肾透明细胞癌患者肾组织干扰素调节因子6基因启动子区甲基化与总生存率的相关性研究

Correlation between methylation of interferon regulatory factor 6 gene promoter in renal tissues and overall survival of patients with Kidney renal clear cell carcinoma

目的探讨干扰素调节因子6(IRF6)基因启动子甲基化在判断肾透明细胞癌(KIRC)患者预后中的价值。方法收集2016年1月至2020年1月在南京医科大学第一附属医院首诊的50例KIRC患者的原发灶组织,采用免疫组化法检测其IRF6蛋白的表达情况,并分析IRF6表达水平与患者生存及肿瘤转移的关系。使用生物信息学方法分别比较KIRC和正常肾组织中IRF6的mRNA及蛋白水平差异,基于大数据库分析癌和癌旁组织中IRF6基因启动子区甲基化率差异;将IRF6基因启动子区甲基化状态与患者总生存率(OS)进行统计分析,筛选出呈现显著负相关的区域,利用细胞系体外验证该启动子区甲基化区域与IRF6转录水平的关系。结果KIRC组织中IRF6 mRNA和蛋白水平均显著低于正常样本(P<0.05),该现象在死亡或发生肿瘤转移的KIRC患者中较为突出。KIRC组织中IRF6基因启动子区甲基化程度远高于临近正常肾组织,并且IRF6基因启动子区甲基化与IRF6 mRNA之间存在显著负相关性(R=-0.52)。IRF6基因启动子区域cg12034118和cg16030177的甲基化程度越高,患者OS越短且预后越差;体外KIRC细胞系实验证实,仅cg12034118中有20个CpG位点呈高度甲基化,使用去甲基化试剂5-Aza-CdR处理KIRC细胞系后IRF6转录水平可呈时间和剂量依赖显著性上调(P<0.05)。结论KIRC患者肾组织中IRF6基因启动子区甲基化与OS密切相关。cg12034118提示可作为供实验室检测的生物标志物,其高甲基化率对KIRC患者的预后判断有一定参考价值。

Objective To assess the prognostic value of methylation of interferon regulatory factor 6(IRF6)gene promoter in patients diagnosed with Kidney renal clear cell carcinoma(KIRC).Methods The primary lesions of fifty KIRC patients who were diagnosed at the First Affiliated Hospital of Nanjing Medical University from January 2016 to January 2020 were collected.The expression of IRF6 protein was determined with an immunohistochemical method.The correlation between the level of IRF6 expression and survival and/or metastasis status was analyzed.The mRNA and protein levels of the IRF6 in KIRC and normal renal tissues were compared by using bioinformatic tools.The difference in the methylation rate of the IRF6 gene promoter between tumor and adjacent tissues was analyzed by searching the online databases.Statistical analysis was carried out for the methylation status of the IRF6 gene promoter region to select those negatively correlated with the overall survival(OS)among the patients.In vitro experiments were conducted with cell lines to verify the correlation between the status of promoter methylation and transcription level of the IRF6 gene.Results The mRNA and protein levels of the IRF6 gene in KIRC tissues were significantly lower than those of the normal controls,and this was more prominent in patients who had died or developed metastasis.The extent of IRF6 gene promoter methylation in the KIRC tissues was much higher compared with that of the adjacent normal renal tissues.There was a significant negative correlation between the methylation of the IRF6 gene promoter and mRNA level of the IRF6(R=-0.52).The higher methylation degree in the IRF6 gene promoter regions cg12034118 and cg16030177,the shorter the OS and worse prognosis in the patients.Only twenty CpG sites in cg12034118 were confirmed to be highly methylated in KIRC cell lines.The transcription level of the IRF6 gene was upregulated in a time-and dose-dependent manner after the treatment with demethylation reagent 5-azadeoxycytidine.Conclusion The methylation of IRF6 gene promoter in the renal tissues of KIRC patients is closely correlated with the OS.Cg12034118 may provide a promising biomarker for laboratory detection,and its high methylation rate has certain reference value for the prognosis.