非内质网应激依赖性的真核生物翻译起始因子2alpha异常磷酸化在胆管癌进展和耐药中的作用及其机制

Stressed microenvironment plays an important role in the occurrence and development of cancer. Moreover, stress response can alter the sensitivity of cancer cells to chemotherapeutic agents. In order to survive, cancer cells initiate specific adaptive signaling events, which are essential for them to adapt to the stressed microenvironment and chemotherapeutic agents. It is important to investigate the role of stressed microenvironment-induced adaptive stress response in the occurrence, development and chemotherapy of cholangiocarcinoma (CCA). Furthermore, the role and molecular mechanism of (AMP-activated protein kinase) AMPK and eukaryotic translation initiation factor 2 alpha (eIF2α) abnormal phosphorylation in the occurrence, development and chemotherapy of CCA remain unknown. In this project, mutagenesis techniques will be used to mimic eIF2α abnormal phosphorylation. CRISPR/Cas9, RT-PCR, western blot, and trans-well assays will be used to investigate the role of stress-mediated AMPK and eIF2α phosphorylation regulation in the occurrence, development and chemotherapy of CCA. Furthermore, we will focus on the molecular mechanism of eIF2α on the occurrence, development and chemotherapy effect of CCA under stress conditions. Importantly, the effect and molecular mechanism of eIF2α on the occurrence, development and chemotherapy effect of CCA under stress conditions will be investigated in vivo. Our research will provide a new theoretical basis for developing new strategies against CCA.

虽然已知应激微环境与肿瘤的过程和化疗效果密切相关，但是其机制还很不清楚。应激反应是否参与了胆管癌的生物学过程及其对胆管癌化疗的影响目前也尚未见报道。认识应激诱导的适应性信号调控在胆管癌发生、发展和化疗中的作用是亟需解决的科学问题。应激重要调控分子AMPK和eIF2α的异常磷酸化在胆管癌发生、发展和化疗中的作用也是尚未解决的科学问题。该项目在利用突变技术模拟eIF2α异常磷酸化的基础上，采用CRISPR/Cas9、RT-PCR、Western blot和Trans-well assays等实验技术，首先明确AMPK对胆管癌细胞eIF2α磷酸化的调控及其对胆管癌细胞生物学行为和化疗效果的影响，进而探讨在应激条件下eIF2α影响胆管癌细胞生物学行为和化疗的机制，并通过体内实验阐述应激微环境对eIF2α的磷酸化调控及其在胆管癌发生、发展和化疗中的作用及其机制，最终为胆管癌的治疗提供新的理论基础。

虽然已经知道应激微环境与肿瘤的发生过程和化疗效果密切相关，但是其分子机制还很不清楚。应激反应是否参与了肝胆肿瘤的生物学过程及其对肝胆肿瘤化疗的影响目前也尚未不清楚。认识应激诱导的适应性信号调控在肝胆肿瘤发生、发展和化疗中的作用是亟需解决的科学问题。本项目重点关注应激重要调控分子eIF2α 的异常磷酸化、mTOR、JNK等在肝胆肿瘤发生、发展和化疗中的作用。该项目利用突变技术模拟eIF2α 异常磷酸化，采用CRISPR/Cas9、RT-PCR、Western blot 和Trans-well assays 等实验技术，探讨eIF2α 磷酸化的调控、mTOR和JNK等的异常活化在肝胆肿瘤细胞生物学行为中的重要作用及其对化疗效果的影响。项目发现ATF4的异常下调在肝脏肿瘤早期损伤中的作用；发现JNK的异常活化通过eIF2α/ATF4促进胆管癌细胞GRP78的表达；发现eIF2α小分子抑制剂对胆管癌细胞的抑制作用及其与mTOR抑制剂联用对胆管癌的抑制的协同效应；发现AMPK抑制剂诱导的自噬不依赖于AMPK和AKT/mTOR，自噬的诱导对Compound C的抑胆管癌效应起抑制作用；发现胆管癌细胞抵抗接触抑制的新机制；发现eIF2α抑制剂调控肝胆肿瘤细胞对不同化疗药物的敏感性。项目结果为肝胆肿瘤的治疗提供了新的理论基础。

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