Overexpression of S100A4 is associated with DNA hypomethylation and enhanced invasiveness in ovarian carcinomas

S100A4蛋白属于钙结合蛋白S100家族，与癌细胞的转移表型有关。在本研究中，我们研究了卵巢癌细胞中S100A4的表达与S100A4第一内含子低甲基化的关系。我们首次用免疫组织化学方法检测了S100A4在113例卵巢上皮性肿瘤中的表达和定位，并分析了其在卵巢癌患者中的预后意义。S100A4强核表达的卵巢癌患者的生存期明显短于未表达S100A4的卵巢癌患者(p=0.0045)。卵巢癌细胞株SKOV3、A2780、A2780/CDDP表达S100A4，而OVCAR3和OSes不表达S100A4。Matrigel侵袭实验显示S100A4的表达水平与卵巢癌细胞的侵袭能力有关。针对S100A4的siRNA抑制了S100A4的表达，抑制了A2780/CDDP细胞的侵袭力。然后，我们用亚硫酸氢盐修饰的DNA测序法研究了卵巢癌细胞系和51例卵巢癌组织中DNA甲基化状态与S100A4表达的关系。我们发现在S100A4阳性的卵巢癌细胞中，S100A4基因第一内含子上的CpG位点发生了低甲基化。相反，在S100A4阴性的卵巢癌细胞中，这些CpG位点甲基化，5-氮-2脱氧胞苷处理后S100A4的表达发生改变。在卵巢癌组织中，24例S100A4强阳性患者S100A4基因第一内含子发生甲基化，14例S100A4阴性患者发生甲基化。

The S100A4 protein which belongs to calcium binding S100 protein family has been suggested to be linked to metastatic phenotypes of cancer cell. In this study, we investigated the association between the expression of S100A4 and hypomethylation of the S100A4 first intron in ovarian carcinoma cells. We first used immunohistochemistry to examine the expression and localization of S100A4 in 113 epithelial ovarian neoplasms and analyzed its prognostic significance in patients with ovarian carcinoma. Ovarian carcinoma patients with strong nuclear S100A4 expression showed a significantly shorter survival than those without (p=0.0045). Three ovarian cancer cell lines (SKOV3, A2780, A2780/CDDP) expressed S100A4, however, OVCAR3 and OSEs did not. Matrigel invasion assay showed that the expression level of S100A4 was associated with invasive ability in ovarian cancer cells. The specific siRNA for S100A4 suppressed S100A4 expression and the invasiveness of A2780/CDDP cells. We then studied the relationship between DNA methylation status and S100A4 expression by bisulfite-modified DNA sequencing in ovarian cancer cell lines and 51 cases of ovarian carcinoma tissues. We found that CpG sites in the first intron of the S100A4 gene were hypomethylated in S100A4 positive ovarian carcinoma cells. In contrast, these CpG sites were methylated in S100A4 negative ovarian carcinoma cells and S100A4 expression was altered by 5-aza-2deoxycytidine treatment. In ovarian cancer tissues, 24 cases of S100A4 strongly positive were hypomethylated in the first intron of S100A4 gene and 14 of all S100A4 negative cases were methylated.