卵巢癌患者RUNX3基因启动子区甲基化状态分析

Methylation of RUNX3 gene in epithelial ovarian carcinoma

目的 探讨人 RUNX3（runt-related transcription factor 3 gene）基因在上皮性卵巢癌组织中的甲基化状态及其在卵巢癌发生发展中的作用。方法用甲基化特异性PCR（MSP）检测32份卵巢癌组织、32份癌旁组织、36份卵巢良性上皮性肿瘤组织及10份正常卵巢组织中RUNX3基因启动子CpG岛的甲基化频率。培养2种卵巢癌细胞系（3AO，SKOV3），MSP法检测加入5-氮-2’-脱氧胞苷（5-aza-2＇-deoxycytidine）前后RUNX3基因在卵巢癌细胞系中的甲基化水平。逆转录（RT）-PCR检测加入5-aza-2’deoxycytidine前后卵巢癌细胞株中RUNX3 mRNA的表达改变，及上述各卵巢组织中RUNX3基因mRNA的表达情况。结果卵巢癌标本中53．1％（17／32）发生了RUNX3基因启动子甲基化；癌旁组织RUNX3基因甲基化率为37．5％（12／32）；良性卵巢肿瘤组织中RUNX3基因甲基化率为16．7％（6／36）；10份正常卵巢组织均未检测到RUNX3基因甲基化。卵巢癌组织中甲基化率与临床分期、细胞分化程度无相关性。卵巢癌与良性卵巢肿瘤和正常组织间的甲基化率的差异有统计学意义（分别X^2=10．060,X^2=8．925，均P〈0．05）。2株卵巢癌细胞系中均发生了RUNX3启动子甲基化，经5-aza-2’deoxycytidine处理细胞株后，可部分或完全逆转RUNX3基因启动子甲基化。RUNX3基因mRNA的表达在卵巢癌中为16．7％（6／32）；癌旁组织为28％（9／32），良性肿瘤中为72％（26／36），正常组织中为80％（8／10），其表达在卵巢癌与良性肿瘤和正常卵巢组织中的差异有统计学意义（分别X^2=19．443,X^2=12．862，均P〈0．05）。经5-aza-2’deoxycytidine处理细胞株后，各细胞株RUNX3基因mRNA较加药前均有不同程度的表达增加。结论 卵巢癌组织中RUNX3基因启动子有较高的甲基化率，且在卵巢癌的发生发展中起重要作用；有可能成为卵巢癌早期诊断的分子生物学指标。RUNX3基因甲基化与其mRNA表达密切相关，是基因表达调节的重要方式之一，这种改变可被甲基化酶抑制剂所逆转。

Objective To access the role of methylated RUNX3 gene in the carcinogenesis and progression of ovarian carcinoma. Methods Sample of 32 epithelial ovarian carcinoma tissues, 32 para- carcinoma tissues, 36 benign epithelial tumors and 10 normal ovarian tissues and 2 cell lines（3AO and SKOV3）, were collected and subject to methylation-specific PCR （MSP）. Promoter methylation status of RUNX3 in two tumor cell lines were analyzed before and after 5-aza-2＇deoxycytidine treatment. In addition, mRNA expression of RUNX3 were investigated by quantitative reverse transcription-PCR. Results CpG island methylation of RUNX3 was observed in 53.1% （17 of 32） of epithelial ovarian cancer, and 37.5% （12 of 32） in corresponding noncancerous tissues, 16. 7% in benign epithelial tumors （6 of 36 ）, and all cell lines, but not in normal control tissues. The prevalence of RUNX3 gene CpG methylation in malignant was significantly higher than those in benign and normal tissues （X^2 = 10. 060 ,X^2 = 8. 925, P 〈 0. 05 ）. Nineteen percent （6 of 32） of ovarian epithelial carcinoma expressed RUNX3 mRNA, while its expression was present in 28% （9 of 32） corresponding noncancerous tissues and 72% （26 of 36） of benign ovarian tumor and 80% （8 of 10） of normal ovarian tissues. The RUNX3 promoter methylation was found in all cell lines tested. The ratio of expression of RUNX3 mRNA in ovarian epithelial carcinoma was significantly lower than those of normal and benign tumors （X^2 = 19. 443 ,X^2 = 12. 862 ,P 〈 0. 05 ）. After 5-aza-2＇deoxycytidine treatment, methylation was partially or completely reversed, and its mRNA expression was increased. The relationship between gene expression and promoter methylation was reversely correlated. Conclusions Our results suggest that promoter hypermethylation of RUNX3 genes is common in ovarian cancer. Therefore, hypermethylation of RUNX3 genes may be involved in the carcinogenesis of ovarian cancer and may serve as an early diagnostic marker for ovarian cancer. The close correlation between RUNX3 methylation of its mRNA suggests that methylation which can be reversed. Thus, it provides a new way for therapy of ovarian