介导结肠癌5-Fu耐药的外泌体miRNA标志物的筛选鉴定

5-Fu-based chemotherapy is the first-line option for patients with advanced colon cancer, but the emergence of drug resistance is the main cause of its treatment failure. Our preliminary results showed that the expression of multiple miRNAs were significantly lower in colon cancer 5-Fu resistant cell lines than the corresponding sensitive cells, both at the exosome and cell levels. Adding exosomes from 5-Fu resistant colon cancer cells or transfecting specific miRNA inhibitors can significantly enhance the 5-Fu resistance of sensitive cells, suggesting that exosomes carring some miRNA may be involved in the formation of 5-Fu resistance in human colon cancer. Therefore, in this study, using 5-Fu resistance and sensitive colon cancer cell lines, the exosome miRNA microarray, nucleic acid transfection, clone formation assay and western blot will be applied to study the mechanism of exosome carrying miRNA regulating 5-Fu resistance in colon cancer cells. Meantime, the expression level of related miRNA in serum exosomes will be determined in clinical samples of human colon cancer, and their correlation with the prognosis of 5-Fu treatment patients with colon cancer will be analyzed. Screening for molecular markers that can effectively predict 5-Fu resistance in colon cancer will provide a new target for clinical treatment of colon cancer.

5-Fu为基础的化疗是晚期结肠癌患者的一线方案，但耐药性的产生是导致其治疗失败的主要原因。我们的前期研究结果表明，在结肠癌5-Fu耐药细胞株及其外泌体中，多种miRNA的表达均明显低于相应的敏感株；加入来自结肠癌5-Fu耐药株的外泌体或转染相应miRNA的抑制剂均可明显增强敏感株5-Fu抗性，由此推测外泌体携带某些miRNA可能参与人结肠癌5-Fu耐药的形成。因此，本研究将在此基础上，以5-Fu耐药性明显差异的结肠癌细胞系作为对象，通过外泌体miRNA芯片、核酸转染、克隆形成、western blot等技术，探求外泌体携带miRNA调控结肠癌细胞5-Fu耐药的作用机制；同时，在人结肠癌临床样本中复核血清外泌体中相关miRNA的表达水平，分析它们与5-Fu治疗结肠癌患者预后的相关性，以期筛选出能有效预测结肠癌5-Fu耐药特征的分子标志物，为结肠癌临床治疗提供新靶点。

5-FU为基础的化疗是晚期结肠癌患者的一线方案，但耐药性的产生是导致其化疗失败的主要原因。筛选结肠癌5-FU耐药特征的分子标志物可预测耐药、提前干预，从而提高结肠癌的临床疗效。在本研究中，我们筛选并验证介导结肠癌5-FU耐药的分子标志物，同时研究其介导耐药的机制。结果表明：（1）加入来自耐药株HCT15/FU的外泌体可明显增强敏感株HCT15的侵袭能力及对5-FU的抗性。（2）对结肠癌5-FU敏感细胞株HCT15及其耐药株HCT15/FU细胞上清外泌体miRNA进行了高通量测序分析，挑选出差异明显的miRNA。结果表明，在耐药细胞上清外泌体中，多种miRNA的表达均明显低于相应的敏感株。（3）从5-FU敏感结肠癌病人及5-FU耐药结肠癌病人的血清中分离其外泌体，测定两组病人miRNA差异表达情况。通过对细胞上清外泌体miRNA与临床病人血清外泌体miRNA双重筛选验证，最终确定重点研究hsa-miR-224-5p在介导结肠癌5-FU耐药中的作用。（4）经细胞miRNA高通量测序及蛋白组学研究，结果显示，包含hsa-miR-224-5p在内的多种miRNA在HCT15/FU细胞的表达明显下调；包含S100钙结合蛋白A4(S100A4)在内的多种钙相关蛋白在HCT15/FU细胞的表达明显上调。RIP结果显示hsa-miR-224-5p可与S100A4结合。（5）体内、外研究结果表明hsa-miR-224-5p通过调控S100A4参与结肠癌5-FU耐药。敏感株HCT15下调hsa-miR-224-5p表达可增加对5-FU的耐药性；耐药株HCT15/FU上调hsa-miR-224-5p表达可增加对5-FU的敏感性。（6）体内、外研究结果表明5-FU联合钙拮抗剂维拉帕米通过调控hsa-miR-224-5p/S100A4通路逆转结肠癌5-FU耐药。（7）雷公藤甲素通过hsa-miR-224-5p/S100A4通路抑制结肠癌HCT15/FU细胞的恶性进展。我们的研究为临床早期预测结肠癌5-FU耐药及提前干预提供理论基础，有望应用于临床作为诊断结肠癌5-FU耐药的分子标志物。