E2F3b/miR-200b负反馈通路参与调控人肺腺癌细胞化疗耐药的分子机制研究

MiRNAs are important epigenetic regulators which negatively regulate gene expression in the post-transcriptional level. Recent studies have highlighted the feedback loop between miRNAs and their target genes in physiological and pathological processes including chemoresistance of cancer cells. Our previous study indicated that the miR-200b/E2F3 axis was closely associated with chemosensitivity of lung adenocarcinoma cells. Moreover, E2F3b was a potential transcriptional regulator on pre-miR-200b gene promoter, suggesting a negative feedback minicircuitry comprising E2F3b and miR-200b. On the previous basis of establishment of docetaxel-resistant lung adenocarcinoma cell line and bioinformational and functional analysis of miR-200b/E2F3 axis, the aim of this study is to investigate the molecular mechanisms of the negative feedback minicircuitry comprising E2F3b and miR-200b by luciferase reporter gene, gel retardation, chromatin immunoprecipitation, site-specific mutation and RNA interference assays, and to illustrate the association of this feedback loop with chemoresistance of lung adenocarcinoma cells. It will provide new insight into clinical reversion of chemoresistance of lung adenocarcinoma.

MiRNA是表观遗传调控体系的重要组分，可在转录后水平负向调控基因表达。近期研究表明miRNA及其靶基因可形成功能反馈性调控通路，广泛参与肿瘤发生发展和耐药形成。前期研究发现：miR-200b/E2F3功能轴表达紊乱与肺腺癌耐药表型形成密切相关，并发现 E2F3b可能对miR-200b产生负向调控。本课题在前期成功建立耐多西他赛人肺腺癌细胞模型、miRNA-200b生物信息学及功能学分析的基础上，拟采用荧光素酶报告基因、凝胶阻滞、染色质免疫共沉淀、位点突变、基因干涉及过表达等实验方法，分析肺腺癌耐药细胞中E2F3b与miR-200b间的负反馈调控关系，并通过体外细胞和活体动物模型分析E2F3b/miR-200b负反馈通路参与调控肺腺癌细胞化疗敏感性的可能机制，为临床逆转肺腺癌耐药提供新的策略。

miRNA是表观遗传调控体系的重要组分，可在转录后水平负向调控基因表达，与肿瘤发生发展和耐药形成密切相关。前期研究发现miR-200b/E2F3功能轴与肺腺癌细胞耐药形成密切相关，而 E2F3b本身作为抑制性转录因子，可能与miR-200b构成双向负反馈通路，共同调控肺腺癌化疗敏感性。通过荧光素酶活性测定，发现miR-200b P2启动子核心区域位于-200～＋34处。进一步生物信息学分析miR-200b核心启动子序列，发现-133～-126处存在有一个潜在的E2F3结合位点（5'-TTTC[A]CGC-3'）。选取两株肺腺癌细胞SPC-A1、H1299及其各自的多西他赛耐药细胞SPC-A1/DTX、H1299/DTX，使用基因干涉及过表达技术，利用染色质免疫共沉淀、荧光素酶报告基因测定启动子活性，证实E2F3b可直接结合于miR-200b基因启动子并负向调控其表达。MTT实验证实E2F3b/miR-200b轴与肺腺癌细胞多西他赛敏感性密切相关；克隆形成实验及流式细胞分析表明，E2F3b可通过影响细胞增殖、凋亡及细胞周期分布负向调控肺腺癌细胞多西他赛化疗敏感性。进一步拯救实验表明，E2F3b通过直接结合并负调控miR-200b基因表达，促进细胞增殖、抑制细胞凋亡、减少细胞周期中G2/M相分布比例，进而对肺腺癌细胞多西他赛化疗敏感性产生负调控作用。最后，通过构建裸鼠皮下移植瘤模型，证实E2F3b/miR-200b负反馈通路同样参与调控了肺腺癌体内化疗敏感性。由此得出结论：肺腺癌中存在E2F3b/miR-200b构成的双向负反馈调控通路，通过影响细胞增殖、凋亡及细胞周期分布等多个途径，在体内外负向调控肺腺癌细胞化疗敏感性。本研究深入分析E2F3b与miR-200b之间的交互调控关系，不仅进一步丰富了肺腺癌化疗耐药表型形成过程中miR-200b的分子调控信号网络，还有望为临床逆转肺腺癌耐药提供新的线索和理论依据。