Institute of Clinical Medicine, National Cheng Kung University Medical College, 138 Sheng-Li Road, Tainan 704, Taiwan.
Carcinogenesis. 2013 Nov;34(11):2452-9. doi: 10.1093/carcin/bgt218. Epub 2013 Jun 17.
Loss of RUNX3 expression is frequently observed in gastric cancer and is highly associated with lymph node metastasis and poor prognosis. However, the underlying molecular mechanisms of gastric cancer remain unknown. In this study, we found that the protein levels of RUNX3 and osteopontin (OPN) are inversely correlated in gastric cancer clinical specimens and cell lines. Furthermore, similar inverse trends between RUNX3 and OPN messenger RNA (mRNA) expression were demonstrated in six out of seven normal-tumor-paired gastric cancer clinical specimens. In addition, low RUNX3 and high OPN expression were associated with poor prognosis in gastric cancer patients. Ectopic expression of green fluorescent protein-RUNX3 reduced OPN protein and mRNA expression in the AGS and SCM-1 gastric cancer cell lines. In contrast, knockdown of RUNX3 in GES-1, a normal gastric epithelial cell line, increased OPN expression. Although three RUNX3-binding sequences have been identified in the OPN promoter region, direct binding of RUNX3 to the specific binding site, -142 to -137bp, was demonstrated by chromatin immunoprecipitation assay. The binding of RUNX3 to the OPN promoter significantly decreased OPN promoter activity. The knockdown of OPN or overexpression of RUNX3 inhibited cell migration in AGS and SCM-1 cells; however, the coexpression of RUNX3 and OPN reversed the RUNX3-reduced migration ability in AGS and SCM-1 cells. In contrast, the knockdown of both RUNX3 and OPN inhibited RUNX3-knockdown-induced migration of GES-1 cells. Together, our data demonstrated that RUNX3 is a transcriptional repressor of OPN and that loss of RUNX3 upregulates OPN, which promotes migration in gastric cancer cells.
RUNX3 表达缺失在胃癌中频繁发生，与淋巴结转移和不良预后高度相关。然而，胃癌的潜在分子机制尚不清楚。在本研究中，我们发现胃癌临床标本和细胞系中 RUNX3 和骨桥蛋白（OPN）的蛋白水平呈负相关。此外，在 7 对正常-肿瘤配对胃癌临床标本中，有 6 对表现出 RUNX3 和 OPN 信使 RNA（mRNA）表达之间相似的反向趋势。此外，低 RUNX3 和高 OPN 表达与胃癌患者的不良预后相关。绿色荧光蛋白-RUNX3 的异位表达降低了 AGS 和 SCM-1 胃癌细胞系中 OPN 的蛋白和 mRNA 表达。相反，在正常胃上皮细胞系 GES-1 中敲低 RUNX3 增加了 OPN 的表达。尽管在 OPN 启动子区域已经鉴定出三个 RUNX3 结合序列，但染色质免疫沉淀分析证实了 RUNX3 与特定结合位点（-142 到-137bp）的直接结合。RUNX3 与 OPN 启动子的结合显著降低了 OPN 启动子活性。OPN 的敲低或 RUNX3 的过表达抑制了 AGS 和 SCM-1 细胞的迁移；然而，RUNX3 和 OPN 的共表达逆转了 AGS 和 SCM-1 细胞中 RUNX3 降低的迁移能力。相反，RUNX3 和 OPN 的敲低均抑制了 GES-1 细胞中 RUNX3 敲低诱导的迁移。总之，我们的数据表明 RUNX3 是 OPN 的转录抑制剂，而 RUNX3 的缺失上调了 OPN，促进了胃癌细胞的迁移。
