印迹抑癌基因ARHI在浆液性卵巢癌中的表达及甲基化研究

Expression and methylation of ARHI gene in ovarian serous carcinoma

目的研究浆液性卵巢癌组织中印迹抑癌基因ARHI mRNA的表达及其3个CpG岛的甲基化状态,以探讨ARHI基因及其甲基化修饰在卵巢上皮癌发生机制中的作用。方法正常卵巢及浆液性卵巢癌标本各20例,采用半定量RT-PCR方法检测ARHI mRNA表达,采用DNA亚硫酸盐修饰、PCR扩增及甲基化敏感性内切酶方法检测ARHI基因3个CpG岛的甲基化状态。结果ARHI/G3PDH在正常卵巢组织中为0.73±0.09,浆液性卵巢癌中为0.43±0.37(P<0.01)。正常卵巢组织的3个CpG岛均同时扩增出ARHI甲基化和非甲基化产物,呈正常半甲基化状态;浆液性卵巢癌的3个CpG岛则存在不同比例的甲基化异常,其中6例(30%)CpGⅠ、8例(40%)CpGⅡ、5例(25%)CpGⅢ处于高度甲基化状态。在浆液性卵巢癌组织中,8例ARHI基因表达缺失者均存在CpG岛的高度甲基化,其余12例中有5例存在CpG岛的高度甲基化,表达缺失者中CpG岛的高度甲基化率显著增高(P<0.01)。结论印迹抑癌基因ARHI启动子及编码区的3个CpG岛在浆液性卵巢癌组织中存在甲基化异常,并抑制了该基因的表达。

Objective To study the expression of ARHI mRNA, an imprinted suppressor gene, in ovarian serous carcinoma and the methylation status of its three CpG islands, and to determine the possible role of aberrant methylation of ARHI gene in pathogenesis of o- varian serous carcinoma. Methods Twenty normal ovarian specimens and 20 ovarian serous carcinoma specimens were analyzed. Total RNA was extracted and semi-quantitative RT-PCR was employed to detect the mRNA expression of ARHI gene. Methylation status of three CpG islands were examined by DNA bisulfite treatment, PCR amplification and methylation specific restriction enzymes, Tagl for CpG Ⅰ and Ⅲ, BstUI for CpG Ⅱ. Results The average of ARHI/G3PDH was 0. 73±0. 09 in normal ovarian specimens, and it was 0. 43 ±0. 37 in ovarian carcinoma specimens （ P〈0. 01）. Both methylated and unmethylated products were observed on three CpG islands in normal ovarian tissue, while different CpG methylation status was observed in ovarian carcinoma. The rates of hypermethylation in CpGI, CpGII and CpGIII was 30%, 40% and 25%, respectively. In ovarian serous carcinoma, hypermethylation status on CpG islands of ARHI gene was detected in all 8 cases with lost expression, while in 5 of 12 cases it showed a down-regulated expression （P〈0. 01）. Conclusion Aberrant methylation of three CpG islands in promoter and coding regions down-regulates the expression of ARHI gene in ovarian serous carcinoma. The result suggests that aberrant methylation of ARHI gene contributes to pathogenesis of ovarian carcinoma, and a theoretical basis is provided accordingly in developing novel treatments for ovarian carcinoma.