MiR-200b靶向Atg12调控自噬并参与人肺腺癌多药耐药表型形成的机制研究

Chemoresistance remains a clinical major obstacle to the patients of lung adenocarcinoma. Autophagy is a cellular self-catabolic process where cytoplasmic components are engulfed and trafficked to lysosomes for degradation. Chemotherapy modulates tumor cell survival programs by regulating autophagic flux. In our previous study, we found that autophagic flux was increased in human chemoresistant lung cancer cell lines and pharmacological inhibitors of autophagy enhanced the cytotoxicity of chemotherapy. Meanwhile miR-200b was found lower lever in SPC-A1/DTX. Upregulating of miR-200b could reduce autophagosome formation and enhance the chemosensitivity of the SPC-A1/DTX. The bioinformatics analysis shows Atg12 may be a candidate target gene of miR-200b, and also Atg12 is a major component of autophagosome formation. So we hypothesize that miR-200b inhibits the autophagy process by downregulating Atg12 and modulates the cytotoxicity of chemotherapy. However the specific molecular mechanism is unknown. In this study, the role of autophagic response modulated by miR-200b/Atg12 in mediating cellular resistance to chemotherapy and the mechanisms would been explored in vitro and in animal models, using a combined bioinformatics approach, gene set enrichment analysis, miRNA target prediction, immunoprecipitation assay and RNA interference et al. Findings of this study would help us better identification of novel approachs for overcoming muti-drug resistance in lung adnocarcinoma.

化疗耐药是肺腺癌临床治疗面临的重大难题。自噬是真核细胞降解长寿命蛋白和细胞器的一种溶酶体途径，化疗药物能通过调节肿瘤细胞自噬水平而影响疗效。我们前期研究发现：肺腺癌耐药细胞的自噬水平高于亲本株，抑制自噬能增强化疗敏感性；耐药细胞中miR-200b表达下调，上调miR-200b可抑制自噬并提高化疗敏感性；参与自噬体膜延长及成熟的Atg12是miR-200b的候选靶基因。推测miR-200b通过靶向Atg12调控自噬并参与肺腺癌多药耐药表型形成，但其机制尚不清楚。本课题拟在前期建立的肺腺癌多西紫杉醇耐药细胞模型、miRNA差异表达筛选、miR-200b功能学与生物信息学分析的基础上，采用电镜、免疫荧光染色、RNA干涉及过表达、免疫共沉淀等实验方法，从细胞和活体动物水平研究MiR-200b靶向Atg12调控的自噬在人肺腺癌多药耐药表型形成中的作用及分子机制，为逆转肺腺癌多药耐药提供新的分子靶点。

肺癌高居全球恶性肿瘤发病率和死亡率的首位，其中肺腺癌仍是主要的病理类型。对于进展期的肺腺癌患者，化疗依然是目前最重要的治疗方式。随着大批化疗新药的广泛应用，化疗的有效率已较过去有所提高，但仍然有半数以上的患者并未能从化疗中获益。化疗耐药的产生仍是限制晚期肺腺癌患者临床疗效的重要障碍。因此，深入阐明肺腺癌化疗耐药的机制并寻找有效分子靶标指导精准化疗已成为提高肺腺癌疗效的重要研究方向。自噬是广泛存在于真核细胞中的生命现象，参与细胞的多种生理及病理过程。自噬的发生与肿瘤耐药之间亦存在密切联系。本项目从三个层次对自噬与肺腺癌化疗耐药的关系展开研究: 其一，首先明确自噬在肺腺癌化疗耐药形成中的作用；其二，从转录后水平对自噬介导肺腺癌化疗耐药的机制进行探讨；其三，分析肺腺癌组织标本中miR-200b表达与临床病理和预后的关系。.本项目取得的主要研究成果如下：1）自噬增加肺腺癌细胞对多西他赛的化疗抵抗，抑制自噬能提高肺腺癌细胞对多西他赛的敏感性，抑制增殖并增加凋亡。2）MiR-200b为肺腺癌细胞中参与自噬调控的miRNA。miR-200b通过直接结合自噬基因ATG12的3’-UTR区抑制自噬，并逆转体内外肺腺癌化疗耐药。3）在多西他赛化疗方案不敏感的肺腺癌组织标本中，miR-200b呈低表达，ATG12呈高表达，两者呈明显负相关；miR-200b低表达患者PFS显著缩短。.本项目在前期成功建立肺腺癌多西他赛耐药细胞模型的基础上，阐明自噬在肺腺癌化疗耐药形成中的作用，并从转录后水平探索miR-200b/ATG12信号通路调控自噬参与肺腺癌化疗耐药形成的分子机制，从而为肺腺癌的个体化治疗和逆转肺腺癌耐药提供新的临床策略。