Gene Regulatory Mechanisms Of Rs684232 In Prostate Cancer

BackgroundGenome-wide association analysis(GWAS)provides an effective method for studying the genetic basis of complex disease.Cancer is a serious threat to human health.GWAS has identified thousands of SNPs in almost all of malignancies.These studies provide a solid basis for elucidating the genetic mechanisms of cancer.Especially,GWAS has identified more than 160 loci associated with prostate cancer susceptible from 2006 to 2018.However,the biological function and mechanism of these loci are poorly understood in cancer.SNPs in the coding region of the gene may alter the structure and function of the encoded protein.SNPs in gene regulatory regions may influence the risk of diseases by affecting the expression of gene.Since most of the SNPs are located in the non-coding region of the genome and they are significantly enriched in the DNase I hypersensitization site and transcription factor binding site,furthermore the regions are annotated as active promoters and strong enhancers.Therefore,these SNPs are most likely to affect the expression of the target gene by altering the activity of regulatory element,which lead to cancer risk.So screening out the SNPs with biological functions and elucidating its mechanism has become an important challenge in the post-GWAS era.GWAS in the European population first reported that rs684232 is strongly associated with prostate cancer risk(OR=1.1,95%CI=1.07-1.14,minor allele frequency(MAF):T=0.4832/2420(1000 Genomes);C=0.4664/58570(TOPMED)).Rs684232 is located in the intergenic region of VPS53 and FAM57A at chromosome 17,and its risk allele is C,and the normal allele is T.However,the biological function and molecular mechanism is still unknown.ObjectiveThis study is to explore the trans-acting factors and target genes association with rs684232.Finally,we hope to provide new molecular markers for early screening and new targets for clinical treatment by elucidating the biological function and molecular mechanism of rs684232 in prostate cancer.Methods1.Screening out SNPs with enhancer regulatory activity based on the new generation of reporter gene analysis system;2.To find a cell line which rs684232 is heterozygous.Use FAIRE to detect the chromosome open state of rs684232 and allele-specific enrichment,and confirm whether it is consistent with the result of next generation sequencing;3.Find possible eQTL gene of rs684232 by database;4.In the heterozygous cell line of rs684232,finding the heterozygous SNP in the exon of the potential target gene by PCR,and further confirmed that whether rs684232 would affect the corresponding allele of target gene exon SNP in cDNA.5.To predict transcription factors binding with rs684232 by RegulomeDB,HaploReg v4.1,PROMO database,and the binding motif of transcription factors was further predicted by JASPAR database;6.Use shRNA to knock down the transcription factors and detect the expression of target gene.Results1.We identified 44 SNPs in 22Rv1 cell line and 7 SNPs in PC3 cell line based on the result of reporter gene analysis system.Importantly,the activity of risk allele C is lower than normal allele T for rs684232 in 22Rv1 cell line.2.In the 22Rv1 cell line,rs684232 is T/C heterozygous.And further FAIRE experiments indicate that T of rs684232 was significantly enriched in the chromatin active region,which is consistent with the result of reporter gene analysis system.3.The potential eQTL gene of rs684232 is GEMIN4,VPS53.4.In the 22Rv1 cell line,we found that rs11558129 is heterozygous in the first exon of VPS53,rs113201579 is heterozygous in the fourth exon of FAM57A,rs3744741 is heterozygous in the second exon of GEMIN4.And only the allele T of rs113201579 is enrichment in cDNA.These results indicated that rs684232 may interaction with FAM57A.5.Rs684232 may bind with transcription factor AR and FOXA1 by predicted with database.6.Using shRNA to knockdown the transcription factor FOXA1,we found that it did not affect the expression of the target gene FAM57A.However,the knockdown of AR caused the down-regulation of FAM57A.Therefore,rs684232 may bind with transcription factor AR.ConclusionIn this study,we found that risk allele C of rs684232 has a low affinity with AR,which lead to the down-regulation of FAM57A.Furthermore,affect the progression of prostate cancer.