Identification of novel metastasis related gene in oral squamous cell carcinoma by means of proteomics analysis

我们分析了原发性口腔鳞状细胞癌(OSCC)和口腔鳞癌来源的细胞系中脾酪氨酸激酶(Syk)基因的突变和甲基化状态以及mRNA和蛋白质水平，并在体外检测了Syk基因在口腔鳞状细胞癌来源的细胞系中的功能。应用实时定量逆转录聚合酶链式反应和免疫荧光技术对7个口腔鳞癌来源的细胞系和正常口腔角质形成细胞(NOK)进行检测，发现Syk基因在所有细胞系中的表达普遍低于NOK细胞。虽然在这些细胞系中没有发现Syk基因编码区的序列变异，但我们在CpG岛区发现了频繁的超甲基化。通过实验性去甲基化恢复SYK的表达。此外，利用伤口愈合实验，我们将Syk基因导入口腔鳞癌来源的细胞系进行功能分析，发现它们显示出显著的运动和侵袭性抑制。在临床标本中，免疫组织化学检测到高频率的Syk下调(33/53[62%])。此外，Syk的表达状态与肿瘤颈淋巴转移显著相关(P=0.0042)。这些结果表明，Syk基因在口腔癌发生过程中经常失活，表观遗传机制可能调节表达缺失，可能导致转移。

We analyzed the mutational and methylation status of the spleen tyrosine kinase (Syk) gene and both mRNA and protein levels in primary oral squamous cell carcinoma (OSCC) and OSCC-derived cell lines and examined the function of the Syk gene in OSCC-derived cell lines in vitro. Using quantitative real-time reverse transcription polymerase chain reaction and immunofluorescence on seven OSCC-derived cell lines and normal oral keratinocytes (NOKs), Syk mRNA and protein expression were commonly down-regulated in all cell lines compared with the NOKs. Although no sequence variation in the coding region of the Syk gene was identified in these cell lines, we found frequent hypermethylation in the CpG island region. Syk expression was restored by experimental demethylation. In addition, using a wound healing assay, we performed functional analysis using Syk transfected into the OSCC-derived cell lines, and they showed significant inhibition of motility and invasiveness. In clinical samples, high frequencies of Syk down-regulation were detected by immunohistochemistry (33 of 53 [62%]). Furthermore, the Syk expression status was correlated significantly (P= 0.0042) with tumor metastasis to cervical lymph nodes. These results suggest that the Syk gene is frequently inactivated during oral carcinogenesis and that an epigenetic mechanism may regulate loss of expression possibly leading to metastasis.