LINE-1 ORF-1p/PXR信号通路在肝癌分子靶向治疗耐药形成中的作用和调节机制研究

Molecular target therapy, as the first-line therapy for advanced Hepatocellular Carcinoma (HCC), has attracted much interest for its drug resistance. However, the molecular mechanism of drug resistance was unclear yet, which made it a hot issue. Human Long Interspersed Nuclear Elements 1 Open reading frame (LINE-1 ORF-1p) can promote the proliferation of tumor cells and induce the tolerance of tumor cells against cytotoxic chemotherapy drugs through a variety of mechanisms. Our previous research showed that the expression of LINE-1 ORF-1p increased in HCC patients with molecular target drug resistance, and can interaction with pregnane X receptor (PXR), which may be the possible cause of drug metabolism and elimination promotion, as well as drug-resistance formation. The purpose of this study is to further confirm the interaction between LINE-1 ORF-1p and PXR, locate the binding sites of this interaction, study the effect of LINE-1 ORF-1p on the activity of PXR, and explore the role, regulating mechanism, and clinical significance of the LINE-1 ORF-1p/PXR signal pathway and PXR downstream genes (CYP450 3A4, et al) in the form of HCC molecular target drug resistance by using GST-Pull down technology, laying the foundation of future higher clinical response rate of HCC molecular target therapy.

分子靶向药物是进展期肝细胞癌的一线治疗方案，其相关耐药性备受关注，但其分子机制尚未阐明,是亟需解决的热点问题。人长散布核元件1编码蛋白1（LINE-1 ORF-1p）可通过多种机制促进肿瘤细胞增殖，诱发肿瘤细胞对细胞毒性化疗药物耐受。我们前期研究结果发现LINE-1 ORF-1p在分子靶向药物治疗HCC耐药患者中表达增加，并能够与核受体PXR相互作用，可能是促进药物代谢与清除、诱发耐药形成的原因。本研究拟通过GST-Pull down等技术进一步证实LINE-1 ORF-1p与PXR存在相互作用，明确二者相互作用的结合位点；检测LINE-1 ORF-1p对PXR 活性的影响；探索LINE-1 ORF-1p与PXR信号通路及PXR下游基因CYP450 3A4等在耐药形成中的作用，调节机制和临床意义。为今后提高HCC患者分子靶向治疗的临床应答奠定基础。

索拉菲尼进展期肝细胞癌的一线治疗方案，其相关耐药性备受关注，但其分子机制尚未阐明,是亟需解决的热点问题。LINE-1 ORF-1p由人前致癌基因LINE-1编码，可通过多种机制促进肿瘤细胞增殖，诱发肿瘤细胞对细胞毒性药物耐受。我们以前的工作表明，LINE-1 ORF-1p可以增强肝细胞癌（HCC）细胞对抗肿瘤药物的耐药性。然而，涉及LINE-1 ORF-1p介导的耐药性的机制仍然很大程度上未知。本课题使用定量PCR（qPCR）检查临床HCC样品中LINE-1 ORF-1p的内源mRNA水平，通过与HCC患者的TTP及OS相关性分析，明确其与HCC患者预后相关。通过荧光素酶报告基因分析、qPCR、染色质免疫沉淀分析和细胞亚分离分析检测孕烯醇酮X受体（PXR）的转录因子活性。利用共免疫沉淀检测LINE-1 ORF-1p和PXR之间的蛋白质相互作用。最后利用体外和体内模型研究LINE-1 ORF-1p对HCC细胞中索拉非尼耐药的影响。结论：1、高水平的LINE-1 ORF-1p表达与接受索拉非尼治疗预后不良相关。2、LINE-1 ORF-1p通过与PXR相互作用增强其转录因子活性，增强其细胞质/核转录，并向其下游基因启动子募集PXR，从而增强索拉非尼耐药相关基因CYP3A4和mdr-1的表达。3、LINE-1 ORF-1p通过增强了PXR的转录因子活化，促进HCC细胞中索拉非尼的清除和耐药性。LINE-1 ORF-1p与索拉菲尼耐药性的发现为肝癌患者个体化精准治疗提供了可能，也为减少耐药延长患者生存期的新药研发提供了理论基础。