Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan.
Int J Oncol. 2010 Dec;37(6):1379-88. doi: 10.3892/ijo_00000789.
We have previously reported a simple technique that combines microarray data from clinical bladder cancer (BC) specimens with those from a BC cell line (BOY) treated with a pharmacological demethylating agent [5-aza-2'-deoxycytidine (5-aza-dC)] to find candidate genes that have tumor suppressive functions. We focused on the cellular retinol-binding protein 1 (CRBP1) gene that was selected by using the microarray data. As CRBP1 regulates intracellular retinoic acid (vitamin A) homeostasis, which is involved in morphogenesis, and cellular proliferation and differentiation, the loss of CRBP1 could cause tumorigenesis in BC. We hypothesized that the inactivation of the CRBP1 gene through CpG methylation contributes to cell viability, including the migration and invasion activity of human BC cells. After the 5-aza-dC treatment, the mRNA and protein expression levels of CRBP1 markedly increased in all BOY and T24 BC cell lines. Combined bisulfite-restriction analysis and bisulfite DNA sequencing revealed that promoter CpG hypermethylation existed in 28 out of the 65 BCs (43%) and in none of the 16 normal bladder epithelia (NBEs). Conversely, CRBP1 mRNA expression in the BCs was significantly lower than that in the NBEs (0.63 ± 0.11 vs. 4.92 ± 0.80, p<0.0001). We found significant inhibition of cell growth (p<0.0001) and migration (p<0.0001) in the CRBP1 stable transfectants compared to the control cell line, in a cell proliferation and wound-healing assay, respectively. In conclusion, the aberrant CpG hypermethylation of the CRBP1 gene promoter could be involved in the development of BC. We demonstrate here for the first time that the CRBP1 gene could have a tumor suppressive function in BC.
我们之前报道了一种简单的技术，该技术将临床膀胱癌（BC）标本的微阵列数据与用药理去甲基化剂[5-氮杂-2'-脱氧胞苷（5-aza-dC）]处理的 BC 细胞系（BOY）的数据相结合，以寻找具有肿瘤抑制功能的候选基因。我们专注于使用微阵列数据选择的细胞视黄醇结合蛋白 1（CRBP1）基因。由于 CRBP1 调节细胞内视黄酸（维生素 A）的动态平衡，这涉及形态发生、细胞增殖和分化，因此 CRBP1 的缺失可能导致 BC 发生肿瘤。我们假设，通过 CpG 甲基化使 CRBP1 基因失活会促进包括人 BC 细胞迁移和侵袭活性在内的细胞活力。在用 5-aza-dC 处理后，所有 BOY 和 T24 BC 细胞系中 CRBP1 的 mRNA 和蛋白表达水平均显著增加。联合亚硫酸氢盐限制性分析和亚硫酸氢盐 DNA 测序显示，在 65 个 BC 中有 28 个（43%）存在启动子 CpG 过度甲基化，而在 16 个正常膀胱上皮（NBE）中则不存在。相反，BC 中 CRBP1 mRNA 的表达明显低于 NBE（0.63±0.11 对 4.92±0.80，p<0.0001）。在细胞增殖和划痕愈合测定中，与对照细胞系相比，CRBP1 稳定转染的细胞生长（p<0.0001）和迁移（p<0.0001）受到显著抑制。总之，CRBP1 基因启动子的异常 CpG 过度甲基化可能参与了 BC 的发生。我们在这里首次证明，CRBP1 基因在 BC 中可能具有肿瘤抑制功能。
