Single nucleotide polymorphism rs13426236 contributes to an increased prostate cancer risk via regulating MLPH splicing variant 4. Single nucleotide polymorphism rs13426236 contributes to an increased prostate cancer risk via regulating MLPH splicing variant 4

A prostate cancer risk single nucleotide polymorphism (SNP), rs13426236, is significantly associated with melanophilin (MLPH) expression. To functionally characterize role of the rs13426236 in prostate cancer, we first performed splicing-specific expression Quantitative Trait Loci (eQTL) analysis and refined the significant association of rs13426236 allele G with an increased expression of MLPH splicing variant 4 (V4) (P= 7.61E-5) but not other protein-coding variants (V1-V3) (P>0.05). We then performed an allele-specific reporter assay to determine if SNP-containing sequences functioned as active enhancer. Compared to allele A, allele G of rs13426236 showed significantly higher luciferase activity on the promoter of the splicing transcript V4 (P 0.05) in two prostate cancer cell lines (DU145 and 22Rv1). Transfection of MLPH splicing variants showed stronger effect of transcript V4 than V1 on promoting cell proliferation, invasion and anti-apoptotic activities. RNA profiling analysis demonstrated that transcript V4 overexpression caused significant expression changes in glycosylation/glycoprotein and metal-binding gene ontology pathways (FDR<0.01). We also found that both transcripts V4 and V1 were significantly up-regulated in prostate adenocarcinoma (P<2.49E-6) but only transcript V4 up-regulation was associated with poor recurrence free survival (P=0.028, HR=1.63, 95%CI=1.05-2.42) in The Cancer Genome Atlas (TCGA) data. This study provides strong evidence showing that prostate cancer risk SNP rs13426236 up-regulates expression of MLPH transcript V4, which may function as a candidate oncogene in prostate cancer. Overall design: To determine effect of MLPH on signaling pathways, we transfected 22Rv1 cells with MLPH transcripts V1 or V4 plasmid for 48 h. Total RNA was isolated using total RNA extraction kit (Zymo Research). RNA-seq was performed in each transfected cell line with a technical replicate. Transfection with empty vector was used as control.

一项与前列腺癌风险相关的单核苷酸多态性（single nucleotide polymorphism, SNP）rs13426236，与黑素亲和素（melanophilin, MLPH）的表达显著相关。为从功能上解析rs13426236在前列腺癌中的作用，本研究首先开展了剪接特异性表达数量性状基因座（expression Quantitative Trait Loci, eQTL）分析，明确了rs13426236的G等位基因与MLPH剪接变体4（V4）的表达上调显著相关（P=7.61×10^-5），而与其他蛋白编码变体（V1-V3）无此关联（P>0.05）。随后，本研究开展了等位基因特异性报告基因实验，以验证包含该SNP的序列是否可作为活性增强子发挥功能。相较于等位基因A，rs13426236的等位基因G在两种前列腺癌细胞系（DU145和22Rv1）中，可使剪接转录本V4的启动子区域的荧光素酶活性显著升高（P=0.05）。对MLPH剪接变体进行转染实验后发现，转录本V4相较于V1，在促进细胞增殖、侵袭及抗凋亡活性方面的作用更强。RNA表达谱分析显示，转录本V4过表达可使糖基化/糖蛋白及金属结合基因本体（gene ontology, GO）通路中的基因表达出现显著变化（FDR<0.01）。本研究同时基于癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据集发现，转录本V4与V1在前列腺腺癌组织中均显著上调（P<2.49×10^-6），但仅转录本V4的上调与较差的无复发生存期显著相关（P=0.028，风险比HR=1.63，95%置信区间CI=1.05-2.42）。本研究提供了强有力的证据，表明前列腺癌风险相关SNP rs13426236可上调MLPH转录本V4的表达，而该转录本可能作为前列腺癌的候选致癌基因发挥作用。

实验整体设计：为解析MLPH对信号通路的影响，本研究将携带MLPH转录本V1或V4的质粒转染至22Rv1细胞，转染时长为48小时。使用总RNA提取试剂盒（Zymo Research）分离总RNA，对每个转染细胞系设置1次技术重复进行RNA测序（RNA-seq）。以空载体转染细胞作为空白对照。