PDF(Pubmed)
Abstract：
Genome-wide association studies along with expression quantitative trait locus (eQTL) mapping have identified hundreds of single-nucleotide polymorphisms (SNPs) and their target genes in prostate cancer (PCa), yet functional characterization of these risk loci remains challenging. To screen for potential regulatory SNPs, we designed a CRISPRi library containing 9,133 guide RNAs (gRNAs) to cover 2,166 candidate SNP loci implicated in PCa and identified 117 SNPs that could regulate 90 genes for PCa cell growth advantage. Among these, rs60464856 was covered by multiple gRNAs significantly depleted in screening (FDR < 0.05). Pooled SNP association analysis in the PRACTICAL and FinnGen cohorts showed significantly higher PCa risk for the rs60464856 G allele (p value = 1.2 × 10-16 and 3.2 × 10-7, respectively). Subsequent eQTL analysis revealed that the G allele is associated with increased RUVBL1 expression in multiple datasets. Further CRISPRi and xCas9 base editing confirmed that the rs60464856 G allele leads to elevated RUVBL1 expression. Furthermore, SILAC-based proteomic analysis demonstrated allelic binding of cohesin subunits at the rs60464856 region, where the HiC dataset showed consistent chromatin interactions in prostate cell lines. RUVBL1 depletion inhibited PCa cell proliferation and tumor growth in a xenograft mouse model. Gene-set enrichment analysis suggested an association of RUVBL1 expression with cell-cycle-related pathways. Increased expression of RUVBL1 and activation of cell-cycle pathways were correlated with poor PCa survival in TCGA datasets. Our CRISPRi screening prioritized about one hundred regulatory SNPs essential for prostate cell proliferation. In combination with proteomics and functional studies, we characterized the mechanistic role of rs60464856 and RUVBL1 in PCa progression.
摘要：
全基因组关联研究以及表达数量性状基因座（eQTL）作图已经确定了数百个单核苷酸多态性（SNP）及其在前列腺癌（PCa）中的靶基因。然而,这些风险位点的功能表征仍具有挑战性.要筛选潜在的监管SNP，我们设计了一个包含9,133个向导RNA(gRNA)的CRISPRi文库,以覆盖2,166个与PCa相关的候选SNP位点,并鉴定出117个能够调节90个基因的SNPs,以获得PCa细胞生长优势.其中,rs60464856被筛选中显著耗竭的多个gRNA覆盖(FDR<0.05)。PRACTICAL和FinnGen队列中汇总的SNP关联分析显示，rs60464856G等位基因的PCa风险明显更高（p值分别为1.2×10-16和3.2×10-7）。随后的eQTL分析揭示G等位基因与多个数据集中的RUVBL1表达增加相关。进一步的CRISPRi和xCas9碱基编辑证实rs60464856G等位基因导致RUVBL1表达升高。此外，基于SILAC的蛋白质组分析证明了在rs60464856区域的粘附蛋白亚基的等位基因结合，其中HiC数据集显示前列腺细胞系中一致的染色质相互作用。在异种移植小鼠模型中,RUVBL1耗竭抑制PCa细胞增殖和肿瘤生长。基因集富集分析提示RUVBL1表达与细胞周期相关通路相关。在TCGA数据集中，RUVBL1的表达增加和细胞周期通路的激活与PCa的低存活率相关。我们的CRISPRi筛查优先考虑了约100个对前列腺细胞增殖至关重要的调节性SNP。结合蛋白质组学和功能研究，我们描述了rs60464856和RUVBL1在PCa进展中的机制作用。
