Objective: To explore the in vitro and in vivo effect of GP73 on the proliferation, invasion and metastasis in hepatocellular carcinoma. Methods: GP73 gene was knocked out using CRISPR/Cas9 gene editing system in H22 and HepG2 cells, and stable knock out strains were constructed. The knockout efficiency was measured by western blot. Colony formation assay was used to detect the effect of GP73 on long-term survival ability. Cells were then highly synchronized in G(1) phase upon treatment with cell synchronization reagents (mimosine), and the percentage of cells in G(2)/M phase at different time points was detected by flow cytometry. The invasive and metastasis abilities of hepatocellular carcinoma cells were detected by Transwell™ assay. Furthermore, the tumor formation abilities in vivo were examined using subcutaneous xenograft models. Results: The stable knock out strains of GP73 in H22 and HepG2 cells were successfully established via puromycin selection. The number of colonies of GP73 knock out groups in HepG2 and H22 cells 10 days after transfection were 400±70 and 248±60, respectively. They were significantly lower than those in the control groups (980±40 and 1 100±50, respectively; P<0.01). In addition, GP73 knockout slowed down the cell cycle progression. Moreover, the cell numbers that had migrated to the underside of the filters were 312±50 and 305±49 in the GP73 knockout groups of HepG2 and H22 cells, respectively, significantly lower than 1 540±87 and 1 270±86 in the controls (P<0.01). For transwell invasion assay, the cell number that had invaded into the underside of the filters were 230±47 and 238±54 in the GP73 knockout groups of HepG2 and H22 cells, respectively, significantly lower than 648±74 and 596±63 in the controls(P<0.01). Furthermore, the tumor volume of GP73 knockout group was (70±170) mm(3,) significantly smaller than (1 200±110)mm(3) of the control guoup (P<0.01). Conclusions: GP73 knockout decreases the proliferation, invasive and migratory abilities of HepG2 and H22 cells in vitro and in vivo. GP73 may contribute to tumorigenesis of hepatocellular carcinoma.
目的： 探讨高尔基体蛋白73(GP73)在肝癌细胞增殖、转移和小鼠肿瘤形成中的作用。 方法： 应用pX459－GP73－sgRNA重组质粒转染HepG2和H22肝癌细胞，通过筛选获得敲除GP73的细胞株。采用Western blot检测GP73在HepG2和H22细胞中的表达，采用平板克隆形成实验检测GP73对细胞生长能力的影响，采用流式细胞仪检测G(2)/M期细胞比例，采用Transwell实验检测GP73对细胞侵袭能力的影响，采用皮下种植实验检测GP73对H22细胞肿瘤形成能力的影响。 结果： 经嘌呤霉素筛选获得稳定敲除GP73蛋白的H22和HepG2细胞。GP73敲除10 d后，HepG2细胞GP73－sgRNA转染组的细胞克隆数[(400±70)个]少于阴性对照组[(980±40)个]；H22细胞GP73－sgRNA转染组的细胞克隆数[(248±60)个]少于阴性对照组[(1 100±50)个]，差异均有统计学意义(均P<0.01)。敲除GP73蛋白后，细胞周期进程减慢。Transwell迁移实验中，HepG2细胞GP73－sgRNA转染组和阴性对照组的穿膜细胞数分别为(312±50)个和(1 540±87)个；H22细胞GP73－sgRNA转染组和阴性对照组的穿膜细胞数分别为(305±49)个和(1 270±86)个，差异均有统计学意义(均P<0.01)。侵袭实验中，HepG2细胞GP73－sgRNA转染组和阴性对照组的穿膜细胞数分别为(230±47)个和(648±74)个；H22细胞GP73－sgRNA转染组和阴性对照组的穿膜细胞数分别为(238±54)个和(596±63)个，差异均有统计学意义(均P<0.01)。GP73敲除后，小鼠的肿瘤体积为(570±170)mm(3)，小于对照组[(1 200±110)mm(3)，P<0.01]。 结论： 敲除GP73蛋白能减弱H22和HepG2肝癌细胞的侵袭和迁移能力，GP73的表达可能与肿瘤侵袭、增殖和转移有关。.
Keywords: Golgi protein 73; Liver neoplasms; Mice; Neoplasm metastasis.