TET1甲基化修饰PDCD4在胆管癌吉西他滨获得性耐药的分子机制研究

A cholangiocarcinoma is generally resistant to gemcitabine. The regulative mechanism remains unclear. Our previous studies have shown that the expression of TET1 and PDCD4 were down-regulated in gemcitabine-acquired cholangiocarcinoma cells using gene chip. Our further studies indicated that TET1 could lead to demethylate the CpG island of target gene and PDCD4 promoter was rich in CpG island. Based on that PDCD4 could negative phosphorylate the AKT/NF-κB pathway, while P-gp is the downstream gene of AKT/NF-κB pathway. Therefore, we hypothesize that TET1 binding to CpG islands in promoter site of PDCD4 leading to demethylation of PDCD4 to phosphorylation activate AKT/NF-κB /P-gp pathway. Through BSP, we will investigate the role of TET1 to methylate regulative PDCD4. Through western blot, we will further explore the molecular mechanism of TET1/PDCD4/AKT/NF-κB/P-gp pathway in acquired chemo-resistant. Via in vivo experiments, combinated the epigenetic drug and gemcitabine to reverse the susceptibility of acquired chemo-resistant cholangiocarcinoma to gemcitabine.

胆管癌对吉西他滨产生获得性耐药的机制尚不明确。我们前期利用基因芯片差异性筛选发现TET1和PDCD4在获得性耐药胆管癌细胞株低表达，TET1可使下游基因CpG岛去甲基化，PDCD4启动子区域富含CpG岛。已知PDCD4可负性磷酸化调控AKT/NF-κB通路，P-gp受AKT/NF-κB通路调控。我们推测：TET1与PDCD4启动子区域CpG岛特异性结合，使PDCD4去甲基化减弱，进而磷酸化激活AKT/NF-κB通路导致P-gp高表达，诱导胆管癌对吉西他滨产生获得性耐药。本项目拟采用BSP等方法检测TET1甲基化修饰PDCD4；用免疫沉淀等方法研究TET1/PDCD4/AKT/NF-κB/P-gp通路调控胆管癌对吉西他滨产生获得性耐药的机制；通过动物实验研究甲基化抑制药物5Aza联合吉西他滨逆转胆管癌获得性耐药的效果，进而明确TET1甲基化修饰PDCD4介导胆管癌细胞获得性耐药的分子机制。

胆管癌预后极差，其5年生存率不足5%。手术根治是延长胆管癌病人长期生存唯一有效的方法。然而术后，胆管癌极易复发和转移，主要是由于胆管癌对吉西他滨产生获得性耐药。目前对胆管癌细胞产生耐药的机制尚不明确。我们主要通过基因芯片，BSP测序，CoIP和ChIP等方法，体内外深入研究TET1通过调解PDCD4/AKT/NF-κB/P.-gp通路调控胆管癌对吉西他滨产生获得性耐药的机制。结果发现，TET1在胆管癌组织低表达，主要是由于本身高甲基化造成。通过大数据分析，TET1与PDCD4存在正相关，可以调控PDCD4的表达，而PDCD4富含CpG岛，而TET1可以结合在PDCD4启动子区域，去甲基化调控PDCD4的表达。而PDCD4的表达可以调节AKT/NF-kB通路，促进多重耐药基因P-gp的表达。对于PDCD4是如何调节AKT/NF-kB通路的表达，我们发现PDCD4可以磷酸化激活AKT。动物体内，我们利用5Aza去甲基化TET1后，引起PDCD4/AKT/NF-κB/P-gp的改变，可以逆战胆管癌细胞对吉西他滨的耐药。通过本研究，不仅可以阐释TET1作为新的耐药分子在胆管癌细胞对吉西他滨产生获得性耐药过程中的重要作用，阐明TET1通过甲基化修饰PDCD4/AKT/NF-κB/P-gp通路介导胆管癌细胞获得性耐药的机制，还可以明确甲基化抑制药物5Aza联合吉西他滨逆转胆管癌获得性耐药的效果，有利于解决胆管癌细胞对吉西他滨产生获得性耐药问题，也为将来的临床试验提供依据。