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

我们以前曾报道，假定的肿瘤抑制基因，APC（腺瘤性息肉病大肠杆菌基因），是突变和/或删除在原发性口腔鳞状细胞癌（OSCC）。本研究的目的是进一步检测人类OSCC中APC基因的蛋白质、mRNA的表达水平以及DNA甲基化状态。对35例原发性口腔鳞状细胞癌及相应的正常口腔黏膜组织进行免疫组化染色，在400倍放大条件下，每一切片至少5个视野观察阳性细胞百分比。采用RT-PCR方法从23例肿瘤组织中提取总RNA，用APC基因外显子1A和外显子2的引物进行PCR扩增。用亚硫酸氢盐修饰的DNA样品进行甲基化特异性PCR检测，证实APC在正常口腔上皮细胞的胞浆中有高水平表达。相反，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%）APC mRNA表达降低。3例（38%）OSCC发生APC异常甲基化。此外，在52例原发性OSCC中，有9例（17%）发现OSCC中E-cadhehn基因启动子的CpG甲基化状态。尤其是9例甲基化病例中，有8例表现为E-cadherin表达减少，组织学上呈弥漫浸润型。我们的数据表明，APC表达下调是人OSCC发生发展中的重要事件，该基因的高甲基化可能是APC基因失活的另一个潜在机制，E-cadherin表达降低与人OSCC的进展有关，E-cadherin基因启动子CpG甲基化导致肿瘤中E-cadherin表达降低。导致获得侵入性pfenotype。

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.