortality worldwide.In recent years,the incidence and mortality of AMI have shown an increasing trend,and the incidence of AMI has begun to increase in young people and women.The traditional risk factors of AMI cannot accurately predict the occurrence of the disease and the main diagnostic index of AMI,troponin,has high sensitivity and low specificity.So the fact is AMI lacks effective early prediction and diagnostic markers.This study explored the key regulatory factors in the pathogenesis of AMI through basic research,and sought theoretical basis for finding new biomarkers of AMI.Methods:(1)The AMI and Sham mouse model were constructed,and RNA-seq was performed on the myocardial tissues of mices to search for the differentially expressed genes of AMI and their alternative splicings,and analyze the differentially expressed genes,differential alternative splicing genes,alternative splicing sites,alternative splicing events,alternative splicing types,etc.GO analysis,KEGG pathway analysis and protein interaction analysis were performed for differential alternative splicing genes.Representative target genes and their alternative splicing were selected and detected by PCR in myocardial tissues of AMI and Sham mices to verify the sequencing results.(2)Patients admitted to the Heart Center of the First Affiliated Hospital of Xinjiang Medical University with definite diagnosis of AMI were included in the study,and the persons without coronary artery disease and atherosclerosis were taken as the normal controls.Baseline datas of them were collected respectively.Their plasma and PBMC samples were collected,CK,CK-MB,troponin I,NT-pro BNP,LDH,ALT,AST,low density lipoprotein,triglyceride,total cholesterol,creatinine,uric acid,blood glucose,CRP,PLT,D-dimer and left ventricular ejection fraction were detected within 24 hours.The plasma samples of AMI patients on the 7th and 30 th day of myocardial infarction were collected.The expression levels of POSTN in plasma and PBMC of AMI patients at different time of myocardial infarction were detected by ELISA and PCR,and the results were statistically analyzed.The working curve of subjects was drawn based on the plasma POSTN level within 24 hours of AMI patients and normal controls to evaluate the diagnostic efficacy for AMI.(3)1)Mouse HL-1 myocardial cell line was purchased,and the OD values of cell growth in 7 days were measured after resuscitation and subculture,and the growth curve was drawn to evaluate the cell growth status.The effects of different hypoxia time(3h,6h,12 h,24h)on OD values of myocardial cells and LDH levels were detected to determine the optimal hypoxia time and establish myocardial cell ischemia hypoxia model.The expressions of POSTN and its alternative splicing in hypoxic cardiomyocytes were detected by PCR,and the expressions of POSTN protein in ischemic hypoxic cardiomyocytes were detected by Western Blot.2)POSTN wild-type,mutant and negative control overexpressed lentiviruses(GV492-Post N-WT,GV492-Post N-MUT,GV492-NC)were constructed and explored the optimal viral transfection conditions.HL-1 cardiomyocytes were transfected with GV492-POSTN-WT,GV492-Post N-MUT and GV492-NC,respectively,and the expression levels of POSTN and alternative splicing in cardiomyocytes were detected by PCR to verify the successful construction of lentivirus.3)Normal and ischemic hypoxic cardiomyocytes were transfected with GV492-POSTN-WT,GV492-POSTN-MUT and GV492-NC,respectively.A total of 8 experimental groups with different intervention conditions were set up,and the proliferation and apoptosis of cardiomyocytes after intervention were detected.To explore the effects of POSTN and alternative splicing on proliferation and apoptosis of normal and ischemic hypoxic cardiomyocytes.Endoplasmic reticulum stress-related apoptosis proteins were selected to detect in 8 intervention groups,respectively,to explore the mechanism of POSTN and alternative splicing on myocardial cell apoptosis.Results:(1)A total of 1367 differentially expressed genes were detected in myocardial tissues of AMI and Sham mices,including 242 up-regulated genes and 1125 down-regulated genes.Non-intron retention accounted for 72.1% and intron retention accounted for 27.9% of all detected alternative splicing events.The levels of 10 common alternative splicing types were similar between AMI and Sham groups,and the most unannotated alternative splicing types were Intron R,A5 SS and 5p MXE.The most AS types detected in all genes in AMI and Sham groups were A5 SS,A3SS and Intron R.The difference was that A5 SS was detected most in AMI mices,while Intron R was detected most in Sham mices.Differential alternative splicing events occurred in 42 differentially expressed genes.GO enrichment analysis of cellular components of the 42 genes are mainly enriched plasma membrane,integral to membrane and extracellular region.The molecular functions are concentrated in protein binding and metal ion binding.The biological processes are mainly concentrated in cell adhesion,immune system process and cell differentiation.KEGG analysis was mainly enriched in Jak-STAT signaling pathway and PI3K-Akt signaling pathway.FHL1,FN1 and POSTN were all low expressed in AMI myocardium,while the alternative splicing of POSTN was high expressed in AMI myocardium,which were consistent with sequencing results.(2)POSTN expressions in plasma and PBMC were significantly lower in AMI patients within 24 hours.The plasma POSTN level of AMI patients was the lowest within 24 h,significantly increased at 7d,and nearly normal at 30 d.The rapid rising period was within 7 days,and the slow rising period was between 7 and 30 days.Compared with normal controls,serum triglyceride,CK,CK-MB,troponin I,LDH,ALT,AST,NT-prob NP,creatinine and D-dimer were significantly increased in AMI patients.There was no significant correlation between plasma POSTN expression levels of AMI patients within 24 hours and troponin I,CK,CK-MB,LDH,AST,ALT,NT-prob NP,total cholesterol,triglyceride,low density lipoprotein,left ventricular EF value,CRP,D-dimer,platelet,creatinine and uric acid.When POSTN was 302.3ng/m L,the sensitivity and specificity to diagnose AMI were 73% and 85%,respectively.(3)The expression level of POSTN decreased in ischemic hypoxic cardiomyocytes,while the alternative splicing of POSTN was increased.POSTN can promote proliferation and inhibit apoptosis of normal and ischemic hypoxic cardiomyocytes.The mechanism of POSTN inhibiting cardiomyocyte apoptosis may be through inhibiting GRP78-e IF2α-ATF4-CHOP pathway of endoplasmic reticulum stress.The alternative splicing of POSTN can inhibit apoptosis of ischemic hypoxic cardiomyocytes,and its mechanism needs further study.Conclusion: POSTN and its exon 17 alternative splicing play an important role in the pathogenesis of AMI.The low expression of POSTN may be related to myocardial apoptosis in early AMI.POSTN may inhibit AMI myocardial apoptosis by inhibiting GRP78-e IF2α-ATF4-CHOP pathway of ERS.Plasma POSTN level within 24 hours has certain diagnostic value for AMI.POSTN is expected to be a new biomarker and potential therapeutic target for AMI.