含有PCa风险性SNP rs10486567的增强子座位在前列腺上皮细胞中直接靶基因的鉴定

Prostate cancer (PCa) is the most commonly diagnosed carcinoma in men. To identify genetic susceptibility predisposing development of aggressive PCa, genome wide association studies (GWAS) examining single nucleotide polymorphisms (SNPs) have identified many loci thought to be linked to disease traits. Through linkage disequilibrium analysis, SNPs with putative functional importance have been found; however, >90% are located in intergenic or intronic regions, making an understanding of SNP variant effects on genes and disease mechanisms nearly impossible. ..Our initial studies beginning to uncover the function of SNPs in non-coding regions used chromatin ChIP-seq to correlate risk SNPs with epigenetic features such as DNAseI hypersensitivity and histone marks H3K27ac and H3K4me. Positive correlation with these epigenetic marks suggested that highly linked “risk” SNPs are located in putative active enhancer-like elements which may be involved in regulating transcription. Of particular interest, risk SNP rs10486567, lies within a transcription factor binding motif for NKX3.1 as the G allele, changes to bind FOXA1 in the A allele form. Putative enhancer activity confirmed by luciferase reporter assay showed allele dependent enhancer activation under dihydrotestosterone treatment. ..We hypothesize that differential binding of transcription factors corresponding to the SNP allele in response to androgen stimulation influences chromatin looping activity of the risk enhancer and resultant transcription profiles. To determine gene targets of the risk enhancer in PCa, we plan to Identify potential interacting genes associating with the risk SNP rs10486567 in LNCaP and RWPE cells. We will accomplish this through performing circular chromosome conformation capture and sequencing (4C-seq) experiments to identify chromatin regions physically interacting with the risk enhancer. Next we will use CRISPR/Cas technology to delete or replace risk SNP rs10486567 enhancer in LNCaP and RWPE cells to determine risk enhancer effect on gene expression. By comparing gene expression differences in wild type and SNP deleted or replaced cells before and after DHT treatment by RNA-seq analysis, we can fully characterize the function of the risk enhancer and effect of the SNP allele in promoting PCa traits. ..Further understanding of PCa etiology is needed not only for treatment of disease, but also prophylaxis and relevant risk factors. We predict that genes identified from our study will be functionally important in PCa cell invasiveness, proliferation, and survival, and will be good candidates for future functional studies.

前列腺癌（PCa）是严重威胁男性生命健康的恶性肿瘤。rs10486567是一个与PCa密切相关的风险性SNP位点，但由于它定于非编码序列，明确它在疾病中的生物学功能是一个艰巨的挑战。前期工作发现，含有该SNP位点的PCa风险性染色体座位是一个受雄激素调节的、等位基因特异性的增强子元件。本项目将采用新近发展起来的环形染色质构象捕获技术和双缺口CRISPR/Cas技术探究含有该SNP的染色体座位在正常前列腺细胞系RWPE和癌细胞系LNCaP中的三维空间组织模式，结合表达谱分析鉴定该增强子的直接靶基因；并对前列腺上皮细胞中该风险性SNP位点中核苷酸残基进行替换编辑，研究协同性顺式调控元件的改变对于增强子直接靶基因表达的影响，明确FOXA1和NKX3.1这两个AR协同转录因子在前列腺细胞中对AR活性的调节作用。研究结果将推进对于PCa发生、发展分子机制的认知，有望发现新的有效肿瘤治疗靶点。

前列腺癌（PCa）是严重威胁男性生命健康的恶性肿瘤。遗传是前列腺癌发生发展的关键因素之一，GWAS研究发现了多个前列腺癌关联的风险SNPs，但多数定于非编码序列，明确它们在疾病中的生物学功能、特别是在精准医学领域是一个艰巨的挑战。本研究以SNP rs10486567位点为切入点，利用CRISPR/Cas9进行染色体片段敲除和SNP位点点突变等基因编辑技术揭示等位基因特异性的增强子元件在肿瘤恶性进展中的作用和信号通路；采用 4C-Seq和Capture C-Seq等方法描绘与含有风险性 SNP rs10486567 的染色体座位相互作用图谱，结合表达谱数据揭示了该风险性增强子直接调控的靶基因，并明确了SNP rs10486567 位点的两种不同碱基形式对靶基因表达的影响以及关键靶基因在肿瘤恶性表型中的作用。在利用建立的研究平台的基础上，课题组同时还揭示了另一个前列腺癌风险性SNP rs55958994关联增强子在前列腺癌细胞中的3D genome组织形式和调控的靶基因。这些研究成果可能为前列腺癌的早期诊断和精准临床干预提供新证据。