长链非编码RNA MALAT-1参与人肺腺癌多药耐药表型形成的分子机制研究

Chemoresistance represents the major obstacle in clinical treatment of lung adenocarcinoma, which results from both genetic and epigenetic dysregulation of tumor multidrug resistance- associated genes. Long non-coding RNAs (lncRNAs) play a key role in the epigenetic, transcriptional and post-transcriptional regulation of physiological and pathological processes. Our previous studies have demonstrated that miR-200b reversed chemoresistance phenotype of human lung adenocarcinoma cells by negatively regulating E2F3. Moreover, LncRNA MALAT-1 was shown to contain 2 potential miR-200b binding sites and its expression level was correlated with that of the E2F3 mRNA. On the basis of the previous LncRNA microarray profiling and biological information analysis, we aim to further investigate the molecular mechanisms via in vitro and in vivo models by which MALAT-1 upregulates E2F3 expression through competitive adsorption of miR-200b, ultimately contributing to the chemoresistant phenotype formation of lung adenocarcinoma. Luciferase activity assay, RNA immunoprecipitation, mutational analysis, RNA interference and exotic gene expression were employed in this study. It not only helps to elucidate the molecular mechanisms of lung adenocarcinoma multidrug resistance, but also provides a novel clinical strategy for reversing lung adenocarcinoma multidrug resistance.

化疗耐药是肺腺癌临床治疗面临的重大难题，其形成涉及肿瘤耐药相关蛋白基因的遗传学及表观遗传学修饰等分子调控过程。长链非编码RNA (lncRNA)可在表观遗传学、转录及转录后水平调控机体生理和病理过程。课题组前期研究已证明：miR-200b可以通过负向调控E2F3表达参与肺腺癌化疗耐药表型的形成，但确切机制尚不清楚。我们新近采用LncRNA芯片筛选和生物学信息分析发现LncRNA MALAT-1含有2个miR-200b潜在结合位点且与E2F3基因mRNA表达存在相关性。本课题拟在前期研究的基础上，采用荧光素酶报告基因、RNA免疫共沉淀、位点突变、RNA干涉及基因过表达等方法通过体外细胞和活体动物模型验证MALAT-1通过竞争性吸附miR-200b进而诱导靶基因E2F3表达水平上调参与肺腺癌细胞化疗耐药表型形成的分子机制，为临床逆转肺腺癌化疗耐药提供新的分子靶点。

肺癌已经成为全球范围内的公共卫生问题，其中肺腺癌是肺癌的主要病理类型。化疗耐药是肺腺癌临床治疗面临的重大难题，其形成涉及肿瘤耐药相关基因的遗传及表观遗传学修饰的分子调控过程。长链非编码RNA（long non-coding lncRNA）可在表观遗传学、转录及转录后水平调控机体的生理和病理过程。本课题前期研究发现，低表达的微小RNA（microRNA，miR）-200b调控靶基因E2F3的异常表达是肺腺癌产生耐药的原因之一，恢复miR-200b表达能显著逆转肺腺癌的化疗耐药表型。本项目在前期成功建立肺腺癌多西他赛耐药细胞模型、lncRNA 差异表达筛选、miR-200b 功能学及生物信息学分析、并明确 miR-200b/E2F3 信号通路紊乱与人肺腺癌化疗抵抗密切相关的基础上，从体外和体内等层面探讨lncRNA介导的miR-200b负向表达调控与肺腺癌化疗耐药之间的关系。证实长非编码RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) 参与调控肺腺癌的化疗耐药表型形成，并进一步采用荧光素酶报告基因实验、位点突变、免疫共沉淀、RNA 干扰及过表达等实验技术证实MALAT-1、miR-200b与E2F3/ZEB1三者之间的调控关系，发现MALAT-1通过竞争性吸附miR-200b诱导E2F3/ZEB1表达水平的上调参与肺腺癌化疗多药耐药表型形成。研究结果表明MALAT-1/miR-200b/E2F3 信号轴和 MALAT-1/miR-200b/ZEB1 信号轴可以在体内、体外层面共同参与调控人肺腺癌的化疗耐药。因此，本课题从转录后水平揭示了lncRNA介导的人肺腺癌耐药细胞中miR-200b基因表达下调的机制。该研究为肺腺癌的个体化治疗以及临床逆转肺腺癌化疗耐药提供了新的策略。