Molecular Mechanism Research Of Insertion-deletion Polymorphisms In Gene Non-coding Regions Associated With Sudden Cardiac Death

Part Ⅰ Association study and mechanism research between Insertion/deletion polymorphism of MIR155HG gene and sudden cardiac deathObjective:our research aims at to explore the association between an insertion/deletion(InDel)polymorphism(rs72014506)in non-coding region of MIR155HG gene and sudden cardiac death(SCD)susceptibility in Chinese populations.Genotype-phenotype correlation analysis,functional experiments and bioinformatics techniques were used to investigate the role of this polymorphism in SCD and its potential mechanism in the pathogenesis of SCD.Methods:(1)we systematically screened the whole region of miR-155 host gene(MIR155HG),expression quantitative trait Loci(eQTL)analysis and histone enrichment annotation from database were used.In final,we identified a 4-bp insertion/deletion variant(rs72014506)resided in intron region of MIR155HG as the candidate polymorphism.(2)The correlation of rs72014506 with SCD susceptibility was evaluated in Chinese populations.By conducting a case-control study,blood samples from 166 SCD cases and 850 matched healthy controls were collected and genotyped.Logistic regression analysis was used to study the association between this polymorphism and SCD susceptibility.(3)Further genotype-expression association study using 19 human myocardium tissue samples via Realtime PCR assay was utilized to investigate the correlation between rs72014506 genotype and MIR155HG/miR-155 expression(4)DNA fragments containing rs72014506 were directly synthesized and subcloned into pGL3-control vector,obtaining the wild type vector containing the insertion allele(pGL3-WT)and the mutant type vector(pGL3-MT)harboring deletion allele.The luciferase activity was measured by dual-Luciferase assay system to explore whether the polymorphism of rs72014506 alleles could regulate the gene expression.To further explore whether the different alleles has different ability in driving gene promoter,the MIR155HG promoter fragments were subcloned into pGL3-Basic vector,and the same restricted enzyme sites were used to subcloned DNA fragments containing the InDel polymorphism,named pGL3-proWT and pGL3-proMT,respectively.(5)Bioinformatic analysis was used to predict the transcription factor which might be recruited by rs72014506,and the predicted results were verified by EMSA and super-shift EMSA.Results:(1)Bioinformatic database annotation showed that the variants rs72014506 is full of H3K4mel or H3K4me3,and hypersensitive to DNase I,eQTL analysis present a expression difference.Thus,rs72014506 was chosen as the candidate variant for the current case-control study.(2)Genotyping results from SCD and healthy controls revealed that the genotypic and allelic frequencies of rs72014506 were in a balanced distribution.Genotypic frequencies observed in the controls were consistent with the Hardy-Weinberg equilibrium(P>0.05).Logistic regression analysis suggested that homozygote del/del genotype significantly decreased the risk of SCD in the overall analysis[odds ratio(OR)=0.55;95%confidence interval(CI)= 0.41-0.75;P=0.0001],(3)Real-time PCR results showed that the deletion allele was intimately linked to lower expression of both MIR155HG and mature miR-155.The MIR155HG mRNA and mature miR-155 expression level of samples which was ins/del or del/del genotype were 0.43-and 0.59-fold lower than that with ins/insgenotype samples.(4)Dual Luciferase Reporter Assay results showed that the group transfected with pGL3-WT presented higher luciferase activity than that of the group transfected with pGL3-MT,indicating that the deletion DNA fragments may have a regulatory function.When use vector contain gene promoter region,group transfected with del allele sequences displayed lower luciferase activity than that of ins allele The results suggested the influence of rs72014506 on MIR155HG promoter and its possible regulatory role on gene transcription.(5)EMSA and super-shift EMSA verified the repression of MIR155HG/miR-155 expression by rs72014506 is mediated by recruiting the transcription factor POU2F1.Conclusion:(1)The rs72014506 variant was highly correlated with SCD susceptibility in Chinese populations.(2)The deletion allele of rs72014506 was associated with decreased MIR155HG/miR-155 transcription.(3)The rs72014506 deletion allele may create a binding site to POU2F1,the binding of POU2F1 might be correlated with the repression of MIR155HG/miR-155,which may finally decrease the risk of SCD.Part II Molecular mechanism of sudden cardiac death induced by an InDel polymorphism in STIM1 geneObjective:To investigate the pathogenic mechanism of rs3061890,an insertion/deletion polymorphism in 5’UTR region of STIM1(Stromal Interaction molecule 1)gene that has been confirmed to be associated with the risk of sudden cardiac death in Chinese population,and reveal its role in the occurrence of sudden cardiac death through functional experiments.Methods:(1)We expand the case-control samples to 157 SCD and 608 healthy control samples,to further verify the association between this polymorphism loci and SCD susceptibility.(2)Functional region analysis and eQTL analysis were used to explore the molecular mechanism of the polymorphism.(3)Bioinformatics methods were used to predict transcription factors binding to rs3061890,EMSA and super-shift EMSA experiments were used to verify the predicted results.Chromatin immunoprecipitation(ChIP)combined with qPCR assay was used to detect the enrichment of transcription factor in different genotypes of cell lines.The transcription factor genes in cells were overexpressed or silenced by constructing the overexpression vector and RNA Interference(RNAi)technology,and the STIM1 protein level was detected by Western blot(WB)to clarify the regulatory relationship.Results:(1)A case-control study with an expanded sample size confirmed the conclusion of the previous study that rs3061890 was significantly associated with SCD susceptibility:the deletion allele was associated with a higher risk of disease(OR=1.60;95%CI=1.23-2.08;P=0.0002.(2)eQTL analysis result confirmed the genotype-phenotype relationships between rs3061890 and STIM1 mRNA level.Functional annotation from ENCODE database demonstrated that the region which rs3061890 located has high enrichment of H3K4me3 and H3K27ac,and has high sensitivity of DNase I.(3)EMSA and super-shift EMSA confirmed that the transcription factor ELF 1 can combined with rs3061890 deletion allele.ChIP-qPCR assays suggested a significant enrichment of ELF 1 in cells which carrying the rs3061890 del/del genotype.And there is a correlation between ELF1 and STIM1,we observed changes in STIM1 protein levels induced by aberrant ELF1 expression.The STIM1 expression level was decreased with overexpression ELF1 and increased when ELF1 was silenced by siRNA in del/del genotype cells.Conclusion:(1)rs3061890 was significantly associated with SCD susceptibility in which the deletion allele enhanced the risk for SCD in its carriers.(2)rs3061890 was located in a high enrichment of histones and high open chromatin region,suggesting that this variant has a prerequisite to participate in the regulation of gene transcription.(3)rs3061890 variant regulates the expression of STIM1 by interfering the binding of ELF1 and may thus considered as a candidate genetic marker for SCD prediction and prevention.Part Ⅲ Genetic risk score predicts the risk of SCD in Chinese populationsObjective:To establish a risk prediction model of sudden cardiac death in Chinese Han population based on eight susceptibility risk loci of SCD confirmed in our previous study.Methods:Genetic risk score(GRS)and weighted genetic risk score(wGRS)were calculated on the basis of previous studies.The combined effect of all loci was analyzed to establish a predictive SCD risk model.The Receiver operating characteristic curve(ROC)was drawn to test and evaluate the reliability of the prediction model.Results:Logistic regression analysis showed that people with GRS>8 or wGRS>5.072 conferred higher risk of sudden cardiac death[GRS,OR=5.81,95%CI(3.52-9.59),P=1.27e-13;wGRS,OR=6.78,95%CI(4.29-10.74),P=2.20e-16].The AUC was 0.761 and 0.722.Conclusion:A total of 8 InDel polymorphic loci have the ability to predict the risk of SCD in Chinese Han population.