Study On The Function And Mechanism Of Prostate Cancer Risk SNPs Based On A Novel High-throughput Reporter Gene Analysis Technology

Background and objectiveProstate cancer is the most common male urinary system tumor,and its worldwide incidence ranks second in male malignancy.Prostate cancer is the fastest-growing type of cancer in China.The rate of increase in morbidity and mortality ranks first among all cancers.In the background of precision medicine,discovering and using effective biomarkers for the identification of prostate cancer and developing targeted drugs are important ways to address the threat of prostate cancer.Prostate cancer is one of the most hereditary diseases.Hereditary factors account for about 42%of the disease risk.The risk SNPs have been found to account for more than 40%.Therefore,the risk genetic variation site is an important candidate biomarker.To date,GWAS has identified more than 200 prostate cancer risk-associated SNPs,but its mechanism remains unclear.Therefore,elucidating the biological functions and mechanisms of these risk SNP loci is significant for the development of prostate cancer precision medicine.Among the SNPs identified by GWAS that are associated with susceptibility to various diseases,more than 95%of the SNPs are not in the coding region of the gene,which brings a huge challenge for the functional study of the mutation.Studies have shown that these risk SNPs are usually enriched in the transcriptional regulatory regions of genes,enhancer for example and regulate the expression of target genes by mediating the binding of transcription factors in these regions.Therefore,the use of a reporter gene analysis system to screen out risk SNP loci with gene regulatory functions is crucial for the systematic interpretation of these sites' biological functions and mechanisms,Due to the large number of risk SNP loci,we urgently need a high-throughput reporter gene analysis system.The traditional luciferase reporter gene analysis system has the disadvantage of low flux,while the existing reporter gene analysis system based on DNA sequence barcodes shows a large systematic error due to the presence of sequence bias.In view of this,we have developed a high-throughput reporter gene analysis system based on second-generation sequencing for the analysis and screening of risk SNP sites with enhancer activity.Compared with the existing report gene analysis system,the system can not only achieve high-throughput reporter gene analysis with high efficiency and low cost,but also combine qPCR technology with the ability to take into account conventional flux level reporter gene analysis and greatly reduce the workload and cost.At the same time,it also reduces the preference of the DNA barcode system and reduces systematic errors.Results1.213 prostate cancer risk susceptibility SNP sites were extracted from the GWAS catalog database and literature search.The risk and normal alleles of each SNP locus were cloned into a high-throughput reporter gene analysis system to obtain a plasmid library containing 426 reporter gene vectors.2.The reporter plasmid library was respectively transfected into prostate cancer cell lines such as 22Rvl,PC3,and LNCaP,and the gene regulation activity of SNP was quantified by second-generation sequencing technology.In the 22Rv1,PC3,and LNCaP cell lines,45,22,and 9 SNP loci were selected respectively.Not only did they have gene regulatory activity,but the risk allele significantly affected the gene expression level.More interestingly,the gene regulatory activities of these potential SNP loci show strong cell specificity,which provides important clues for further elucidating the function of risk SNPs.3,The FAIRE-qPCR analysis revealed that the chromatin with a gene-regulated activity in 22Rvl cells was highly open in the 25 SNPs.ChIP-qPCR analysis of the chromatin activity markers H3K4me3 and H3K27ac indicated that the two SNP loci of rs684232 and rs887391 had higher enrichment folds and showed strong selectivity for highly transcriptionally active alleles.We hypothesize that the risk allele of rs684232 disrupts the activity of normal enhancer elements,resulting in decreased expression of downstream target genes,while the risk allele of rs887391 greatly enhances the activity of enhancer elements and enhances the expression of downstream target genes level lead to prostate cancer risk.ConclusionsIdentification of risk-related SNPs associated with susceptibility to prostate cancer with gene regulatory activity through high-throughput reporter gene analysis system and studying their molecular functions,we hope to help to interpret the functional mechanisms between risk SNPs and diseases in the post-GWAS era,and provide help for risk assessment and accurate diagnosis and treatment of prostate cancer.