胰岛素抵抗肝癌细胞耐药过程中自噬作用及相关microRNA的筛选与机制研究

Insulin resistance(IR) is involved in hepatocarcinogenesis, development and poor prognosis of hepatocarcinoma, while IR in hepatocarcinoma always be accompanied with drug resistance. Several studys showed that autophagy might participate in the fomation of drug resistance in hepatocarcinoma cells of IR, which mechanism is still unknown. Our research has showed that the high autophagy activity is the most important factor which mediate multidrug resistance in hepatocarcinoma cells of IR, and there might be some microRNAs participate in the regulation of autophagy and drug resistance. However, it remains unclear whether microRNA mediate drug resistance in hepatocarcinoma cells of IR by regulating the autophagy signaling pathway. Our issue proposed to employ high concentration of insulin inducing hepatocarcinoma cells to establish IR hepatocarcinoma cells model, to definite the relationship between drug resistance in hepatocarcinoma cells of IR and autophagy as well as the grade of hepatocarcinoma. Adopting biochip technology to compare miRNA expression differences in hepatocarcinoma cells of IR, to screen the common difference of microRNAs from these various degree cells, to predict and validate the microRNAs related autophagy and to verify in IR hepatocarcinoma patients, eventully, to make sure the mechanism of drug resistance mediated by autophagy which regulated by microRNAs in hepatocarcinoma cells of IR.

胰岛素抵抗(IR)与肝癌发生、发展及不良预后密切相关，伴发IR的肝癌常具有药物耐受性，研究表明自噬可能参与IR肝癌细胞耐药性形成，然而机制不明。我们前期研究发现高自噬活性是介导IR肝癌细胞多药耐药性的重要因素，且有miRNA分子可能参与自噬活性和耐药性的调控，但尚不明确miRNA是否通过调控自噬通路介导IR肝癌细胞的耐药性。本课题拟在前期研究基础上采用胰岛素诱导高、低度恶性肝癌细胞建立IR细胞模型，通过调控自噬活性后检测IR肝癌细胞对化疗药物敏感性的变化，明确IR、耐药性、自噬活性和恶性度的关系；应用miRNA芯片从IR肝癌细胞中筛选和比较与耐药及自噬有关的共同差异表达miRNA分子，运用生物信息学和分子生物技术预测及验证可能参与调控上述通路的关键miRNA及作用靶点，并在临床IR肝癌患者中验证，以阐明miRNA调控IR肝癌细胞自噬活性介导其耐药性的分子机制，为肝癌的临床干预提供理论依据。

胰岛素抵抗(IR)与肝癌发生、发展及不良预后密切相关，伴发IR的肝癌常具有药物耐受性。课题在前期研究基础上采用胰岛素诱导高、低度恶性肝癌细胞建立IR细胞模型，通过调控自噬活性后检测IR肝癌细胞对化疗药物敏感性的变化，明确IR、耐药性、自噬活性和恶性度的关系，我们发现高自噬活性是介导IR肝癌细胞多药耐药性的重要因素，在胰岛素抵抗介导的肝癌细胞耐药中自噬作为调控子调控内质网应激反应促进内质网的稳态从而发挥促进化疗药物耐受的作用。为了进一步阐述参与调控耐药的microRNAs分子，我们应用miRNA芯片从IR肝癌细胞中筛选和比较与耐药及自噬有关的共同差异表达miRNA分子，运用生物信息学和分子生物技术预测发现这些差异的miRNAs可能作为癌基因或抑癌基因促进肿瘤细胞的进展、复发、转移从而导致药物耐受。有趣地是GO以及KEGG分析发现这些差异miRNA的靶基因功能富集在生物合成、代谢、修饰等生物过程，相关通路富集在肿瘤相关的Ras通路和Hippo通路，并且在此基础上我们建立了互作图进一步明确miRNA以及靶基因的相互作用，并在细胞以及临床患者标本中进行验证，阐明导致胰岛素抵抗肝癌细胞耐药的机制。