Retinoid X Receptor(RXR)α启动子甲基化在结直肠癌发生发展中的作用

Cancer has been traditionally considered a genetic and cytogenetic disease, but recent years have brought epigenetics to the forefront of cancer research. Altered DNA methylation is nowadays considered a hallmark of neoplasia, including two different phenomena in cancer cells: global hypomethylation and CpG-promoter hypermethylation of tumor suppressor genes.The importance of epigenetic alterations in cancer is further highlighted by their use in diagnosis and by the development of new therapeutic strategies aiming at correcting them.Our previous study showed that expression of retinoid X receptor alpha (RXRα) was down-regulated in colorectal carcinoma (CRC) tissues. RXRα could directly interact with β-catenin and suppressed β-catenin expression. Moreover, the expression of RXRα was closely associated with the histological differentiation degree and TNM stages of CRC. Epigenetic inactivation of RXRα in non-small cell lung cancer which also down-regulated RXRα expression has been reported. It suggests that methylation-associated downregulation of RXRα may play a key role in carcinogenesis and progression of CRC. However, the mechanism of methylation suppressing the expression of gene is unclear. Methyl-CpG-binding domain(MBD) proteins selectively bind to methylated DNA and recruit chromatin remodelling and transcriptional repressor complexes, thereby establishing a repressive chromatin state.MBD2 which is a member of the MBD protein family,binds to methylated promotor CpG islands and acts as a methylation-dependent transcriptional repressor.Taken together,we make a hypothesis that (1)CpG-promotor hypermethylation of RXRα can be observed in CRC.(2）β-catenin may escape the degradation from RXRαby recruiting MBD2 ,which could bind to CpG-promotor hypermethylation of RXRα and further suppress the expression of RXRα；（3）RXRα expression downregulation and β-catenin abnormal upregulation associates with carcinogenesis and progression of CRC. Our current project is to detect CpG-promoter hypermethylation of RXRαin CRC cell lines and tissues, and to study MBD2 expression and its interacting with hypermethylated CpG island of RXRα by upregulating/down-regulating β-catenin expression in CRC cell lines. Our objective is to clarify the role of RXRα promotor hypermethylation in carcinogenesis and progression of CRC.

肿瘤表观遗传学是目前肿瘤研究的热点。我们前期研究发现，在结直肠癌组织中RXRαmRNA和蛋白均呈低表达，并与结直肠癌的组织分化程度和临床分期相关，同时发现RXRα可抑制β-catenin的表达。但RXRα低表达的机制未明。有报道在非小细胞肺癌中RXRα存在甲基化导致其低表达。甲基化CpG结合蛋白2（MBD2）与启动子甲基化的CpG岛序列直接结合，抑制甲基化序列转录并下调基因的表达。我们提出假设：结直肠癌中存在RXRα启动子甲基化,使RXRα对β-catenin的负向调控作用降低，β-catenin募集MBD2进一步抑制RXRα转录，上调β-catenin表达促进结直肠癌的发生发展。本项目拟通过检测结直肠癌细胞和组织中RXRα启动子甲基化，进而高/低表达β-catenin检测结直肠癌细胞中MBD2表达及其与甲基化的RXRα的CpG岛结合情况，以探讨RXRα甲基化在结直肠癌发生发展中的作用。

项目背景：结直肠癌是全球常见的恶性肿瘤之一。结直肠癌的发病年龄多在40-60岁，高峰在50岁左右。结直肠癌的预后主要与临床TNM分期密切相关，部分病人在确诊时就已经发生了远处转移。尽管随着放疗、化疗、新辅助化疗和外科手术的发展，使得大肠癌的生存期有了明显提高，遗憾的是，一些特异性的靶向药物只对部分病人有效，还有较高的耐药率。目前结直肠癌患者的5年总生存率很低，且复发率比较高。.主要研究内容：我们前期研究发现，在结直肠癌组织中RXRαmRNA和蛋白均呈低表达，并与结直肠癌的组织分化程度和临床分期相关，同时发现RXRα可抑制β-catenin的表达。但RXRα低表达的机制未明。本项目通过检测结直肠癌细胞和组织中RXRα的甲基化状态、在分析RXRα甲基化状态对结直肠癌细胞生物学行为影响的基础上以探讨RXRα甲基化在结直肠癌发生发展中作用的分子机制。.重要结果及关键数据：1、完成对结直肠癌细胞株SW480、HCT116、HT29、SW620、LOVO和正常结肠黏膜上皮细胞NCM460中RXRα的mRNA和蛋白质表达水平的检测，发现与正常结肠黏膜上皮细胞相比，RXRα的mRNA和蛋白质表达水平在结肠癌细胞株中是高低不一的。2、在所有标本中均可检测到RXRα的启动子的甲基化，但水平高低不一。RXRα启动子高甲基化的结直肠癌细胞其RXRα的表达是下调的，包括mRNA和蛋白质表达水平。3、使用去甲基化药物处理结直肠癌细胞后，结直肠癌细胞的高甲基化状态能得到逆转且能观察到其RXRα的表达发生相应的变化。4、明确了RXRα表达及其启动子甲基化对结直肠癌细胞生物学行为的影响。5、完成β-catenin在结直肠癌中的表达的研究，并阐明其与RXRα的关系。5、明确不同甲基化程度的结直肠癌病人的预后不同。.科学意义：RXRα在结直肠癌的发生发展中起关键作用并与 Wnt/β-catenin信号通路存在相互关系。探索这两者之间的关系对结直肠癌早期发现、早期诊断、新治疗方案确定和判断患者预后的有价值的分子标记物具有重要的理论和临床意义。