AEG1诱导自噬促进细胞失巢凋亡抵抗在肝癌转移中的作用及机制研究

Anoikis resistance, an important factor of tumor metastasis, protects tumor cells from apoptosis when cells intravasate into vessels. Our previous study found that the metastasis potential of hepatocellular carcinoma cells is closely related to anoikis resistance and AEG1 could enhance the ability of anoikis resistance in HCC cells. Recent studies suggest that autophagy could enable tumor cells survival under unbeneficial conditions. Up to now, there were data about the relationships among AEG1, autophagy, and anoikis. Our researches demonstrated that AEG1 induces autophagy upregulation confirmed by LC3 I/II transform in suspension cultured HCC cells, and inhibition of autophagy significantly promotes anoikis. Therefore, we pose a hypothesis that AEG1 enhances anoikis resistance by upregulating autophagy in HCC cells, which promoting HCC metastasis. Our data also suggested that AEG1 inhibits the phosphorylation of mTOR, a key regulator of autophagy. Based on the present theories and our experiments, this project plans to up/down-regulate AEG1 expression in HCC cells, and confirm that AEG1 promotes anoikis resistance though upregulating autophagy in HCC cells under ECM-detachment conditions. We will further explore the pathways and autophagy-related genes involved in this process. In animal experiments, we plan to verify the effects of autophagy induced byAEG1 on anoikis resistance and metastasis. In conclusion, this project which is aimed at clarifying the relationships among AEG1, autophagy, anoikis resistance, and HCC metastasis, will be benefit for exploring potential intervention targets of HCC metastasis.

肿瘤细胞渗入循环系统后失巢凋亡抵抗是促进转移的重要因素，我们前期研究发现，肝癌细胞的转移潜能与失巢凋亡抵抗明显相关；AEG1能降低肝癌细胞的失巢凋亡率。自噬对肿瘤细胞在不利条件下生存多起保护作用，肿瘤转移中AEG1、自噬、失巢凋亡之间的关系目前尚无研究,我们证实抑制自噬能促进肝癌细胞失巢凋亡。预实验还发现，肝癌细胞体外失巢模型中过表达AEG1可使自噬小体膜LC3蛋白Ⅰ/Ⅱ转变，提示AEG1诱导自噬增强。据此推测，AEG1通过提高自噬水平增强肝癌细胞失巢凋亡抵抗，促进肝癌转移；我们进一步研究发现AEG1可抑制细胞自噬信号通路mTOR磷酸化。基于此，本课题拟通过过表达及沉默AEG1，探索失巢条件下自噬对细胞凋亡的影响，及AEG1调控自噬的通路和自噬基因变化，动物水平观察AEG1上调自噬水平促进肝癌转移。阐明AEG1、自噬、失巢凋亡与肝癌转移之间的关系及分子机制，以期寻找抑制转移的潜在干预靶点。

肝癌在我国高发，早期及术后均易发生血管转移，严重影响患者预后。肿瘤细胞脱离原癌巢渗入循环系统称为失巢，而失巢凋亡抵抗是促进转移的重要因素。在失巢状态下，循环肿瘤细胞失去基质粘附，处于不利环境中，有可能会发生自噬，以促进肿瘤细胞存活。我们前期研究发现，肝癌细胞的转移潜能与失巢凋亡抵抗明显相关，抑制自噬能促进肝癌细胞失巢凋亡。AEG-1在肝癌细胞中高表达，与肿瘤转移和失巢凋亡抵抗密切相关，且可能诱导自噬。本课题探讨AEG-1、自噬、失巢凋亡与肝癌转移之间的关系及分子机制，以期寻找抑制转移的潜在干预靶点。..本课题建立了肝癌细胞体外失巢培养模型，利用质粒和小分子干扰RNA，分别在肝癌细胞系SMMC-7721和HCC-LM3中上调和下调AEG-1的表达，western blot和流式细胞术检测细胞凋亡的改变；同时western blot、RFP-GFP-LC3双荧光系统检测自噬水平。利用siRNA干扰ATG探索自噬在失巢凋亡中的作用，以及探索AEG-1诱导自噬的关键信号通路及其对失巢凋亡的影响。最后将SMMC-7721细胞通过慢病毒转染AEG-1和ATG7 shRNA，注入裸鼠尾静脉，建立转移癌模型，探索抑制自噬对AEG-1促进肝癌转移的影响。..结果发现：1）AEG-1能增强肝癌细胞失巢状态下凋亡抵抗，促进细胞存活，抑制AEG-1则取得相反效果；2）在失巢状态下肝癌细胞自噬流增加，而自噬被ATG5或ATG7 siRNA分子阻断后，细胞失巢凋亡增加，细胞存活率下降；3）AEG-1在失巢状态下诱导自噬，干扰自噬可降低AEG-1的促失巢凋亡抵抗作用，证实AEG-1通过诱导自噬促进失巢凋亡抵抗；4）AEG-1在失巢状态下可激活PERK-eIF2α-ATF4-CHOP内质网应激信号通路，干扰此信号通路可抑制AEG-1诱导的自噬，降低AEG-1的促失巢凋亡抵抗；5）体内转移癌模型中，AEG-1促进肝癌细胞在裸鼠肝和肺中的转移，而干扰ATG7抑制自噬可部分逆转AEG-1 的促转移作用。..综上所述，AEG-1可通过诱导自噬，促进肝癌细胞失巢凋亡抵抗，促进肝癌转移，在此过程中PERK-eIF2α-ATF4-CHOP信号通路可能参与其中，AEG-1可能是肝癌治疗的一个潜在干预靶点。