HDAC/Sp1复合物负向调控microRNA-200b表达参与人肺腺癌化疗耐药表型形成的分子机制研究

Chemoresistance is the most significant obstacle towards lung cancer treatment, and this process involves genetic and epigenetic regulation of chemoresistance-related genes. Histone acetylation is one of the most important epigenetic mechanisms controlling gene expression, and it can regulate various physiological and pathological processes by transcriptional regulation of gene expression. Our previous study showed that miRNA-200b was significantly downregulated in chemoresistant lung adenocarcinoma cells and its overexpressiou could reverse the chemoresistance of human lung adenocarcinoma. However, the molecular mechanisms of miRNA-200b downregulation in chemoresistant lung adenocarcinoma cells remain unclear. On the previous basis of establishment of docetaxel-resistant lung adenocarcinoma cell line and functional and bioinformational analysis of miRNA-200b, the aim of this study is to investigate the molecular mechanisms of HDAC/Sp1 complexes induced-negative regulation of miRNA-200b expression by chromatin immunoprecipitation, electrophoretic mobility shift, site-specific mutation and RNA interference assays, to illustrate the association of histone acetylation regulation of miRNA-200b expression with chemoresistance of lung adenocarcinoma. It will provide a novel strategy for reversing chemoresistance of lung adenocarcinoma in the future.

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。组蛋白乙酰化是表观遗传修饰调控基因表达的重要方式，通过对基因表达的转录水平调控作用参与调控各种生理及病理过程。前期研究发现：miRNA-200b在肺腺癌耐药细胞中呈显著的低表达，其表达水平升高能显著逆转肺腺癌的化疗耐药表型，并且发现HDAC处理与miRNA-200b表达水平的变化相关。本课题拟在前期成功建立耐多西他赛人肺腺癌细胞模型、miRNA-200b功能学及生物信息学分析的基础上，拟通过染色质免疫共沉淀、凝胶阻滞、位点突变、RNA干涉等实验方法分析HDAC/Sp1负向调控miRNA-200b表达的分子机制，从细胞和活体动物水平探讨miRNA-200b表达的组蛋白乙酰化调控与肺腺癌化疗耐药相关的功能学分析，从而为临床逆转肺腺癌耐药提供新的策略。

肺癌严重危害人类健康，已经成为全球范围内的公共卫生问题。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所面临的重大难题。化疗耐药与肿瘤耐药相关基因的遗传学及表观遗传学修饰的分子调控密切相关。组蛋白乙酰化是表观遗传修饰调控基因表达的重要方式之一，调控基因表达参与多种生理及病理过程。本课题前期研究发现：低表达的微小RNA（microRNA，miR）-200b是肺腺癌耐药的原因之一，恢复miR-200b表达能显著逆转肺腺癌的化疗耐药表型；抑制组蛋白去乙酰化酶（histone deacetyltransferases，HDACs）可恢复miR-200b的表达。本课题在前期成功建立人肺腺癌细胞多西他赛耐药模型、miR-200b功能学以及生物信息学分析基础上，通过免疫共沉淀、染色质免疫共沉淀、位点突变、荧光素酶活性试验等实验方法探索HDACs负向调控miR-200b表达的分子机制，从体外和体内等层面探讨miR-200b表达的组蛋白乙酰化调控与肺腺癌化疗耐药之间的关系。证实组蛋白乙酰化而非DNA甲基化参与了人肺腺癌耐药细胞miR-200b的表达调控，进而参与调控了人肺腺癌的化疗抵抗。进一步研究确定在HDAC1-11的亚型中，HDAC1/4参与调控耐药株miR-200b的表达，HDAC1/4和miR-200b表达水平与肺腺癌患者预后密切相关。HDAC1/4/miR-200b信号通路可以在体内、体外层面参与调控人肺腺癌的化疗耐药。总之，HDAC1/4/Sp1/miR-200b信号轴通过进一步调控E2F3/Aurora-A和E2F3/Survivin的表达可能是HDAC1/4/miR-200b信号通路参与调控人肺腺癌化疗耐药的重要分子机制。本课题首次发现HDAC1/4/Sp1/miR-200b/E2F3/Aurora-A/Survivin信号轴参与调控人肺腺癌化疗耐药；揭示了在临床标本中HDAC1/4与miR-200b表达水平密切相关，并且与肺腺癌患者预后密切相关；因此，本课题从组蛋白乙酰化层面揭示了人肺腺癌耐药细胞中miR-200b基因表达下调的原因，为临床肺腺癌的个体化治疗及逆转人肺腺癌的化疗抵抗提供了新的分子靶点。