RNA结合蛋白Musashi2增强CDC42mRNA稳定性促进EMT和结肠癌转移的机制研究

RNA binding protein Musashi2 is closely related to the progression of colon cancer, however, its molecular mechanism for promoting metastasis has not yet been reported. Our previous study showed increased expression of Musashi2 in stage IV colon cancer tissue and liver metastases. Transcriptome sequencing found that Musashi2 could regulate the expression of CDC42 in the signaling pathway of actin cytoskeleton. Moreover, bioinformatics analysis showed that RNA binding protein Musashi2 could combine with CDC42 mRNA 3' untranslated region. Cell experiments showed that TGF beta could increase the expression of Musashi2. Similarly, knockdown of Musashi2 gene could reduce the expression of CDC42 mRNA level. Therefore, we put forward the hypothesis that TGF beta increases the expression of Musashi2 in colon cancer cells. The combination of Musashi2 and CDC42 mRNA 3' untranslated region would enhance the mRNA stability of CDC42. Conceivably, high expression of CDC42 could promote the formation of filopodia in cancer cells and improve its invasiveness, which eventually leads to epithelial-mesenchymal transition and metastasis of colon cancer. This study will adopt phalloidin staining, RNA immunoprecipitation, dual luciferase reporter and other experiments to elucidate the regulation mechanism of TGF beta -Musashi2-CDC42 signal axis in the epithelial-mesenchymal transition and metastasis of colon cancer from three aspects of clinical study, cell and animal experiment. This study is expected to provide important theoretical and experimental basis for inhibiting the metastasis of colon cancer.

RNA结合蛋白Musashi2与结肠癌进展密切相关，其促进转移的分子机制尚未见报道。我们前期研究证实Musashi2在IV期结肠癌和肝转移灶中高表达；预实验转录组测序发现此蛋白可调控细胞骨架通路CDC42基因的表达；生信分析显示此蛋白可与CDC42 mRNA 3’UTR结合；细胞实验发现TGFβ可促进结肠癌细胞Musashi2上调，敲降Musashi2可下调CDC42 mRNA水平。为此，我们提出假说:TGFβ上调结肠癌细胞Musashi2表达并促进此蛋白与CDC42 mRNA结合以增强其稳定性，高表达CDC42可促进癌细胞丝状伪足形成并提高其侵袭能力，最终导致EMT和结肠癌转移。本研究拟采用鬼笔环肽染色、RNA-IP、双荧光素酶报告等实验，从细胞、动物和临床三水平阐明TGFβ-Musashi2-CDC42信号轴在EMT和结肠癌转移中的调控机制，为靶向阻断结肠癌转移提供新的理论和实验依据。

RNA结合蛋白Musashi2的表达与结肠癌不良预后密切相关，而具体的分子机制未见报道。. 本项目主要通过构建稳定上调结肠癌SW480细胞和下调HCT116细胞Musashi2基因表达的体外细胞模型，检测外源性干预Musashi2的表达对SW480和HCT116细胞体外增殖、细胞形态变化、迁移和侵袭能力的影响，并在构建好的稳定上调Musashi2的SW480体外细胞模型中进一步敲降CDC42基因表达，通过Rescue实验证实CDC42是Musashi2影响结肠癌EMT和转移能力的主要调控分子，最后构建TGF-β诱导的结直肠癌细胞EMT模型，进一步全面阐明了TGFβ -Musashi2-CDC42信号轴在EMT和结直肠癌转移过程中对下游靶标基因和效应通路的影响。此外，通过小鼠皮下荷瘤实验并辅以免疫组化也证明了Musashi2、CDC42基因表达对结直肠癌细胞致瘤性和转移能力的影响。. 该研究完善了Musashi2的下游调控网络的干预靶点，拓宽了相关理论认识并为阻断结肠癌侵袭和转移提供实验依据，也为阻断TGF-β参与EMT和结肠癌转移过程提供新的治疗靶点，最终有望为实现结肠癌患者的临床治愈提供新的思路和理论依据。