Molecular Mechanism Of G6PC On Invasion And Metastasis Of Cervical Cancer Hela Cells

Objective: To explore the effect of glucose-6-phsophatase,catalytic subunit(G6PC)on the proliferation,migration and invasion,apoptosis of cervical cancer Hela cells and the possible molecular mechanism,provide a new direction and theoretical basis for G6 PC as a new target for clinical treatment of cervical cancer.Methods: Human Protein Atlas(HPA)database were performed to analyze the protein expression of G6 PC in cervical cancer.The expression of G6 PC was confirmed by western blot in normal cervical epithelial cells and different cervical cancer cell line.Then,the cells with high expression of G6 PC were screened for further study.RNA interfering(RNAi)was used to knockdown the expression of G6 PC in cervical cancer Hela cells,and the silencing effect of protein was confirmed by western blot.The MTT method were performed to detect the effect of knocking down G6PC(24h,48 h,72h)on the proliferation of cervical cancer Hela cells.The colony forming ability of cells were observed by colony formation assay.Scratch healing experiment were applied to observe the effect of G6 PC knockdown on lateral migration ability of Hela cells.Transwell chamber experiment were used to detect the effect of knocking down G6 PC on the longitudinal migration and invasion ability of cervical cancer Hela cells.The angiogenic ability of human umbilical vein endothelial cells(HUVECs)cultured in the supernatant of cervical cancer Hela cells treated with knockdown of G6 PC was detected by the angiogenesis experiment.The cell apoptosis was evaluated by Annexin-V/PI double staining and Hoechst 33342 fluorescence staining.Last,the Vascular endothelial growth factor,EMT related proteins,apoptosis related proteins expression were detected by western blot.The expression of total and phosphorylated AKT and m TOR were determined by western blot analysis.Results:1.G6 PC expression was overexpressed in cervical cancer tissues and cells:HPA database retrieval found that G6 PC was overexpression in cervical cancer tissues.Westeren blot experiments showed that the expression of G6 PC in normal cervical epithelial cells was significantly lower than that in cervical cancer cell lines.And the expression was higher in Hela cells,so Hela cells are selected for subsequent experiments.2.Knockdown of G6 PC inhibits the proliferation and induced apoptosis of Hela cells: MTT and colony formation results showed that knockdown of G6 PC expression significantly decreased the proliferation and colony formation ability of the Hela cells(P<0.01 or <0.05).The cell morphology was observed by Hoechst33342 fluorescence staining.The results showed that the cervica cancer Hela cell with knockdown of G6 PC gene shoued obvious apoptotic features such as nuclear pyopysis and fragmentation.Annexin V-FITC/PI double staining test results showed that compared with the control group,knockdown of G6 PC expression cause apoptosis rate increased,Western blot also show that Cleaved-caspase 3,Cleaved-caspase 9 expression are raised(P<0.05),and resistance to apoptosis protein expression of Bcl-2 is lower(P<0.05),the ratio of Bax/Bcl-2 elevated(P<0.01),the expression of PARP lowered(P<0.05).3.Knockdown of G6 PC inhibits the migration,invasion,angiogenesis and EMT process Hela cells: Wound healing and transwell assay showed that knockdown of G6 PC expression significantly inhibited the migration and invasion of Hela cells(P<0.05 or <0.01).Compared with the control group,the angiogenesis ability of human unbilical vein endothelial cells cultured in the supernatant of Hela cells was significantly reduced after knockdown of G6 PC expression(P<0.01).Western blot results showed that knockdown of G6 PC up-regulated the expression of epithelial maker E-cadherin(P<0.05)and down-regulated the expression of interstital cell maker Vimentin and transcriptional factors Snail(P < 0.05).4.Knockdown of G6 PC inhibits the PI3K/ Akt /m TOR signaling pathway: Western blot assay was used to detect the expression levels of related proteins such as Akt /m TOR signaling pathway,and the results showed that the expression levels of p-m TOR and p-Akt were significantly decreased(P < 0.05).Conclusion: 1.Knocking down G6 PC gene can inhibit the proliferation of cervical cancer He La cells and induce their apoptosis.2.Knocking down G6 PC gene inhibited the invasion and metastasis of He La cells by reversing EMT and inhibiting angiogenesis.3.The effect of knockdown of G6 PC gene on the phenotype of cervical cancer He La cells may be related to the inhibition of PI3K/ Akt /m TOR signaling pathway.