内质网应激激活PI3K通路诱导肝癌细胞耐药的分子机制

Endoplasmic reticulum stress (ERS) could trigger a series of cyto-protective responses and helps the cells escape from being hurt, which renders the tumor cells less sensitive to the chemotherapy. Calreticulin (CRT) is a kind of molecular chaperon and could translocate to the cell membrane, which is associated with the activation of PI3K pathway. Our previous data showed that ERS strikingly activated PI3K pathway and induced chemo-resistance in hepatocellular carcinoma cells. Inhibition of PI3K could partly abrogated ER stress induced chemo-resistance. However, it is not clear about how ERS activates PI3K pathway. We will investigate whether PI3K pathway is activated via CRT translocation upon ERS, which subsequently decreases the sensitivity of hepatocellular carcinoma cells to the chemotherapeutic agent, adrimycin as follows: 1. It will be explored whether ERS could induce CRT translocation and the correlation of CRT translocation and PI3K pathway. 2. CRT translocation will be inhibited or induced to study its effects on the activation of the PI3K pathway. 3. It will be studied whether ERS-induced chemo-resistance will be overcome after inhibition of CRT translocation, which will provides new insights into the molecular mechanism of ERS-induced chemo- resistance in hepatocellular carcinoma cells.

内质网应激反应是细胞逃避损伤的一种自我保护反应，可诱导肿瘤细胞对化疗药物产生耐药。钙网蛋白属于分子伴侣，可参与内质网应激反应，并可发生膜转位，膜转位后的钙网蛋白与PI3K通路的激活有关。我们前期研究结果显示内质网应激可通过激活PI3K/Akt通路诱导肝癌细胞对阿霉素产生耐药。但内质网应激激活PI3K的分子机制尚不清楚。本研究拟从以下几方面探讨内质网应激是否通过诱导钙网蛋白膜转位来激活PI3K通路，进而引发肝癌细胞对阿霉素耐药：1.检测内质网应激可否诱导肝癌细胞内钙网蛋白发生膜转位及其转位后是否与PI3K发生免疫共沉淀。2.干扰肝癌细胞内钙网蛋白的膜转位，观察它对PI3K通路活化状态的影响。3.检测钙网蛋白膜转位受阻能否逆转内质网应激诱导的肝癌细胞对阿霉素的耐药现象，揭示内质网应激通过诱导钙网蛋白膜转位来激活PI3K通路，引发肝癌细胞对阿霉素产生耐药的机制。

肝癌是全球范围内第五大常见恶性肿瘤，患者就诊时已属于中晚期，失去手术根治的机会，化疗药物治疗在治疗中晚期肝癌扮演重要角色，然而，肝细胞癌对化疗药物敏感性下降是导致肝癌治疗效果不佳的重要原因之一。内质网（Endoplasmic reticulum）在一些理化因素如缺血，缺氧，营养缺乏，酸中毒刺激下，导致未折叠蛋白在内质网堆积，引发细胞一系列适应性反应，称为内质网应激(Endoplasmic reticulum stress ERs)，当未折叠蛋白的堆积超过内质网的承受能力时，内质网通过激活其下游分子ATF（activating transcription factor 6）、RNA-dependent protein kinase(PKR)- likeendoplasmic reticulum kinase( PERK）、IRE1(inositol-requiring enzyme 1)与免疫球蛋白重链结合蛋白(immunoglobulin heavy chain bingding protein Bip)诱发细胞产生耐药、增殖、转移等自我保护。本研究的主要内容分为三个部分：（1）：观察内质网应激可否引起内质网分子伴侣钙网蛋白（calreticulin CRT）发生膜转位并引起肝癌细胞耐药的发生；（2）：CRT膜转位后与PI3K作用机制；（3）:CRT/PI3K/Akt通路在肝癌细胞耐药的作用及分子机制。本研究发现内质网应激可激活PI3K/Akt信号通路并诱导肝癌细胞SMMC-7721、HepG2对阿霉素产生耐药，利用PI3K特异性阻断剂后可抑制内质网应激介导的耐药，提示PI3K/Akt通路参与了内质网应激介导的肝癌细胞对阿霉素的耐药。内质网应激后促进CRT发生膜转位，同时利用免疫共沉淀技术发现CRT通过与PI3K相互结合的机制参与了内质网应激介导的肝癌细胞的耐药，利用小干扰RNA技术沉默CRT在肝癌细胞的表达后证实了CRT在肝癌细胞增殖、凋亡、转移中的的作用，阐述了CRT/PI3K/Akt信号通路参与肝癌细胞耐药的机制，为肝癌细胞耐药的现象奠定了机制基础，为中晚期肝癌患者的治疗提供新的治疗思路。