GSE135888
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 <0.03) but not on promoter of transcript V1 (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.