The role of restored expression of RECK gene in regulating carcinogenesis and invasion by demethylation agent in oral squamous cell carcinoma cell lines

我们以前曾报道MMP-2活性形式与淋巴结转移有关。RECK基因是近年来发现的一种新的肿瘤抑制基因，它可以调节MMPs和MT 1-MMP的活性。RECK基因的下调在肿瘤细胞的恶性进展中起重要作用。此外，超甲基化被称为与癌症发展相关的表观遗传变化。因此，检测超甲基化在肿瘤发生过程中具有重要意义。然而，RECK基因在口腔鳞癌中的甲基化状态的研究尚未见报道。我们研究RECK基因甲基化逆转可能与抑制MMPs活性和肿瘤侵袭能力有关。此外，通过RT-PCR证实了RECK mRNA的表达增强，并且在用5-氮杂-2 '-脱氧胞苷处理后，这些细胞系中的RECK高甲基化逆转与mRNA水平明显相关。此外，MMP-2和MMP-9的活性在用去甲基化剂处理后的癌细胞中也观察到抑制。有趣的是，5-aza-2 '-deoxycytidine通过减少三维胶原侵袭模型中的侵袭灶数量而显著抑制癌细胞的侵袭能力，我们的研究结果支持了去甲基化剂可能通过MMPs抑制剂如RECK的去甲基化作用而在抑制MMPs活性和细胞侵袭能力中起关键作用的结论。

We have previously reported that MMP-2active form correlated with lymph node metastasis. RECK, a novel tumor suppressor gene, was recently found to regulate the activity of MMPs and MT1-MMP. Down regulation of RECK gene plays an important role in malignant progress of cancer cells. Furthermore, hypermethylation is known as the epigenetic change that is associated with cancer development. Therefore determination of hypermethylation is important in carcinogenesis process. However, study on methylation status of RECK gene in oral squamous cell carcinoma has not yet been reported. We investigate that reversal of hypermethylation of RECK gene may relate to inhibition of MMPs activity and cancer invasive ability.The present results showed that hypermethylation occurred in RECK gene promoter in oral squamous cell carcinoma cell lines. In addition, enhanced expression of RECK mRNA was confirmed by RT-PCR and mRNA levels were clearly associated with reversal of hypermethylation of RECK in these cell lines after treatment with 5-aza-2'-deoxycytidine. Furthermore, the inhibition of MMP-2 and MMP-9 activities was also observed in cancer cells after treatment with demethylation agent. Interestingly, 5-aza-2'-deoxycytidine significantly suppressed cancer cell invasive ability by decreasing the number of invasive foci in three-dimensional collagen invasion model.Our findings support the conclusion that demethylation agent plays a crucial role in inhibiting MMPs activities and cell invasive ability possibly by demethylation effect of MMPs inhibitors such as RECK.