Analysis of cell adhesion factor and metastasis-related gene, E-cadherin APC, in oral squamous cell carcinoma

我们之前已经报道了假定的肿瘤抑制基因APC（腺瘤性大肠息肉病基因）在原发性口腔鳞状细胞癌（oscc）中发生突变和/或缺失。本研究的目的是进一步研究人类OSCCs中apc基因CpG岛的蛋白、mRNA和DMA甲基化状态的表达水平。采用免疫组化染色法对35例原发OSCCs及匹配的正常口腔组织进行了研究。在400倍放大镜下，在每个切片至少5个视场内测定阳性肿瘤细胞的平均百分比。用23个肿瘤的总rna进行RT-PCR生成CDNA。用APC外显子1a和2的引物进行PCR扩增。甲基化特异性PCR检测来自8oscc来源细胞系的亚硫酸酯修饰的DMA样品。我们已经证实APCis在正常口腔上皮细胞的细胞质中有高水平表达。相比之下，在检查的肿瘤中，8例（23%）APC蛋白显著下调。与RT-PCR结果相似，13例（57%）患者APC mRNA表达降低。3例OSCCs发生APC甲基化异常（38%）。此外，在52例原发性oscc中，有9例（17%）的oscc中发现了E-cadhehn基因启动子的CpG甲基化状态。特别是，9例甲基化病例中有8例显示e -钙粘蛋白表达降低和组织学上弥漫性侵袭型肿瘤。我们的数据表明，APC表达下调是人类OSCC发展过程中的一个重要事件，该基因的高甲基化可能是口腔癌变中APC基因失活的另一个潜在机制，E-cadherin表达的减少与人类OSCC的进展有关，E-cadherin基因启动子的CpG甲基化导致肿瘤中E-cadherin表达的减少，从而获得侵袭性表型。

We previously have reported that the putative tumor suppressor gene, APC (adenomatous polyposis coli gene), is mutated and/or deleted in primary oral squamous cell carcinomas (OSCCs). The aim of the present studywas to further examine the expression level of protein, mRNA, and also DMA methylation status on the CpG islands of the APCgene in human OSCCs. Thirty-five primary OSCCs and matched normal oral tissues were investigated by immuno-histochemical staining.A mean percentage of positivetumor cells was determined at least 5 fields at 400X magnification in each section. RT-PCR was performed using total RNAof 23 tumors to generate CDNA.These cDNAwere amplified by PCR with primers for exon1A and 2 of APC. Methylation specific PCR assay was performed in bisulfite-modified DMA samples derived from 8OSCC-derived cell lines.we have comfirmed that APCis expressed at high levels in the cytoplasm of normal oral epithelial cells. In contrast, among the tumors examined, 8 (23%) significant down-regulation of APC protein. Similar to the results of RT-PCR, reduced APC mRNA expression was detected in 13 (57%) cases. Aberrant APC methylation occurred in 3 OSCCs (38%). In addition, the CpG methylation state of E-cadhehn gene promoter in OSCCs was found in 9 (17%) of52 primary OSCCs. In particular, 8 of the 9 methylated casesshowed reduced expression of E-cadherin and histologically diffuse invasion type of tumor. Our data suggest that down-regulation of APC expression is an important event in the development of human OSCC, and that hypermethylation of the gene may be another potential mechanism for inactivation of the APC gene in oral carcinogenesis, and reduction of E-cadherin expression is associated with the progression of human OSCCs, and CpG methylation of E-cadherin gene promoter causes reduction of E-cadherin expression in the tumor, resulting in acquisition of the invasive pfenotype.