Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
Int J Mol Med. 2010 Aug;26(2):241-7. doi: 10.3892/ijmm_00000458.
We 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 pharmacologic demethylating agent (5-aza-dC). We focused on the human four-and-a-half LIM domains 1 (FHL1) gene which was selected on the basis of previous microarray data analysis. Because LIM domains provide protein-protein binding interfaces, FHL genes play an important role in cellular events, such as focal adhesion and differentiation, by interacting with the target protein as either a repressor or activator. We hypothesized that inactivation of the FHL1 gene through CpG methylation contributes to cell viability including migration and invasion activity of human BC. After 5-aza-dC treatment, the expression levels of FHL1 mRNA transcript markedly increased in all cell lines tested, as shown by real-time reverse transcription-polymerase chain reaction (RT-PCR). The methylation index of FHL1 in our samples was significantly higher in 70 BC specimens than in 10 normal bladder epithelium (NBE) specimens (63.9+/-25.5 and 0.3+/-0.2, respectively; p=0.0066). Conversely, FHL1 mRNA expression was significantly lower in the BC specimens than in the NBE ones (0.331+/-0.12 and 2.498+/-0.61, respectively; p=0.0011). In addition, significant inhibitions of wound healing (45.78+/-6.2, and 100+/-0, respectively; p=0.009) and of cell invasion (18.5+/-2.3 and 95.2+/-2.4, respectively; p=0.02) were observed in stable FHL1-transfected cells than in the control BC cells. In conclusion, we found that the mechanism of FHL1 down-regulation in BC is through CpG hypermethylation of the promoter region. FHL1 gene inactivation by CpG hypermethylation may thus contribute to migration and invasion activity of 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-aza-dC）处理的 BC 细胞系（BOY）的微阵列数据结合起来。我们专注于人类四个半 LIM 结构域 1（FHL1）基因，该基因是基于先前的微阵列数据分析选择的。由于 LIM 结构域提供蛋白质-蛋白质结合界面，因此 FHL 基因通过与靶蛋白相互作用作为抑制剂或激活剂，在细胞事件（如粘着斑和分化）中发挥重要作用。我们假设 FHL1 基因通过 CpG 甲基化失活有助于包括人膀胱癌的迁移和侵袭活性在内的细胞活力。在用 5-aza-dC 处理后，所有测试的细胞系中 FHL1 mRNA 转录本的表达水平均明显增加，如实时逆转录-聚合酶链反应（RT-PCR）所示。我们的样本中 FHL1 的甲基化指数在 70 个 BC 标本中明显高于 10 个正常膀胱上皮（NBE）标本（分别为 63.9+/-25.5 和 0.3+/-0.2；p=0.0066）。相反，FHL1 mRNA 在 BC 标本中的表达明显低于 NBE 标本（分别为 0.331+/-0.12 和 2.498+/-0.61；p=0.0011）。此外，与对照 BC 细胞相比，稳定转染 FHL1 的细胞的伤口愈合（分别为 45.78+/-6.2 和 100+/-0；p=0.009）和细胞侵袭（分别为 18.5+/-2.3 和 95.2+/-2.4；p=0.02）的抑制作用明显。总之，我们发现 BC 中 FHL1 下调的机制是通过启动子区域的 CpG 过度甲基化。因此，CpG 过度甲基化导致 FHL1 基因失活可能有助于 BC 的迁移和侵袭活性。
