p62/IMP2促进食管鳞癌化疗耐药的分子机制

Development of drug resistance is the main clinic feature against successful chemotherapy for esophageal squamous cell carcinoma (ESCC). It is imminent to study its molecular mechanism. Our previous results showed that the tumor associated expression of p62/IMP2 was significantly up-regulated, comparing with adjacent tissues in ESCC patients. Overexpressed of p62/IMP2 in a ESCC cell line promoted cell proliferation, migration and invasion of, inhibition against apoptosis and increased sensitivity to chemotherapeutics.On the other hand, knock-down of p62/IMP2 had a negative effect on ESCC cells. Moreover, known p62/IMP2 target DcR2 reduces the sensitivity of ESCC cells to TRAIL mediated apoptosis. Therefore, we hypothesize that p62/IMP2 up-regulates DcR2 to inhibit TRAIL-mediated apoptosis and promote resistance to chemotherapy in ESCC. Our proposal will verify this hypothesis from three independent aspects: elucidating the molecular mechanisms of p62/ IMP2 up-regulation in ESCC cancer tissue; confirming the molecular mechanism of p62/IMP2 the development of chemotherapy resistance of ESCC cell with cell and animal models; verifying the correlation of p62/IMP2, DcR2, TRAIL and long-term response to chemotherapy in ESCC patients. This project will be of great significance for the further elucidations of drug resistance mechanism of ESCC.

食管鳞癌（ESCC）细胞耐药性产生是影响化疗效果的主要原因，研究其耐药机制迫在眉睫。我们的前期结果显示p62/IMP2在ESCC患者癌组织中的表达显著高于癌旁组织，在Eca109细胞中过表达p62/IMP2促进细胞增殖、迁移和侵袭能力，并抑制其凋亡和对化疗药物的敏感性，在KYSE150细胞中干扰p62/IMP2表达对细胞起反作用。并且p62/IMP2上调DcR2降低ESCC细胞对TRAIL的敏感性。由此我们提出假说：p62/IMP2上调DcR2抑制TRAIL介导的细胞凋亡，促进ESCC产生耐药性。本课题将从三方面验证假说：1.阐释p62/IMP2在ESCC患者癌组织中表达上调的分子机制；2.在细胞和动物水平上证实p62/IMP2在ESCC细胞生物学功能和耐药中的分子机制；3.在人群样本中验证p62/IMP2、DcR2和TRAIL的相关性。本课题对于深入研究ESCC的耐药机制具有重意义。

在我国，食管鳞状细胞癌（ESCC）是最主要的食管癌类型，而EAC的发病率较低。ESCC患者确诊时往往处于晚期，且获得性耐药在很大程度上影响了化疗疗效，研究其耐药机制迫在眉睫。p62(IGF2BP2，IMP2)是胰岛素样生长因子2 mRNA结合蛋白家族的一员，作为m6A阅读器增强其靶基因RNA的稳定性。在多种肿瘤中高表达并参与肿瘤的发生发展过程，但其在肿瘤治疗中的作用机制尚不清楚。本课题深入探究p62在ESCC对化疗药物敏感性中的作用机制。.本项目主要研究内容分为以下三个部分：（1）通过生物信息学分析、免疫组化、蛋白质免疫印迹、qRT-PCR探究p62在ESCC肿瘤组织和多种ESCC细胞系中的表达特征及对甲基化水平的影响；（2）通过克隆形成实验、CCK-8增殖实验、细胞划痕实验、Transwell迁移实验、Transwell侵袭实验、凋亡实验、CCK-8耐药实验、裸鼠荷瘤实验研究p62/IMP2在ESCC细胞生物学功能、化疗药物敏感性及体内肿瘤生长中的影响；（3）利用RNAseq、KEGG通路富集分析、免疫共沉淀（CoIP）和RNA结合蛋白免疫沉淀（RIP）等实验研究p62在ESCC对肿瘤坏死因子相关凋亡配体（TRAIL）敏感性中的作用及对该通路的调控机制；结果表明p62在ESCC细胞及组织中显著过表达，p62的表达水平与患者组织中甲基化呈正相关；低表达p62促进了ESCC细胞的凋亡，抑制了ESCC细胞的增殖、迁移、侵袭、耐药及肿瘤体内生长，而p62高表达时则影响相反；p62通过与TRAIL介导的凋亡通路的诱饵受体DcR2和死亡受体DR4、DR5互作，影响其稳定性，进而抑制TRAIL诱导的细胞凋亡。综上所述，我们的研究表明p62抑制ESCC细胞对化疗药物敏感性，为ESCC细胞耐药机制及分子靶向治疗提供新的研究思路。