miR-873的下调促进结直肠癌细胞增殖的分子机制

Previously, we demonstrated lipid raft protein FLOT1 promoted the aggressiveness in esophageal squamous cell carcinoma (Gastroenterology, 2012, 143 (4), 995-1005). However, the expression and regulatory mechanism of FLOT1 remain largely unclear in colorectal cancer. We identified that miR-873 was significantly downregulated in colorectal cancer tissues compared to normal colorectal tissues, Using a microRNA chip assay. Real-time PCR results demonstrated that miR-873 expression inversely correlated with the clinical stage of colorectal cancer. Furthermore, our recent preliminary data showed that ectopic expression of miR-873 inhibited the proliferation of colorectal cancer cells via inducing G1/S arrest. Moreover, intergration of the results of bioinformatic analysis and biological experiments revealed that miR-873 regulated FLOT1, FLOT2 and STOML2 by directly targeting its 3` untranslated region (3`-UTR) and inactivates of NF-κB and EGFR pathway signalling. Additionally, we found that the oncogene c-myc could repress the expression of miR-873. Taken together, these results indicate that miR-873 might be a key factor in the prevention of colorectal cancer tumorigenesis. Herein, in the current project, we aim to employ immunofluorescence, chromatin immunoprecipitation (ChIP) and luciferase reporter assays in both in vivo and in vitro systems, and use clinical samples to further investigate the mechanism by which downregulation of miR-873 activates NF-κB and EGFR pathway and promotes the proliferation of colorectal cancer cells. This work may identify novel targets for the diagnosis and treatment of colorectal cancer.

我们前期已发表文章证明脂筏蛋白FLOT1促进食管鳞状细胞癌的发展，但FLOT1在结直肠癌中的表达及调控机制仍不清楚。通过分析microRNA芯片，我们发现miR-873在结直肠癌组织中表达下调，且表达水平与结直肠癌恶性程度呈负相关。预实验结果显示高表达miR-873可导致结直肠癌细胞G1/S停滞并抑制细胞增殖；高表达miR-873下调脂筏蛋白FLOT1、FLOT2和STOML2的表达，抑制NF-κB和EGFR信号通路；而c-Myc可下调miR-873。本项目承前启后，将以高表达或抑制miR-873表达的细胞为模型，通过免疫荧光、染色体免疫共沉淀及荧光素酶报告基因分析等方法，研究miR-873的下调导致脂筏蛋白上调，激活NF-κB和EGFR信号通路的分子机制；通过体内体外系统，并结合临床样品，探讨miR-873的下调促结直肠癌细胞增殖的分子机制，为结直肠癌的诊治提供新的靶标。

结直肠癌是常见的消化系统恶性肿瘤之一，其全球发病率居恶性肿瘤的第三位。世界上每年结直肠癌新增病例达100万，且每年约有50万人死于结直肠癌。MicroRNA-873 (miR-873)被认为在多种恶性肿瘤中表达失调，然而，其在结直肠癌中的生物学功能和分子机制尚不清楚。本项目旨在研究miR-873的下调促结直肠癌细胞增殖的生物学功能及分子机制。首先，我们应用Real-time PCR技术检测miR-873在结直肠癌细胞系和结直肠癌患者组织中的表达情况，发现miR-873在结直肠癌细胞系和患者组织中均表达下调。我们采用MTT和克隆形成实验研究miR-873在结直肠癌细胞增殖方面的生物学功能，结果显示miR-873可以抑制结直肠癌细胞的增殖。运用TargetScan、Pictar和miRANDA软件分析预测，我们发现FLOT1、FLOT2、STOML2、TRAF5和TAB1 mRNA 3'UTR区域均具有miR-873的结合位点。Western blotting实验和荧光素酶双荧光报告系统实验结果显示miR-873可以靶向作用于FLOT1、FLOT2、STOML2、TRAF5和TAB1。我们应用荧光素酶双荧光报告系统和Real-time PCR技术进一步研究miR-873的下调促结直肠癌细胞增殖的分子机制，结果表明miR-873可以抑制NF-κB信号通路的激活。此外，我们检测了125例结直肠癌患者癌组织中miR-873的表达情况，统计学分析结果显示miR-873的表达水平与结直肠癌Dukes'分期、临床分期和病理分级密切相关(P=0.003，P=0.003，P=0.014)。Kaplan-Meier生存曲线分析结果显示miR-873高表达组的结直肠癌患者总体生存预后显著好于miR-873低表达组患者(P<0.001)。单因素和多因素Cox比例风险模型的预后分析结果提示miR-873可作为判断结直肠癌患者生存预后的独立风险因素。最后，我们在结直肠癌临床样本中，发现miR-873表达水平与细胞增殖的标志性分子Ki67呈显著负相关（r=-0.785，P=0.007），进一步表明miR-873的表达水平与结直肠癌细胞增殖能力密切相关。综合全文，我们的研究阐明了miR-873的下调促进结直肠癌细胞增殖的生物学功能及分子机制，为结直肠癌的预后评估及分子靶向药物的研发提供了新的靶点。