Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, P.R. China.
Suzhou Key Laboratory for Molecular Cancer Genetics, Suzhou 215123, P.R. China.
Int J Oncol. 2014 Mar;44(3):874-82. doi: 10.3892/ijo.2014.2255. Epub 2014 Jan 10.
Decorin (DCN) has been suggested to display an anti-metastatic role by antagonizing bioactive TGF-β in advanced human cancers. However, the epigenetic mechanisms by which defective expression of DCN causes cancer metastasis remain unclear. We focused on non-small cell lung cancer (NSCLC) cell lines with low metastatic potential (95C) and high metastatic potential (95D), which share a similar genetic background. Quantitative PCR and clonal bisulfite sequencing indicated that the methylation levels of the +58CpG site in the DCN 5'-UTR region was significantly higher in 95D cells with low expression of DCN mRNA compared to 95C cells with high expression of DCN mRNA. In silico prediction and ChIP assay showed a correlation between +58CpG site and AhR, which was reported as a transcriptional activator. Importantly, EMSA and luciferase reporter gene assays suggested that +58CpG methylation specifically diminished the recruitment of AhR to DCN 5'-UTR sequence and caused a reduction of approximately 50% in transcriptional activity. At baseline, 95D cells exhibited higher p-Smad3 levels and lower E-cadherin expression when compared with 95C cells. The demethylating agent 5-Aza significantly led to restoration of DCN expression, reduced level of p-Smad3, increased expression of E-cadherin in 95D cells. Taken together, we identified the methylated +58CpG in DCN 5'-UTR associated with reduced expression of DCN mRNA, and revealed that +58CpG methylation may be one of the mechanisms accounting for reduced recruitment of the transcriptional activator AhR to DCN 5'-UTR, and suggest that this mechanism promotes TGF-β/Smad signaling by enhancing the phosphorylation of Smad3, thereby downregulating E-cadherin in NSCLC cells with high metastatic potential.
核心钙黏蛋白（DCN）被认为通过拮抗晚期人类癌症中的生物活性 TGF-β 发挥抗转移作用。然而，DCN 表达缺陷导致癌症转移的表观遗传机制仍不清楚。我们专注于具有低转移潜能（95C）和高转移潜能（95D）的非小细胞肺癌（NSCLC）细胞系，它们具有相似的遗传背景。定量 PCR 和克隆亚硫酸氢盐测序表明，DCN 5'-UTR 区域的+58CpG 位点的甲基化水平在低表达 DCN mRNA 的 95D 细胞中明显高于高表达 DCN mRNA 的 95C 细胞。计算机预测和 ChIP 实验表明，+58CpG 位点与 AhR 之间存在相关性，AhR 被报道为一种转录激活剂。重要的是，EMSA 和荧光素酶报告基因检测表明，+58CpG 甲基化特异性地减少了 AhR 与 DCN 5'-UTR 序列的募集，并导致转录活性降低约 50%。在基线水平上，与 95C 细胞相比，95D 细胞表现出更高的 p-Smad3 水平和更低的 E-钙黏蛋白表达。去甲基化剂 5-Aza 显著导致 DCN 表达的恢复，降低 p-Smad3 水平，增加 95D 细胞中 E-钙黏蛋白的表达。总之，我们确定了与 DCN mRNA 表达降低相关的 DCN 5'-UTR 中的甲基化+58CpG，并揭示了+58CpG 甲基化可能是减少转录激活剂 AhR 募集到 DCN 5'-UTR 的机制之一，并表明该机制通过增强 Smad3 的磷酸化，从而下调高转移潜能 NSCLC 细胞中的 E-钙黏蛋白，从而促进 TGF-β/Smad 信号转导。
