血清miRNA预测结直肠癌奥沙利铂耐药的价值及相关机制

Oxaliplatin-based chemotherapy regimen is the main treatment for advanced colorectal carcinoma (CRC), however, CRC patients initially respond to oxaliplatin-based chemotherapy ultimately will develop progressive disease because of drug resistance. Therefore, how to select the potentially responsive population to provide individualized therapy and effectively reverse drug resistance to oxaliplatin is crucial for the improvement of clinical therapeutic efficacy. Our previous study identified a group of serum miRNAs which is closely related with chemosensitivity to oxaliplatin, moreover, we found that miR-661 was involved in the drug resistance to oxaliplatin by regulation of excision repair cross-complementation group 1 (ERCC1). In order to further understand the mechanism of drug resistance to oxaliplatin and the function of related miRNAs, our project will increase sample sizes to validate these serum miRNAs, and then to obtain a group of serum miRNA which is related with oxaliplatin therapeutic efficacy but differentially expressed between patients who were identified as chemosensitive and chemoresistant. Subsequently, with clinical sample detection and in vivo and in vitro experiment, we will investigate if miR-661 could induce oxaliplatin resistance by regulation of ERCC1 to inhibit the covalent cross-linking between oxaliplatin and DNA. And further to explore effective method for reversing drug resistance. Taken together, with this project we would establish the bimarkers and predictive model for oxaliplatin treatment responsive population, and to elucidate the mechanism of miRNA involved in resistance to oxaliplatin, hopefully to find an effective approach to regulate the process, and therefore provide the theoretical base for the improvement of advanced colorectal carcinoma treatment.

奥沙利铂为主的化疗方案是晚期结直肠癌的主要治疗手段，但如何筛选优势人群给予个体化治疗，并通过对耐药机制的深入研究有效逆转耐药，对于提高其临床治疗效果具有重要意义。我们在前期工作中筛选出与奥沙利铂敏感性相关的一组血清miRNA，并发现其中miR-661通过影响切除修复交叉互补基因（ERCC1）参与了奥沙利铂耐药的形成。本项目拟扩大样本量验证前期所筛选的血清miRNA，得到一组稳定差异表达的结直肠癌奥沙利铂化疗疗效相关的血清miRNA；并通过临床标本检测以及体内外实验，验证miR-661在奥沙利铂耐药中的作用及其机制，进一步探索有效的调控手段以逆转耐药。通过本项目的研究，能够建立奥沙利铂治疗优势人群筛选的分子标志物和预测模型，并阐明miRNA参与奥沙利铂耐药的作用机制，建立有效的调控手段，为提高晚期结直肠的治疗效果奠定理论基础。

结直肠癌的发病率高，预后差；晚期结直肠癌患者多数接受奥沙利铂或伊利替康等药物为主的化疗方案。但临床治疗中，部分病人表现出对某种化疗方案的耐药性或反应不敏感。因此，如何在对患者实施化疗前选择有益的治疗方案，并且寻找可以预测结直肠癌化疗耐药的特异性血清标志物就成了亟待解决的临床问题。微小核糖核酸（microRNA）是长度在22 bp左右的微小非编码RNA，近年来，血清microRNA被发现在多种肿瘤中表现出不同的特征性的表达谱，被认为是肿瘤诊断的新型标志物。本课题着眼于血清microRNA在预测结直肠癌患者奥沙利铂耐药中的潜在价值和应用，寻找可以用于指导临床化疗方案选择的血清标志物，并将探讨这些特异性的microRNA在介导奥沙利铂耐药中的分子机制。本研究收集IV期结直肠癌患者的化疗前血清，记录患者化疗效果，通过采用高通量测序和定量PCR验证的方法，筛选出来一组在结直肠癌奥沙利铂耐药和伊利替康耐药病人中特异的血清microRNA。其中血清miR-135b和miR-208b在奥沙利铂耐药的病人中增高显著；而血清miR-135b，miR-10a和miR-627在伊利替康耐药的病人中明显升高。且两组miRNA均表现出较好的灵敏度和特异度。在对奥沙利铂化疗敏感的病人中，血清miR-135b和miR-208b在化疗后普遍下降；而在奥沙利铂耐药的病人中，miR-135b/208b在化疗后均表现出一定水平的增高。对伊利替康方案的研究也得出类似的结果。高水平的miR-135b/208b也被发现与患者的较差的无进展生存期（PFS）密切相关。实验发现miR-135b通过直接靶向TGFR2基因，负向调控TGFR2的蛋白表达，减少了肿瘤细胞的凋亡。而结肠癌奥沙利铂耐药细胞产生的外泌体中富集了miR-208b，这些miR-208b被外泌体运输到T细胞，抑制T细胞中PDCD4基因的表达，诱导T细胞分化为Tregs。因此，外泌体miR-208b可以介导结肠癌细胞躲避免疫监视，产生免疫逃逸。本课题为预测结肠癌患者奥沙利铂和伊利替康耐药提供了潜在的血清miRNA标志物，并证明了这组miRNA在减少癌细胞凋亡，诱导癌细胞免疫逃逸中的分子机制，具有重要的临床意义。