miR-27a下调RXRα通过β-catenin参与结直肠癌发生和转移的分子机制

β-catenin signaling pathway plays important roles in carcinogenesis of colorectal cancer(CRC) and the study on molecular mechanism of β-catenin signaling pathway is the hot spot of the current research. Our previous study showed that retinoid X receptor α (RXRα) expression suppression and β-catenin abnormal expression was found in CRC. RXRα knockdown dramatically increased β-catenin expression and it's transcriptional activity in CRC cells. However, the mechanism of RXRα expression suppression in CRC has not been reported in the literature. Our preliminary data showed that miR-27a might target RXRα in CRC. So we creat a hypothesis that miR-27a targeting RXRα and further regulating β-catenin signaling pathway might be crucial in cacinogenesis and progression of CRC. Our current project includes the following contents: 1) the expression characteristics of miR-27a in different stages of CRC; 2)the effect on celluar biological behavior and RXRα expression and β-catenin transcriptional activity and its downstream genes'expression by upregulating or downregulating miR-27a expression in CRC cell lines; 3) the pattern of miR-27a targeting RXRα and regulating β-catenin signaing pathway to clarify the molecular mechanisms of miR-27a/RXRα/β-catenin signaling pathway in carcinogenesis and metastasis of CRC. Our object is to provide the new idea and valuable experiment evidence for illustrating the mechanism of carcinogenesis and progression of CRC and to find a novel target gene for CRC treatment.

申请者前期研究发现结直肠癌中RXRα低表达和β-catenin高表达；下调RXRα表达可明显增加结直肠癌细胞中β-catenin的蛋白表达和转录活性。但RXRα在结直肠癌组织中低表达的机制尚未见报道。申请者通过生物信息学分析和初步实验提示miR-27a可能靶向调控结直肠癌中RXRα表达。本课题拟利用临床结直肠癌组织对miR-27a表达进行动态检测，探讨miR-27a与RXRα表达和结直肠癌发生和侵袭转移的关系；体内外实验分别上调和下调结直肠癌细胞miR-27a表达，检测其对癌细胞生物学行为及RXRα和β-catenin通路活性及下游基因表达的影响；进一步研究miR-27a下调RXRα并调控β-catenin通路的作用模式，探明miR-27a/RXRα/β-catenin信号通路促进结直肠癌发生和转移的分子机制，为阐明结直肠癌发生发展机制提供理论和实验依据，并为结直肠癌基因治疗寻找新的靶点。

本项目为阐明miR-27a下调RXRα通过β-catenin参与结直肠癌发生和转移的分子机制。我们检测结直肠癌组织中miR-27a-3p 表达及其与RXRα表达和结直肠癌发生和侵袭转移的关系；探讨miR-27a-3p 表达对结肠癌细胞生物学行为及RXRα和β-catenin 通路活性及下游基因表达的影响及其分子机制。结果发现：.1）结直肠癌细胞和组织中mir-27a-3p表达明显高于正常结肠粘膜上皮细胞和癌旁结肠粘膜组织；结直肠癌组织中mir-27a-3p和RXRα表达存在明显的负相关性。miR-27a-3p表达与结直肠癌组织分化程度、临床分期和远处转移存在明显相关性。单因素Cox回归分析显示，组织学分化程度、临床分期、N分期、M分期和miR-27a-3p表达水平是结直肠癌患者的独立预后因子。多因素Cox回归分析显示，临床分期和miR-27a-3p表达水平是结直肠癌患者的独立预后因子。.2）上调或下调结肠癌中miR-27a-3p表达可明显促进或抑制结肠癌细胞的增殖、克隆、迁移和侵袭能力，并抑制或促进癌细胞凋亡。体内裸鼠动物实验发现上调结肠癌细胞中miR-27a-3p表达可明显促进结肠癌移植瘤的生长和肺转移。.3）荧光素酶活性实验提示miR-27a-3p通过靶向结合RXRα-mRNA影响RXRα表达。上调SW480细胞中的miR-27a-3p 后，RXRα表达明显下降，Wnt/β-catenin信号通路相关蛋白表达显著上升；下调miR-27a-3p的基础上敲低RXRα后，Wnt/β-catenin信号通路相关蛋白表达上调，提示敲低RXRα可部分逆转HCT116细胞下调miR-27a-3p后引起的RXRα上调和Wnt/β-catenin信号通路相关蛋白的下调。研究结果为阐明miR-27a-3p通过抑制RXRα而激活Wnt/β-catenin通路继而促进结直肠癌的发生发展机制提供了重要实验依据。