表达谱基因芯片筛选子宫颈癌甲基化差异基因的研究

目的筛选人乳头瘤病毒（HPV）阳性宫颈癌细胞系中由于HPV感染诱导沉默的特异抑癌基因，探讨HPV感染可能导致抑癌基因发生甲基化的机制。方法选取HPV阳性HeLa和Caski宫颈癌细胞系和HPV阴性C-33A和HT-3宫颈癌细胞系，分别经去甲基化药物5-氮杂-2′-脱氧胞苷（5-Aza～CdR，Aza）处理，采用Agilent人类全基因组表达谱芯片检测Aza处理前后细胞全基因组的表达水平，采用实时荧光定量PCR（RTqPCR）验证芯片结果，亚硫酸氢盐-基因组测序法（BGS）检测目的基因甲基化水平。结果①芯片结果显示：HPV阳性和阴性细胞系共721条基因表达存在差异。②用药后表达显著上调的基因：HeLa细胞系825条，Caski细胞系815条，c_33A细胞系1023条，HT-3细胞系1196条。HeLa和Caski细胞系共表达上调的基因为182条，剔除阴性细胞系共表达上调基因，筛选出阳性细胞系HPV相关表达上调基因97条，与阴性细胞系基因表达比较，差异有统计学意义（P〈0．01），最终筛选出13条差异表达基因，其中具有功能者7条。③RT-qPCR结果：筛选基因在宫颈癌细胞系中的表达与芯片检测结果一致；HPV阳性宫颈癌细胞系中甲基化频率明显高于阴性细胞系，支持芯片结果。结论筛选出的7条新的潜在抑癌基因可能由于HPV感染诱导发生甲基化而沉默，为进一步探讨HPV诱导抑癌基因发生甲基化的机制提供实验基础。

Screening of methylated gene from cervical carcinoma with expression gene chip

Objective To screen specific silent tumor suppressor genes which were induced HPV infection in cervi- cal cancer cells lines and explore the mechanisms of tumor suppressor gene methylation leading by HPV infections. Methods Exposed the HPV-positive cervical cancer cells （HeLa and Caski） and HPV-negative cervical cancer cells （C-33A and HT-3） to 5-Aza-CdR, Agilent human whole genome expression profiling chips were used to detect the gene expression levels before and after the treatment. RT-qPCR and BGS was used to validate the results of microarray. Results ① There were signifi cant differences in expression profiles between HPV-positive cervical cancer cells with negative cervical cancer cells, 1 436 genes were detected. ② The array test results showed as below： Significantly up-regulated genes： HeLa, 825 genes; Caski, 815 genes; C-33A, 1 023 genes; HT-3, 1 196 genes. The high expression genes of HPV-positive ceils was 182, removed up-reg- ulated genes of negative cell lines, 97 HPV-associated up-regulated genes were screened out from HPV-positive cell lines; the different genes of HPV-positive and HPV-negative ceils was 1436. Compared these 97 genes with differentially expressed genes, 13 genes, seven of which have functions were screened. ③ The expression levels of 7 differential genes （CFTR, FAM9C, SERPINF1, NMB, PLIN5, LO388588, HEATR7B1） in 5-Aza-CdR treated ceils were confirmed by real-time qPCR, which were consistent with the results of microarray. The result of PLIN5 BGS showed that promoter was hypermeth- ylated in HPV positive cell lines and hypomethylated in HPV negative cell lines. Conclusion Seven new potential tumor sup- pressor genes, which may be silenced by HPV-associated methyiation, were screened out. This provides an experimental basis for the further study of HPV-induced tumor suppressor gene methylation mechanism.