ZNF772基因启动子区DNA甲基化及其表达与宫颈癌的相关性

Correlation between DNA Methylation of ZNF772 Promoter Region and Cervical Cancer

目的研究ZNF772基因启动子区DNA甲基化的状态及其mRNA、蛋白的表达,分析ZNF772基因DNA甲基化在宫颈癌中的临床意义。方法选取3例宫颈鳞癌组织(SCC)和年龄相匹配的3例正常宫颈组织作对照,采用全基因组重亚硫酸盐测序(WGBS)联合RNA测序技术筛选DNA高甲基化和低表达的转录子,采用实时荧光定量PCR(RT-qPCR)和亚硫酸盐化测序PCR(BSP)方法在40例SCC患者宫颈组织和45例正常宫颈组织中检测ZNF772的DNA甲基化状态和mRNA的表达,免疫组织化学方法检测其蛋白表达量。在SCC组中分析ZNF772基因启动子DNA甲基化及其mRNA的表达与宫颈癌病理参数的关系。结果联合WGBS和RNA测序技术检测显示,异常DNA甲基化基因ZNF772与其mRNA表达关联;RT-qPCR方法验证,与对照组相比,SCC组中ZNF772 mRNA的表达下调(t=8.351,P=0.016);免疫组织化学分析进一步证实SCC组中ZNF772蛋白的阳性表达下调(t=3.802,P=0.005);BSP结果证实,与对照组比较,SCC组中ZNF772基因启动子区(-420、-422位点)DNA甲基化率显著升高(χ^2=8.566,P=0.038;χ^2=6.332,P=0.043);Spearman相关性分析显示,SCC组ZNF772启动子DNA甲基化和mRNA的表达呈负相关(r=-0.351,P=0.045;r=-0.349,P=0.032),且与HPV16/18型感染、肿瘤大小、世界卫生组织病理分级及国际妇产科联合会临床分期相关(P=0.018,P=0.012,P=0.009,P=0.035)。结论ZNF772基因启动子区DNA高甲基化与宫颈癌的发生、发展相关。

Objective To investigate the DNA methylation in ZNF772 promoter region and its mRNA and protein expressions and analyze the clinical significance of DNA methylation of ZNF772 gene in cervical cancer.Methods Cervical squamous cell carcinoma(SCC)tissues were harvested from three patients(SCC group),and normal cervical tissues from healthy individuals of the same age were used as the control group.Hyper-methylation and lower transcripts were screened by whole-genome bisulfite sequencing(WGBS)and RNA sequencing.Furthermore,in 40 cervical tissue samples in SCC group and 45 normal cervical tissues in the control group,DNA methylation status and mRNA expression of ZNF772 were measured by using real-time quantitative polymerase chain reaction(RT-qPCR)and bisulfite sequencing polymerase chain reaction(BSP).The protein expression was detected by immunohistochemistry.In the SCC group,the potential relationships of DNA methylation status in ZNF772 promoter and mRNA expression with the clinicopathological parameters of cervical cancer were analyzed.Results As shown by WGBS and RNA sequencing,the abnormal DNA methylated gene ZNF772 was associated with mRNA expression.RT-qPCR verified that the mRNA expression of ZNF772 was significantly lower in SCC group than in control group(t=8.351,P=0.016).Immunohistochemistry further confirmed that the positive expression of ZNF772 protein was down-regulated in SCC group(t=3.802,P=0.005).BSP showed that the DNA methylation rate of ZNF772 promoter region(-420,-422 locus)in SCC group was significantly higher than that in control group(χ^2=8.566,P=0.038;χ^2=6.332,P=0.043).Spearman correlation analysis showed that,in SCC group,DNA hypermethylation in ZNF772 promoter was negatively correlated with the mRNA expression(r=-0.351,P=0.045;r=-0.349,P=0.032)and was significantly correlated with HPV16/18 infection,tumor size,World Health Organization pathological grade,and International Federation of Gynecology and Obstetrics clinical stage(P=0.018,P=0.012,P=0.009,and P=0.035,respectively).Conclusion The DNA hypermethylation in the promoter region of ZNF772 gene is involved in the occurrence and development of cervical cancer.