从Sec62调节内质网应激角度探讨三石猫颗粒抗肝癌术后复发转移的作用机制

The recurrence and metastasis after surgery is one of the leading causes of death for hepatocellular carcinoma patients. We have found that SEC62 in PBMC and HCC tissues of early recurrence patients were both higher than those in late recurrence patients, which was an independent risk factor for recurrence. Sec62 is involved in endoplasmic reticulum stress (ERS),and ERS play a part in the prognosis of tumor and treatment sensitivity, suggesting that high expression of Sec62 maybe critical to recurrence of HCC after surgical resection. Additionally,the ratio of recurrence after liver surgery with Sanshimao treatment was lower than that with TACE treatment in a multi-center, prospective clinical study, but the mechanism is not clear. In ex vivo study, we have found that the containing serum of Sanshimao could inhibit expression of Sec62 in huh7 cell. A question is proposed: If Sanshimao destroy the protection of cell from death by down-regulation of Sec62 regulated ERS to inhibit recurrence of HCC after surgery? We will confirm the relationship between Sec62 and early recurrence after HCC surgery by a prospective clinical study.Taking advantage of a orthotopic xenograft model of HCC, we will observe the effect of Sec62 in HCC recurrence and the role of Sanshimao on Sec62 in the prevention of HCC recurrence in vivo.Furthermore, we will study the effct of Sec62 in regulation of Eendoplasmic reticulum stress (ERS) of HCC cells in vitro and ERS related anoikis, migration and signaling parthway by Flow cytometry, transell, Western-blot,pull-down and RNAi. The study will confirm the potential effect of Sec62 in HCC recurrence after surgery and in treatment of Sanshimao after surgical resection.By this study, we will provide an experimental evidence for traditional chinese medicine (TCM) against HCC recurrence after surgery by Sec62 regulation of ERS and a new strategy for study of TCM anti-tumor recurrence and metastasis.

肝癌术后复发转移是其预后差的主要原因。课题组前期发现，Sec62在肝癌术后早期复发患者中的表达显著高于晚期复发者。Sec62可调节内质网应激（ERS），而后者与肿瘤的预后及治疗敏感性有关，提示高表达的Sec62可能是促进肝癌术后复发的关键基因之一。我们认为"余毒未尽"是肝癌术后重要病机，以清热解毒为主的三石猫颗粒经多中心临床研究证实可显著减少肝癌术后复发，效果优于TACE；三石猫体外可抑制Sec62表达。那三石猫是否通过调节Sec62/ERS途径抑制肝癌术后复发？我们拟通过前瞻性临床研究，进一步证实Sec62与肝癌术后早期复发间的关系，采用裸鼠肝癌术后复发模型，观察Sec62在体内对肝癌术后复发的影响及三石猫的干预作用，体外利用western-blot、pull-down、RNAi等技术从Sec62调控ERS角度探讨三石猫抗肝癌术后复发的分子机制。本研究试图为中药抗肿瘤复发转移提供新思路。

目前,手术仍然是早期肝癌的主要治疗方式，但是大约有30-50%的患者会在术后2年内出现复发。术后复发是肝癌患者的主要死亡原因。我们在以前的临床回顾性研究中发现，Sec62可能是影响肝癌术后复发的独立危险因素。本研究利用Sec62low和Sec62high-Huh7肝癌细胞稳转株，建立裸鼠肝癌原位移植瘤模型，手术切除，小动物活体成像仪观察不同水平Sec62对小鼠肝癌术后复发的影响，发现高表达的Sec62会促进术后复发转移，低表达的Sec62则抑制术后复发，证实了Sec62对肝癌术后早期复发的促进作用；体外采用细胞划痕和Transwell小室对两种肝癌细胞株Huh7和MHCC97H研究发现，高表达的Sec62可能通过促进细胞的迁移、侵袭促进术后转移复发；芯片结果显示，Integrin a通路可能是Sec62调节细胞运动促进细胞迁移侵袭的靶通路；上调该通路上游的Integrin a/CAV1可以逆转Sec62敲除引起的细胞迁移能力下降，证实Integrin a通路在Sec62促进术后复发转移中的作用，免疫共沉淀结果显示，Sec62通过与Integrin a5和 a Ⅴ直接相互作用调节Integrin通路。前瞻性临床研究进一步证实Sec62促进肝癌患者术后早期复发转移，Western-blot检测癌组织中Sec62和Integrin 通路相关蛋白表达升高，与动物细胞实验结果一致，Kaplan-Meier生存分析认为Sec62是肝癌术后复发转移的独立危险因素。.中药三石猫是我科一直以来用于防治肝癌术后复发的院内制剂，经过几十年的临床验证，疗效确切。本研究在证实Sec62促进术后复发作用的基础上，根据上述方法建立人肝癌Huh7细胞原位移植瘤裸鼠模型，手术切除原位瘤3天后，分别使用三石猫水煎剂提取物[18g/(kg·d)]及生理盐水灌胃14 d后，观察肝脏肿瘤复发率，发现三石猫治疗组肿瘤术后复发率（2/6, 33.3%）较对照组（6/6，100%）明显降低，治疗组复发肿瘤组织中Sec62蛋白表达低于对照组; 体外实验中三石猫提取物可以显著抑制肝癌细胞Huh7侵袭、迁移，但是将Huh7细胞中的Sec62过表达后，这种抑制作用明显减弱。认为三石猫可以抑制人肝癌Huh7细胞原位移植瘤手术切除后复发肿瘤的生长，其机制可能与通过降低Sec62表达抑制细胞的侵袭迁移有关。