Effect Of Aqu Aporin9 On The Proliferation, Apoptosis, Invasiveness And Migration Of HepG2 Cell Line

Objective To explore the impact of aquaporin9 on the proliferation, apoptosis, invasiveness and migration of HepG2 cell line.Methods Lentiviral vector targeting the coding region of human AQP9 was constructed. The recombinant lentiviral vector was harvested from 293T cell and was utilized to transfect HepG2 cell line, and resistant cell clones were selected with puromycin. Transfection efficiency was validated by Laser confocal. Expression of AQP9 in HepG2 cell line was detected by western blot and real-time qPCR at protein and mRNA levels. There were three group:CC group without lentiviral vector、PWPI group with empty carrier slow virus、overexpression of AQP9 group with gene expression AQP9 slow virus vector. Plate colony formation assay, the wound healing assay, transwell assay and flow cytometry detection were applied to evaluate the change in proliferation, migration and invasion ability of HepG2 cell line which overexpressed AQP9 as well as in apoptosis and cell cycle.Results Green fluorescen protein was showed by confocal laser which distributed in the membrane. Overexpressed of AQP9 was observed in overexpression of AQP9 group at mRNA and protein levels compared with PWPI group and CC group. There were statistically significant differences(p<0.01).Plat clone experiments showed that AQP9 was capable of reducing the cells clone formation rate from (23.53±2.10)% in CC group and (23.00±2.02)% in PWPI group to(16.93±3.19)% in overexpression of AQP9 group. There was statistically significant difference among the three groups(F=6.46,P=0.032<0.05).FCM showed the overall apoptosis rate in overexpression of AQP9 group was(44.96±3.53)%, PWPI group was (24.37±2.38)% and CC group was(19.7±2.49)% respectively. There was statistically significant difference among the three groups(F=66.88, P<0.01),The apoptosis rate was increased in overexpression of AQP9 group compared to PWPI group and CC group with statistical significant differences (p<0.01).Likewise, Flow cytometry detection found that AQP9 increased cell number in G1 stage and decreased cell number in S stage (G1=(66.58±0.99)% and S=(15.25±1.81%).It was statistically significant differences compared with other groups. The wound healing assay showed the cell migration distance in overexpression of AQP9 group was less than PWPI group and CC group with statistically significant differences among groups(P=0.010.05). Transwell experiments showed that the invasion of HepG2 cells were significantly suppressed by AQP9 (P=0.01<0.05). Numbers of cell permeating septum was 17±8 in overexpression of AQP9 group, meanwhile 95±11 in PWPI group and 109±9 in CC group. Statistically significant difference was existed (p<0.05).Conclusion AQP9 significantly inhibits the capabilities of migration and invasiveness of HepG2 cells, induces cell apoptosis and inhibits cell proliferation via causing cell cycle arrest at Gl/S phases.