SLCO4C1 promoter methylation is a potential biomarker for prognosis associated with biochemical recurrence-free survival after radical prostatectomy

Abstract

BackgroundProstate cancer (PC) is a commonly diagnosed malignancy in males, especially in the western hemisphere. The extensive use of multiple biomarkers plays an important role in the diagnosis and prognosis of PC. However, the accuracy of biomarkers for PC prognosis needs to be urgently improved. This study aimed to identify a novel prognostic biomarker for PC.Materials and methodsDifferentially methylated CpG sites were identified from the GSE76938 dataset (https://www.ncbi.nlm.nih.gov/geo/) using R software version 3.1.4. Four significant CpG sites on the SLCO4C1 gene were found to be closely associated with prognosis in PC. Data downloaded from The Cancer Genome Atlas (TCGA) were used for validation. Co-expression and functional enrichment analyses were used to explore the roles of SLCO4C1 in molecular functions, biological processes and cellular components. Total RNA extraction and qRT-PCR were used to reveal the difference in SLCO4C1 expression between tumour and normal tissues. Bisulfite amplicon sequencing (BSAS) was used to identify methylation levels at the CpG sites.ResultsIn the GSE76938 cohort, 10,206 CpG sites were identified to be differentially methylated in tumour versus normal prostate tissues. Among the CpG sites, four sites (cg06480736, cg19774478, cg19788741 and cg22149516) located in the promotor region (TSS200-1500) of SLCO4C1 were found to be significantly hypermethylated in tumour tissues. The results were validated in an independent dataset (TCGA PRAD cohort). In the cohort from TCGA, SLCO4C1 expression was negatively correlated with methylation levels at the four sites. The results of qRT-PCR validated that tumour tissues had a relatively lower expression of SLCO4C1. Bisulfite amplicon sequencing (BSAS) further confirmed a higher methylation level at the SLCO4C1 promoter in tumour tissues. SLCO4C1 (cg06480736, cg19774478, cg19788741 and cg22149516) was identified as a significant promising biomarker for biochemical recurrence-free survival in Kaplan-Meier analysis (P < 0.01) and univariate Cox proportional hazards analysis: cg06480736 (HR 15.914, P < 0.001), cg19774478 (HR 9.001, P < 0.001), cg19788741 (HR 10.759, P = 0.003) and cg22149516 (HR 17.144, P = 0.006). However, three sites, namely, cg06480736 (HR 1.809, P = 0.049), cg19774478 (HR 1.903, P = 0.041) and cg22149516 (HR 2.316, P = 0.008), were confirmed in multivariate analysis.ConclusionsSLCO4C1 promoter methylation, including that at three CpG sites, namely, cg06480736, cg19774478 and cg22149516, is a potential biomarker for risk stratification and might offer significantly relevant prognostic information for PC patients after radical prostatectomy.