抑癌基因IRF8在肾细胞癌中的表观遗传学改变及分子机制研究

The epigenetic alterations of the promoters in tumor suppressor genes have been a hotspot in basic cancer research. A great number of tumor suppressor genes harboring aberrant methylation have been confirmed to play a significant role in tumor developing and progression. We have figured out that IRF8, a candidate tumor suppressor gene, have aberrant methylation by high thoroughout microarray analysis and plays a significant role in renal cell carcinoma(RCC). In our subject, IRF8 was highly expressed in normal kidney tissues from human adults and children, but was greatly down-regulated or transcriptional silenced in several RCC cell lines which could be reversed by drug-induced demethylation .The methylation specific PCR revealed that IRF8 promoter was methylated in 50% RCC cell lines, and in 31.25% renal primary tumors by analyzing 48 RCC samples. The silencing of IRF8 in RCC, due to aberrant DNA methylation, could be reversed by demethylation agent AZA. Ecotopic expression of IRF8 inhibited RCC cell lines (786-O, A498) colony formation, and caused G2/M arrest, suggesting its tumor-suppressive function in RCC cell lines. In addition, Dab2, a target gene of IRF8, was up-regulated 3 times in IRF8-transfected RCC cell lines. Further reseach will be carried out on the basis of the preliminary experiments. First,we will enrich the clinical RCC samples ,detect the expression of IRF8 and its promoter methylation status in more clinical samples, and define whether IRF8 is a candidate epigenetics biomarker or not. Second, we will continue to screen candidate tumor suppresser genes of RCC and optimize the spectrum of tumor suppressor genes harboring aberrant methylation associated with renal cell carcinoma. At last, the molecular mechanism of IRF8 or the other TSGs relevant to RCC will be studied in-depth.

抑癌基因启动子高度甲基化已成为目前肿瘤病理学基础研究的热点，研究证实，DNA甲基化与肾癌的发生发展密切相关。本项目组成员通过高通量微距阵基因芯片技术在肾细胞癌中筛选出具有异常甲基化的抑癌基因IRF8, 并在预实验中发现， IRF8在正常成人及婴儿肾组织中表达，而在多个肾癌细胞株中表达沉默，通过药物去甲基化处理能诱导其重新表达。我们利用MSP检测了48例肾癌组织标本，发现IRF8启动子区甲基化频率达31.25%。另外，我们在肾癌细胞株中证实IRF8能抑制肾癌细胞生长并上调IRFs靶基因Dab2的表达。本项目拟在预实验的基础上继续如下研究：1）进一步扩充临床样本，检测IRF8在肾癌标本及血液、尿液标本中的表达及甲基化情况，明确其能否作为肾癌早期诊断的表观遗传学分子标志物。2）继续筛选肾癌相关的异常甲基化的抑癌基因，扩充肾细胞癌基因异常甲基化谱。3）探索IRF8及其他抑癌基因的作用机制。

抑癌基因在肾癌发生发展中发挥着重要作用，IRF8作为IFN-γ信号通路中一个重要的元素在肿瘤中发挥着抑癌作用。然而其在肾癌中的表达情况及相关分子机制尚未明确指出。在本课题中，我们检测了IRF8在肾癌细胞系及肾癌组织中的表达水平和甲基化，并进一步验证其抑癌作用。我们发现IRF8在人的正常组织中表达，而在肾癌细胞系中其表达明显下调，通过研究分析我们验证引起其表达下调的主要机制为启动子区的甲基化。此外，我们在25%的肾肿瘤中检测到该启动子区的甲基化，但在癌旁正常肾组织中我们没有检测出甲基化。通过统计分析结果显示，IRF8启动子区的甲基化与肿瘤的核分级相关。接下来的细胞功能实验证明过表达IRF8能够抑制肿瘤细胞的克隆形成，转移能力；介导细胞凋亡和细胞周期的阻滞（G2/M）。进一步研究发现，IRF8能够抑制癌基因（YAP1、Survivin）的表达，并且能够促进CASP1，p21和PTEN的表达。总之，我们的研究发现IRF8在肾癌中作为抑癌基因，并且常发生甲基化。