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

我们以前报道过，在口腔鳞状细胞癌(OSCCs)中，可能的抑癌基因APC(腺瘤性息肉病结肠基因)发生突变和/或缺失。本研究的目的是进一步检测口腔鳞状细胞癌中APC基因CpG岛上蛋白、mRNA的表达水平以及DNA甲基化状态。对35例原代口腔鳞状细胞癌及其配对的正常口腔组织进行免疫组织化学染色，在400倍放大倍数下，每张切片至少5个视野测定平均阳性肿瘤细胞百分率。用23例肿瘤组织的总RNA进行RT-PCR扩增，扩增产物为APC的外显子1和2。对8OSCC细胞亚硫酸盐修饰的DMA样品进行甲基化特异性PCR检测，证实APCis在正常口腔上皮细胞胞浆中高水平表达。相比之下，在所检测的肿瘤中，8例(23%)APC蛋白显著下调。与RT-PCR结果相似，13例(57%)APC基因表达降低。3例口腔鳞状细胞癌(38%)发生APC异常甲基化。此外，52例原发口腔鳞癌中有9例(17%)发现E-cadhehn基因启动子CpG甲基化状态。在9例甲基化病例中，有8例E-钙粘附素表达降低，组织学呈弥漫浸润型。我们的研究结果提示，APC基因表达下调是口腔鳞癌发生发展过程中的重要事件，APC基因高甲基化可能是口腔鳞癌发生发展过程中APC基因失活的另一种潜在机制，E-钙粘蛋白表达降低与口腔鳞状细胞癌的发生发展有关，E-钙粘蛋白基因启动子CpG甲基化导致E-钙粘蛋白在口腔鳞癌中表达降低，从而导致口腔鳞癌获得侵袭性亚型。

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.