癌蛋白DEPDC1调控肝癌细胞自噬和凋亡的机制及其靶向干预研究

Hepatocellular carcinoma (HCC) is still threatening people’s life in our country although significant progresses have been made during last several decades, which demands further efforts to reveal its mechanisms and discover more effective treatments. DEPDC1 is recently discovered oncoprotein in bladder cancer cells, which promotes carcinogenesis by inhibiting apoptosis. More recently, it was reported that chemotherapy drug vincristine induces apoptosis via targeting DEPDC1. In our previous work, we detected DEPDC1 mRNA levels in 205 cases of HCC samples, and found whose expression is upregulated in 70% samples. Moreover, its expression could be independent predictor for bad prognosis. In this project, we will perform experiments to study the role of DEPDC1 on apoptosis and autophagy in HCC cells by siRNA techniques and Western blot, and then elucidate the mechanisms by which DEPDC1 regulates apoptosis and autophagy by using interfering peptides that is capable of disrupting the complex of DEPDC1 and ZNF224 and by NF-kB and JNK inhibitors. Finally, we will perform experiments in nude mice by injecting HCC cells in which DEPDC1 expression is silenced by shRNA to animals to confirm the vitro findings and unveil the role of DEPDC1 on tumorigenesis and the efficacy of peptides on HCC tumor interference.

癌蛋白DEPDC1在多种癌组织中表达，它在膀胱癌中与ZNF224形成复合物抑制A20表达，导致NF-κB激活而发挥抗凋亡作用；在HeLa和MCF-7细胞中长春新碱通过DEPDC1激活JNK促进细胞凋亡。但不清楚DEPDC1是否调控自噬。我们发现DEPDC1在肝癌中表达，但是它在肝癌中的作用及机制尚无报道。本项目用siRNA技术将肝癌细胞DEPDC1基因沉默，然后用Western blot等方法检测细胞凋亡指标和自噬指标，揭示DEPDC1对细胞凋亡和自噬的影响；使用干扰多肽、NF-κB和JNK的抑制剂等处理细胞后，检测细胞凋亡和自噬的变化来阐明DEPDC1的调控机制；在动物水平检测DEPDC1沉默对肿瘤生长及细胞凋亡和自噬的影响，以及DEPDC1-ZNF224复合物干扰多肽对肿瘤的干预效果。本项目的完成不仅有助于了解DEPDC1在肝癌发生中的作用机制，而且可能发现新的干预靶点和药物。

过往的研究表明，含DEP结构域的蛋白质1（DEPDC1）在多种癌组织中高表达。在膀胱癌细胞中，它与锌指蛋白224（ZNF224）形成复合物以抑制A20表达，从而激活了核因子（NF）-κB信号通路。本项目组的前期结果显示DEPDC1在肝癌中高表达，且与患者不良预后相关。但它在肝癌中的作用机制尚未见报道。.本项目的研究目的是阐明DEPDC1在肝癌发病中的作用机制以及探索其是否可以作为肝癌治疗的靶标。我们首先检测了几种肝癌细胞中DEPDC1的表达水平，发现在HepG2细胞中DEPDC1表达最高，于是用HepG2细胞进一步实验。用siRNA敲低DEPDC1之后 ，用流式细胞仪测定细胞凋亡结果显示：敲低DEPDC1表达可以显著地诱导HepG2细胞凋亡。用siRNA敲低DEPDC1之后 ，用流式细胞仪测定细胞自噬结果显示：敲低DEPDC1表达可以显著地诱导自噬标志分子p62蛋白降解以及LC3-I向LC3-II转变。此结果说明敲低DEPDC1表达可以诱导HepG2细胞自噬。将HepG2细胞分别用对照多肽（3uM）和 11R-DEP:611-628多肽（3uM）处理，之后用TUNEL法和western blot法测定细胞凋亡，结果显示11R-DEP:611-628多肽诱导细胞凋亡。将HepG2细胞分两组分别用对照多肽（3uM）处理和11R-DEP:611-628多肽（3uM）处理。处理后用real-time PCR测定A20表达，用免疫荧光染色测定NF-κB胞内定位，结果显示：11R-DEP:611-628多肽诱导A20表达升高，抑制NF-κB核转位。11R-DEP:611-628多肽联合JNK抑制剂处理后，检测对HepG2细胞凋亡。结果显示：11R-DEP:611-628多肽诱导细胞凋亡；抑制JNK活性能够增强11R-DEP:611-628多肽的效果。裸鼠成瘤实验结果显示：敲低DEPDC1之后，HepG2细胞成瘤能力显著减弱；11R-DEP:611-628多肽具有抑制HCC肿瘤生长的功效。.总之，本项目的研究阐明了DEPDC1在肝癌发病中的机制是通过与ZNF224形成复合物以抑制A20表达，从而激活了NF-κB信号通路。并证明DEPDC1可以作为肝癌治疗的靶标，11R-DEP:611-628多肽具有临床应用前景。