中国人群中胃癌患者5-Fu化疗敏感性相关miRNA的研究

Gastric cancer ranks as the highest leading cause of cancer death in China, especially in north- western China. Chemotherapy remains the primary treatment for advanced gastric cancer. However, drug resistance remains a major clinical obstacle to successful treatment. Better understanding of the drug sensitivity mechanisms is needed in order to improve the current chemotherapy regimens. Recently, increasing studies are revealing an important role of miRNAs in anticancer drug sensitivity. So, exploiting the emerging knowledge of miRNAs for understanding the mechanism of drug sensitivity and development of new human therapeutic applications for overcoming anticancer drug resistance will be important. Our plan is to carry out a study of candidate gene association in combination with functional analysis based on the results of microRNA microarray analysis. Firstly, differentially expressed miRNA which involve in 5-fluorouracil resistance phenotype will be verified in clinic samples by RT-PCR and a prediction system of 5-fluorouracil response on gastric cancer specimens will be established. Then, the miRSNPs/polymorphisms will be selected with the Bioinformatics methods. The miRSNPs/polymorphisms will be tested in clinic samples of gastric cancer patients treated with 5-fluorouracil followed by functional test and further identification. And a prediction system of 5-fluorouracil response on gastric cancer specimens will be established based on miRSNPs/polymorphisms. At last, above two systems will be validated in clinic sample. These results will help us better understand the mechanism of 5-fluorouracil resistance and also be of great help to predict the sensitivity and efficacy in the gastric cancer patients with 5-fluorouracil therapy and may be helpful to a certain extent for evaluating the prognosis of advanced gastrointestinal cancer patients who received 5-fluorouracil therapy.

胃癌在我国西北地区属于高发的恶性肿瘤，化疗是晚期胃癌患者的主要治疗方式，但化疗耐药仍然是制约临床治疗的关键。目前许多证据揭示miRNAs与化疗敏感性有关。本项目拟采用基于microRNA芯片结果分析的候选miRNA基因序列多态关联研究策略与功能研究结合的方法进行研究。首先对microRNA芯片结果筛选、验证，并确定相关microRNA组合，建立基于microRNA生物标志物的胃癌化疗个体化用药检测体系；然后利用生物信息学方法筛选出差异表达microRNA基因序列中的多态位点，采用两步病例-对照的研究方法，在大规模胃癌化疗临床样本中进行敏感性关联分析，对阳性位点进行功能性分析及鉴定，建立基于miRNA多态性的胃癌化疗个体化用药检测体系；最后，对上述两个检测体系进行临床有效性评价。从而为了解miRNA自身受到调控的因素，探讨胃癌化疗敏感性的分子机理，指导临床合理化用药奠定研究基础。

目前许多证据揭示miRNAs在抗癌药物耐药性中扮演着重要角色。本项目采用基于microRNA芯片结果分析的候选miRNA基因序列多态关联研究策略与功能研究结合的方法进行研究。首先针对胃癌氟尿嘧啶耐药细胞株利用芯片共筛选出29个差异miRNA。应用qRT-PCR验证差异miRNA中的hsa-miR-125b、hsa-miR-125a、hsa-miR-100、hsa-miR-130a、hsa-miR-221、hsa-miR-194、hsa-miR-192 ；应用生物信息学分析预测miR-125b的靶基因有ERBB2、ERBB3。进一步双荧光素酶报告基因实验证实miR-125b分别与ERBB2、ERBB3基因的3'UTR区有直接的靶向结合作用。在胃癌耐药细胞株中，过表达miR-125b可以使胃癌耐药细胞对5-Fu的药物敏感性显著增强(P<0.05)；转染miR-125b inhibitors，耐药细胞内miR-125b的表达进一步下调，耐药性增强。针对上述研究中得到的7个候选miR-125b-5p、miR-125a-5p、miR-100-5p、miR-130a、miR-221-3p、miR192-5p和miR-194-5p，确立了重要的功能SNP和指标组合群，选择了ERBB3基因rs1810132、rs2271194 ，BMPR1B rs1434536等29个位点在临床样本中进行验证。研究发现ERBB2 rs1810132等5个位点的等位基因频率分布在敏感组和耐药组之间存在显著性差异，有统计学意义。同时，还发现对于使用FOLFOX化疗方案的患者ERBB2基因rs1810132位点的等位基因频率分布在敏感组和耐药组之间有显著差异，而ERBB3基因rs2271194位点的等位基因频率分布无显著差异。上述结果有望成为临床区分敏感患者和耐受患者的参考指标之一，有助于指导个体化用药。