miR-7受长链非编码RNA FENDRR调控介导胃癌多药耐药的功能与机制研究

Multi-drug resistance is the major cause for the failure of the chemotherapy against gastric cancer. The applicant has conducted a series of studies concerning miR-7 in gastric carcinogenesis and recently found that miR-7 is decreased in gastric cancer multi-drug resistant cell lines and that overexpression of miR-7 in those drug resistant cells could restore the sensitivity to chemotherapy drugs. Through lncRNA microarrays between the gastric drug-resistant cell lines and their parental cell line, we discovered that lncRNA FENDRR is significantly upregulated in gastric cancer multi-drug resistant cell line. Moreover, bioinformatic analysis indicated that FENDRR harbors multiple binding sites for miR-7 and miR-7 expression was increased after FENDRR silencing. In the current project, we aim to clarify the role of miR-7 in gastric cancer multi-drug resistance through in vitro and in vivo functional studies. FENDRR functions as the ceRNA targeting miR-7 will be clarified by luciferase reporter gene assay and UV cross-linking and immunoprecipitation. Additionally, we will screen out the crucial downstream effectors and signaling pathways regulated by FENDRR/miR-7 interaction through cDNA microarray and iTRAQ technique. Through studying the new mode of lncRNA-miRNA interaction, we will deepen the understanding of non-coding RNA regulatory network and provide novel strategy for reversing multi-drug resistance in gastric cancer.

多药耐药是胃癌化疗失败的主要原因，但非编码RNA在其中的调控功能与机制仍不清楚。申请人前期发现miR-7在胃癌耐药细胞中表达降低，上调miR-7能增强胃癌细胞化疗敏感性。我们通过比对胃癌耐药细胞与亲本细胞的lncRNA表达谱，发现FENDRR在耐药细胞中显著增高；生物信息学分析显示其含多个miR-7结合位点，且下调FENDRR能促进miR-7表达。我们推测FENDRR可作为ceRNA，与miR-7共同调控胃癌多药耐药。本研究拟通过体内外功能实验，阐明miR-7和FENDRR在胃癌耐药中的功能；通过萤光素酶报告基因实验和紫外交联免疫共沉淀，阐明miR-7与FENDRR的相互关系；联合转录组和蛋白组学技术，筛选miR-7和FENDRR共同调控的耐药相关效应分子和信号通路。本项目通过研究lncRNA-miRNA相互作用新模式，将深化对非编码RNA调控胃癌耐药机制的认识，为逆转胃癌耐药提供新靶点。

我国胃癌的发病率和死亡率均位居恶性肿瘤前列。除手术外，进展期胃癌的治疗主要依靠化学药物治疗（化疗），而多药耐药的产生是化疗失败最主要的原因。近年来，非编码RNA尤其是微小RNA（miRNA）和长链非编码RNA（lncRNA）在肿瘤恶性行为中的作用逐步受到人们关注。项目负责人前期发现miRNA miR-7在胃癌耐药细胞中表达降低，上调miR-7能增强胃癌细胞化疗敏感性。通过比对胃癌耐药细胞与亲本细胞的lncRNA表达谱，发现FENDRR在耐药细胞中显著增高，生物信息学分析显示其含多个miR-7结合位点，且下调FENDRR能促进miR-7表达，推测FENDRR可能与miR-7共同调控胃癌多药耐药。基于此，本项目着重研究FENDRR在介导胃癌多药耐药中的生物学功能，并探讨其与细胞内基因表达调控分子（转录因子和包括miR-7在内的众多miRNA）的相互作用，进而影响胃癌多药耐药表型的分子机制。通过本项目研究，我们较为系统地阐明了FENDRR的表达模式与功能：（1）FENDRR在耐药细胞表达升高，同时在胃癌耐药PDX模型中表达升高；（2）体外MTT实验、平板克隆形成实验、流式细胞仪检测凋亡和体内裸鼠移植瘤实验证实FENDRR可以促进胃癌多药耐药表型形成。我们还较为完整地揭示了FENDRR的分子机制：（1）一方面，FENDRR在转录水平促进FOXC2的表达，发挥增强子样作用；（2）另一方面，FENDRR作为内源性竞争RNA结合miR-4700-3p（经实验验证并非通过miR-7）。miR-4700-3p对其下游靶分子FOXC2的抑制作用降低，FOXC2蛋白表达增加，促进胃癌的多药耐药表型形成；（3）FENDRR上游还受到FOXL1的转录调控，FENDRR可促进FOXL1表达，从而构成一个正反馈调节环路。综上，通过本项目研究，我们揭示了FOXL1-FENDRR-miR-4700-3p-FOXC2通路在胃癌多药耐药中的功能与机制，深化了对非编码RNA在胃癌多药耐药表型中发挥功能和机制的认识，将为发现逆转胃癌多药耐药新靶点提供理论和实验依据。