Depletion of GP73 inhibits invasion and metastasis of hepatocellular carcinoma cells / 中华肿瘤杂志

ABSTRACT

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 G1 phase upon treatment with cell synchronization reagents (mimosine), and the percentage of cells in G2/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) mm3, significantly smaller than (1 200±110)mm3 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.