CPG HYPERMETHYLATION OF COL1A2 CONTRIBUTES TO PROLIFERATION AND MIGRATION ACTIVITY OF BLADDER CANCER

Abstract

INTRODUCTION AND OBJECTIVE: Gene silencing due to aberrant DNA methylation plays an important role in carcinogenesis. For genome-wide screening for methylated genes in bladder cancer (BC), we performed cDNA microarray analysis of the BC cell line (BOY) treated with demethylating agent (5-Aza-dC). Collagen type 1 alpha 2 (COL1A2) gene was identified as the most up-regulated one of the 30,144 genes screened. We hypothesize that inactivation of the COL1A2 gene through CpG methylation contributes to proliferation and migration activity of human BC by changing extra-cellular matrix (ECM). METHODS: To test this hypothesis, we extracted DNA and RNA from 67 BC samples and 10 normal bladder epitheliums (NBEs) from cystectomy and transurethral resection. The CpG hypermethylation of COL1A2 promoter was analyzed by quantitative methylation-specific PCR (QMSP) and confirmed by bisulfite-modified DNA sequencing. Real-time RT-PCR was carried out to evaluate COL1A2 mRNA expression. We established a stable COL1A2 transfectant for gain of function studies, for which cellular proliferation and migration were examined by MTT assay and wound healing assay, respectively. The protein secretion of type 1 collagen in culture medium was detected by immunoblot. RESULTS: Bisulfite DNA sequencing demonstrated that the promoter hypermethylation of COL1A2 was a frequent event in clinical BCs. The methylation index was significantly higher in the 67 BCs than in the10 NBEs (42.3 ± 28.9 vs. 0.07 ± 0.01, p = 0.0011). Conversely, COL1A2 mRNA expression was significantly lower in the BCs than in the NBEs (15.8 ± 3.8 vs. 32.0 ± 22.0, p = 0.0052). In vitro, after 5-aza-dC treatment, the COL1A2 mRNA expression was markedly increased in BOY. Our cell proliferation assays consistently demonstrated a significant growth inhibition in the COL1A2 transfectant compared with control and wild-type BOY cells (on day 5; 0.791 ± 0.020, 1.056 ± 0.034, and 1.107 ± 0.027, respectively, p < 0.0001). Wound healing assays also showed a significant wound healing inhibition in the COL1A2 transfectant compared to the counterparts (% of wound closure; 75.5 ± 0.2, 94.3 ± 2.9, and 96.9 ± 3.1, respectively, p = 0.0016). Immunoblot demonstrated that type 1 collagen protein markedly increased in culture medium of the COL1A2 transfectant. CONCLUSIONS: Our data suggest that inactivation of the COL1A2 gene through CpG methylation may change an ECM binding on epithelial collagen and contributes to proliferation and migration activity of human bladder cancer.