DNA羟甲基化缺失通过调节胆固醇代谢促进肾透明细胞癌进展的机制研究

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer and has the highest propensity to manifest as metastatic disease. ccRCC is characterized by accumulation of lipids such as cholesterol, however, the mechanisms and tumor promotion effect are still unclear. Our previous results proved that 5-hydroxymethylation of cytosine in genome DNA is defect in ccRCC tissues, and the loss of 5-hydorxymethylcytosine is related to higher tumor stage and grade and worse prognosis. The latest studies showed that upregulation of 5hmC could inhibit the expression of cholesterol metabolic genes, and regulated accumulation of lipids such as cholesterol. So we assumed that loss of 5hmC may promote ccRCC progression by regulating cholesterol metabolism. Firstly this project will analyze the results of RNA-seq and TAB-seq and carry out molecular biologic experiments such as chromosome immunoprecipitation (ChIP) to identify the target gene of 5hmC regulating cholesterol metabolism. Secondly we will interfere the expression of the target gene and evaluate the effects on cholesterol metabolism and cell growth, apoptosis and invasion. Thirdly we will establish the Patient-Derived tumor Xenograft model of ccRCC and test the influence of 5hmC and cholesterol metabolism interference on tumor growth and metastasis. This study will clarify the mechanism of 5hmC loss promoting ccRCC progression by regulating cholesterol metabolism and provide theoretical basis for precision targeting therapy on ccRCC.

肾透明细胞癌是肾癌中最主要的病理亚型，易发生术后复发和转移。肾透明细胞癌以胆固醇等脂质堆积为主要特征，然而其具体机制尚不清楚。本课题组前期研究发现基因组羟甲基化（5hmC）修饰可调节肾透明细胞癌中胆固醇代谢相关基因表达，调节胆固醇含量；而5hmC在肾透明细胞癌组织中缺失，且缺失程度与肿瘤分期分级和不良预后密切相关。以此我们推测，5hmC缺失可能通过调节胆固醇代谢促进肾透明细胞癌进展。本项目拟①整合分析RNA-seq和TAB-seq测序结果并开展分子生物学等实验，鉴定5hmC调节胆固醇代谢的关键靶基因；②干预靶基因表达，检测其对5hmC调节胆固醇代谢和细胞增殖、凋亡和浸润能力的影响；③构建人源化肾透明细胞癌移植瘤模型，检测干预5hmC含量和胆固醇代谢对移植瘤生长转移情况的影响。以此解析5hmC缺失通过调节胆固醇代谢促肾透明细胞癌的具体机制，为肾透明细胞癌的精准靶向治疗提供理论基础。

肾透明细胞癌（clear cell Renal Cell Carcinoma, ccRCC）是肾癌中最常见的病理亚型，具有复杂的生物学特征，对传统放疗和化疗均不敏感，虽然外科疗效确切，但仍有20%~40%的患者术后出现局部复发或远处转移，预后较差。本课题组早期通过多种组学研究发现ccRCC中基因组羟甲基化胞嘧啶（5-hydromethylcytosine, 5hmC）水平明显减少，且5hmC缺失的患者生存期缩短，是患者预后的独立预测因素，然而肾癌5hmC含量下降的机制尚不清楚，阻碍了以5hmC为靶点的肾癌治疗转化研究。为探究肾癌5hmC含量下降的机制，我们通过生物信息学方法分析了5hmC相关通路及代谢过程中的基因集在肾癌组织和细胞中的表达水平，发现TDG在肾癌组织中比正常肾皮质表达增高，且与患者不良预后密切相关。通过构建TDG稳定敲低的肾癌细胞系，发现细胞基因组的5hmC水平含量增加，细胞迁移侵袭能力明显下降，表明TDG可通过降低5hmC含量促进肾透明细癌进展转移。抗坏血酸（维生素C）能够激活TET酶催化5mC转变为5hmC，我们使用抗坏血酸磷酸酯镁（APM）处理肾癌细胞，发现APM能够抑制肾癌细胞的3D成球能力；将APM注射入肾癌骨转移模型小鼠体内能够明显抑制肾癌细胞的骨转移能力，表明抗坏血酸磷酸酯镁可能通过上调细胞内5hmC水平发挥抑制肾癌转移的作用，可能是未来治疗晚期肾癌的潜在药物。