Hint1与Girdin/Akt及Src信号通路串话在肝癌细胞增殖中的调控机制

In our previous study, we found that the Histidine triad nucleotide-binding protein (Hint1) function as a tumor suppressor gene in hepatoma and colon cancer. However, the detailed mechanism of HINT1 function as a tumor suppressor gene is still unknown. Girders of actin filaments (Girdin) is a new funding protein which can increase the cell growth through Akt/JNK signaling pathway and crosstalk with Src signaling pathway. There is no report on Girdin affecting on the up regulating hepatoma cell growth. In this study, we will detect the detailed mechanism of Hint1 down regulating Src/Girding/Akt signaling pathway and inhibiting the cell growth in hepatoma cells. First, the function and mechanism of Girdin/Akt signaling pathway on cell growth in hepatoma cells will be detected. The shRNA plasmid which block the expression of Girdin and Src will be constructed and transferred into HepG2 cells. The growth inhibition affects both in hepatoma cells and tumors in nude mice model which is transplanted hepatoma cells will be detected by shRNA-Girdin and shRNA-Src transduction. Western-blot and Real-time PCR will be used to detect the expression of phospholaration of Akt and Girdin and other proteins down stream. The con-focal microscope and co-imunoprecipitation will be used to detect the formation of Girdin/Akt protein complex in hepatoma cells. HepG2 cells will be co-transfected with NF-κB/Luc report gene and shRNA Girdin or and shRNA Src, the transcription factor activity will be detected by luciferase report assay. Second, HINT1 down regulate Src/Girdin/Akt signaling pathway by forming an immune protein complex in hepatoma cell. pHA-hint1 will be transfected into HepG2 cells to over-expression Hint1. Western blot and Real-time PCR will be used to detect the expression of phospholaration of Girdin, Akt and Src Expression in hepatoma cells and nude mice transplanted with hepatoma cell. Then, the con-focal microscope and co-imunoprecipitation detection will be used to detect the formation of Hint1/Girdin protein compress in vivo. HepG2 cells will be co-transfected with NF-κB/ report gene and pHA-Hint1 or pFlag-Girdin, the transcription factor activity will be detected by luciferase report assay. Finally, the clinical samples of Hepatocellular carcinoma from surgical operation will be collected. The expression of Hint1, Girdin and Akt or other proteins downstream will be detected by immuno histochemistry. Also, the expression, co localization of GIRDIN, AKT and HINT1 will be detected by con-focal microscope. The relationship between clinical manifestation of the hepatocellular carcinoma and HINT1/AKT signaling pathway will be analyzed. Thus, we will conclude that activation of Src/Girdin/Akt signaling pathway will increasing the cell growth in hepatoma cells. Hint1 function as a tumor suppressing gene in HCC cells is, at least in part, by the inhibition of Girdin/Src signaling pathway.

课题组前期研究发现三组氨酸核苷结合蛋白1（Hint1）通过与POSH, JNK2，USF2等蛋白结合而调控肝癌细胞增殖，但其抑癌机制仍未阐明。新近报道肌动蛋白细丝桥梁蛋白 (Girdin)可促进肿瘤细胞增殖，并参与经典Src信号通路调控。综合前期研究，Hint1可能通过与Src/Girdin通路串话调控肝癌细胞增殖。本项目通过体外、体内及临床相关研究，探索上述机制：① Girdin/Akt通路激活调控肝癌细胞增殖的作用及机制；Src对Girdin/Akt磷酸化、下游蛋白表达、转录因子活性影响； ② Hint1抑制 Src/Girdin信号通路而抑制肝癌细胞增殖的作用及机制；Hint1对Src及Girdin磷酸化及蛋白复合体形成的影响；Hint1与Src及Girdin蛋白复合体检测。上述研究，国内外尚无文献报道。本项目研究对探索发现抗肝癌药物新的靶向性作用位点及研发抗肝癌生物制剂有重要意义。

课题组前期研究发现三组氨酸核苷结合蛋白1（Hint1）通过与POSH, JNK2，USF2等蛋白结合而调控肝癌细胞增殖，但其抑癌机制仍未完全阐明。新近报道肌动蛋白细丝桥梁蛋白(Girdin)可促进肿瘤细胞增殖，并参与经典Src信号通路调控。综合前期研究，Hint1可能通过与Src/Girdin通路串话调控肝癌细胞增殖。本项目通过临床标本、细胞培养、基因转染、敲减、Western blot、免疫共沉淀、免疫荧光共聚焦显微镜、MTT、集落形成、Transwell小室、裸鼠移植瘤等研究手段，研究Hint1、Src、Girdin、Akt在肝细胞癌生长、迁移、侵袭中功能和分子间相互作用/信号转导通路。有以下主要发现/关键数据：（1）免疫荧光共聚焦显微镜：人肝细胞癌手术标本较癌旁标本Girdin表达明显增高；（2）肿瘤集落形成实验：肝癌细胞敲减Girdin表达，抑制集落形成；（3）Western blot：肝癌细胞敲减Girdin表达，Akt磷酸化受抑制；（4）Western blot：肝癌细胞过表达Hint1质粒，Girdin及其下游Akt磷酸化水平受抑制；敲减Hint1表达，Girdin及其下游Akt磷酸化水平增高；（5）肝癌细胞Hint1与Girdin蛋白免疫共沉淀，且这些共沉淀的Girdin未在pSer1416、pTyr1764、及pTyr1798等关键位点磷酸化；人肝细胞癌手术标本及肝癌细胞Hint1与Girdin共定位；（6）肝癌细胞敲减Hint1，迁移、侵袭能力增加，依赖于下游Girdin表达；肝癌细胞过表达Hint1，凋亡增加；敲减Hint1，凋亡减少，依赖于下游Girdin表达；（7）云南特色中草药灯盏花活性成分灯盏花乙素（Scutellarin）通过抑制STAT3/Girdin/Akt活性抑制肝细胞癌生长、迁移、侵袭。本项目科学意义：（1）提出并验证了Hint1抑制肝癌增殖、侵袭、转移新的分子机制，即Hint1抑制Girdin/Akt通路活化，该抑制可能通过Hint1与Girdin形成蛋白复合物实现；（2）初步验证了灯盏花乙素抗肝癌作用，并发现其新的靶点Girdin/Akt，有可能成为进展期/晚期肝癌病人治疗药物选择。