维甲酸诱导表达RAI2基因抑制结肠癌干细胞样特性的分子机制研究

The poor outcome of patients with colorectal cancer (CRC) is attributed to recurrence of the disease after curative treatment and the resistance of CRC cells to conventional chemotherapy, which are associated with stem cell-like characteristics of CRC cells. A deep understanding of the underlying mechanisms will be of considerable value to the progress of treatment and prognosis prediction of patients with CRC. RAI2 is a tumor suppressor gene in breast cancer, and we confirmed the tumor suppressor role of RAI2 in CRC, including inhibition of proliferation，induction of apoptosis and suppression of migration and invasion. The loss of RAI2 expression is associated with methylation of promoter region, late TNM stage, lymph node metastasis and poor survival rate. Based on the previous study, we confirmed that RAI2 can interact with CtBP2 and down-regulate the expression of CtBP2 in CRC. It was reported that CtBP2 can promote the activation of Wnt signaling by inhibiting the conformation of degradation complex (Axin-APC-GSK-3β). The Wnt signaling is tied to the stem cell-like properties of cancer cells. It was also shown that re-expression of RAI2 suppresses the activity of Wnt/β-catenin signaling, which has been implicated in the stemness maintenance of cancer cells. Therefore, it is hinted that RAI2 may serve as a tumor suppressor gene via inhibiting Wnt signaling-induced stem cell-like properties of CRC cells. And we hypotheses that RAI2 inhibit the Wnt signaling via reducing the binding of CtBP2 to degradation complex, promoting the degradation of β-catenin and decreasing the nuclear translocation of β-catenin. To test the hypothesis, the effect of ectopic expression or silencing of RAI2 on the stem cell-like phenotypes of CRC cells is detected using sphere formation assay, flow cytometry and in vivo tumorigenicity etc. It will be investigated how RAI2 regulates the activation of β-catenin by molecular biological technology, laser scanning confocal microscopy and co-immunoprecipitation. We will further investigate the role of RAI2 in chemo-sensitivity by cell proliferation assay and Patient-derived tumour xenograft (PDTX) mice model. The main theme of the proposed research is tantamount to elucidate the role of RAI2 in inhibiting stem cell-like characteristics of CRC cells and the underlying mechanisms. This study may demonstrate a better understanding of the role of RAI2 in inhibiting CRC, and favor overcoming recurrence and chemoresistance of CRC by novel therapy based on RAI2 protein.

肿瘤复发和耐药是结肠癌患者预后差的主要原因，肿瘤干细胞样特性与此密切相关。前期研究提示RAI2基因在结肠癌中扮演抑癌基因的作用，能下调与干性紧密相关的Wnt通路的活性。CtBP2可参与抑制β-catenin降解复合体的形成，从而促进Wnt通路的活性。基于文献报道和前期实验验证，我们发现RAI2可与CtBP2相互作用，并可下调其表达。本项目将通过慢病毒过表达、shRNA敲降/CRISPR基因敲除技术、流式细胞术、成瘤实验及球体形成实验等研究RAI2对结直肠癌干性相关表型的影响，应用激光扫描共聚焦及免疫共沉淀等方法探讨RAI2抑制结肠癌Wnt通路的机制，我们推测结肠癌中RAI2可能通过抑制CtBP2基因转录或与CtBP2竞争结合等途径促进β-catenin的降解，进而抑制Wnt通路以及结肠癌干细胞样特性。利用细胞增殖实验、PDTX人源肿瘤模型探讨RAI2对奥沙利铂、氟尿嘧啶等药物敏感性的影响。

背景：Wnt/β-catenin信号通路的异常激活在肠癌干细胞样特性的维持中起着重要作用。我们之前的研究中发现维甲酸诱导表达基因RAI2（Retinoic acid-induced 2）在肠癌中扮演抑癌基因的角色，在新的研究中，我们进一步研究了RAI2在 Wnt信号通路中的重要作用及对肠癌肿瘤干细胞样特性、化疗敏感性等的影响。.方法:作为一个转录共调控因子，CtBP2 (C-terminal Binding Protein 2)被报道可通过多种复杂方式参与调控 Wnt号通路。在本研究中，我们利用TCGA数据库中的RNA测序数据分析了RAI2和CtBP2在肠癌中的表达相关性，通过免疫共沉淀方法（Co-IP）分析了二者间的相互作用。通过双荧光素酶报告基因检测系统和免疫荧光染色分别探索RAI2对Wnt信号通路和β-catenin的定位的影响。利用Western blot方法检测RAI2、CtBP2、Wnt信号通路下游基因的表达。球体形成实验用于检测RAI2对肠癌细胞干细胞样特性的影响；细胞活性检测实验用于检测RAI2对化疗敏感性的影响。.结果：RAI2与CtBP2间的相互作用通过Co-IP实验得到证实，另外，我们通过TCGA数据分析发现RAI2与CtBP2的表达间存在负相关，在肠癌细胞LoVo和HCT116中恢复RAI2表达后可抑制Wnt信号通路活性，促进β-catenin磷酸化，抑制β-catenin核转位，同时可抑制Wnt下游靶分子的表达，例如c-Myc, CyclinD1, ASCL2和LGR5，相比之下，将RAI2与CtBP2相结合的位点突变后，上述作用却会消失。我们在肠癌组织中发现RAI2在 33.89% (101/298)的肠癌组织中表现为低表达或表达缺失，与β-catenin在这些组织中的磷酸化水平呈正相关（r=0.8866, P＜0.0001）。除此，RAI2的低表达被发现与较差5年OS（P = 0.0102）和RFS（P = 0.0029）相关。在LoVo和HCT116肠癌细胞中恢复RAI2表达可提高肠癌细胞对氟尿嘧啶和奥沙利铂的化疗敏感性。.结论：RAI2表达状态可作为独立的肠癌预后预测指标，RAI2可通过与CtBP2相互作用或者下调CtBP2表达抑制Wnt信号通路活性，抑制肠癌细胞干细胞样特性，提高肠癌细胞对化疗药物的敏感性。

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