miR-19a下调THBS1促进结肠癌转移的作用及机制研究

The role of MiR-17-92 cluster in tumor angiogenesis was very important. In recent years, some researches showed that they were associated with lymphatic metastasis of malignant tumor, but the mechanisms have not be found. Some scholars put forward that their target is THBS1, but it has not been confirmed. Calculated with TargetScan, there is a conserved site for miR-19 in THBS1 sequence, which is a member of MiR-17-92. It's subtype miR-19a is in high expression in colon cancer. Our researches showed that miR-19a expression was in great relation with lymph node metastasis in colorectal cancer, and co-immunoprecipitation of THBS1 and VEGF-C were positive in colon cancer cell lines. To sum up, we speculate that there is a mechanism in colon cancer: miR-19a can downregulate THBS1 expression to promote tumor angiogenesis and Lymphangiogenesis. For this reason, we plan to prove the regulating function of miR-19a on THBS1 with gene-expression control experiment and luciferase Assay, to prove binding reaction of THBS1 and VEGF-C with Co-Immunoprecipitation and fluorescence resonance energy transfer(FRET), and to prove this regulating pathway at last to reveal the mechanism of neoplasm metastasis by blood-vessel and lymph-vessel.

miR-17-92家族在肿瘤血管生成中发挥了重要作用，而且近年来有研究表明其与肿瘤淋巴转移相关，但其具体作用机制尚未阐明。有研究报道THBS1是其作用靶点。查询TargetScan发现THBS1与此家族中的miR-19存在保守结合位点，其亚型miR-19a在结肠癌组织中呈高表达。我们前期研究发现miR-19a与结肠癌淋巴结转移密切相关，且在结肠癌细胞株中THBS1与VEGF-C的免疫共沉淀呈阳性。综上所述，我们推测，结肠癌中可能存在miR-19a通过抑制THBS1的表达而促进肿瘤的血管及淋巴管生成。为此，我们拟用表达调控实验和双荧光素酶实验验证miR-19a对THBS1的调控，用免疫共沉淀实验和荧光共振能量传递技术验证THBS1与VEGF-C的结合反应，最终证明这条调控途径，进一步揭示肿瘤血管和淋巴转移的机制。

目的 通过对结肠癌组织和细胞株中miR-19a表达情况及其对THBS1调控作用的研究，揭示miR-19a下调THBS1促进结肠癌转移的作用以及机制。 方法 通过对LoVo, SW480和 HT29等细胞株中miR-19a表达情况的检测，筛选表达最强的细胞株，构建并转入miR-19a的siRNA质粒载体，检测转入前后该细胞株中THBS1和VEGF的 mRNA表达及蛋白表达情况、增殖转移能力的变化，并用双荧光素酶实验和双荧光素酶突变实验验证miR-19a 与THBS1的结合与调控作用，最后用免疫共沉淀实验验证THBS1与VEGF的结合反应。 结果 结肠癌组织中，miR-19a表达明显升高，并且在淋巴结转移组中的表达明显高于无淋巴结转移组。结肠癌细胞株中，miR-19a表达程度由高到低为LoVo，SW480和HT29 ，这与结肠癌细胞株的恶性程度一致。抑制LoVo细胞中miR-19a的表达后，THBS1的表达明显升高，细胞的增殖转移能力均明显下降。 结论 结肠癌细胞中， miR-19a的表达与细胞的增殖转移能力密切相关，并且通过下调THBS1表达，提升细胞中VEGF的蛋白水平，促进结肠癌细胞增殖、转移。