Study On The Effect Of Knockdown CZNF292 On Radiosensitivity Of Hypoxic Hepatoma Cells And Its Mechanisms

Objective:This research aims to establish a hepatoma cell line that stably knocks down circRNA cZNF292 to investigate the effect and its mechanisms of knockdown cZNF292 on radiosensitivity and the biological functions of hypoxic hepatocellular carcinoma cell,such as cell cycle,proliferation,apoptosis and vasculogenic mimicry.Methods:Real-time fluorescent quantitative PCR?qRT-PCR?was used to detect the expression of four circRNAs induced by hypoxia in hepatoma cells.The cZNF292 shRNA lentiviral expression vector was constructed and the SMMC7721 cell line stably knocked down cZNF292 was established after transfection.The control and knockdown cells were cultured in normoxic and hypoxic?1%O₂?environments,respectively.The cell proliferation ability was detected by CCK8 assay;the cell cycle and apoptosis were detected by flow cytometry;the expression levels of?-catenin,CyclinD1,C-myc,and p21 protein were detected by Western blot;The formation of tube-like structures of SMMC7721 cells in vitro was detected by vasculogenic mimicry?VM?;the effects of knockdown cZNF292 on the radiosensitivity and DNA damage repair ability of SMMC7721 cells were detected by colony formation assay and?H2AX immunofluorescence staining,respectively.The co-immunoprecipitation of RNA binding protein,qRT-PCR and Western bolt were used to detect the interaction of cZNF292 with the corresponding protein and its regulation mechanism of Wnt/?-catenin signaling pathway.Results:The expression level of circRNA cZNF292 in SMMC7721 cells gradually increased with prolonged hypoxic culture time.The stably transfected cZNF292 shRNA constructed by lentiviral vector can effectively down-regulate the expression of cZNF292.Under normoxic and hypoxic culture conditions,knockdown of cZNF292 induced G1 arrest and significantly decreased proliferation.Underhypoxic culture conditions,knocked-down cZNF292 significantly decreased the expression of?-catenin,CyclinD1 and C-myc protein in hypoxic SMMC7721 cells and increased the expression of p21 protein in hypoxic conditions.Under normoxic and hypoxic cultures,the ability to form tube-like structures in the knockdown group was inhibited.Under hypoxic conditions,knockdown of the cZNF292 group associated with vasculogenic mimicry of target proteins including?-catenin,Twist1 and VE-cadherin protein levels decreased significantly.Under normoxic or hypoxic culture,the radiosensitivity of the knock-down group was significantly higher than that of the control group.The number of?H2AX foci in the knock-down group was significantly higher than that of the control group.The level of DNA-PKcs?S2056?in the knock-down cZNF292 group was significantly increased under hypoxic conditions,and there was no significant change in the level of ATM?S1981?protein.cZNF292 is located in the cytoplasmic,and can be pulled down by SOX9 antibody through RIP.Knockdown of cZNF292 significantly reduced pull-down SOX9 protein level,but did not affect the expression level of SOX9 protein.Conclusion:Hypoxia induces the expression of cZNF292 in hepatocellular carcinoma cell line SMMC7721.Knockdown of cZNF292 leads to cell cycle arrest and proliferation inhibition in both normoxic and hypoxic hepatocellular carcinoma,reduced formation of tube-like structures,and enhanced radiosensitivity.The mechanism may be related to the knockdown of cZNF292 to increase the nuclear translocation of SOX9 and significantly reduce the activity of Wnt/?-catenin pathway.