A Study Of Cif Inhibiting Colorectal Cancer Cell Proliferation By Targeting Cullin-RING Ubiquitin Ligase

Objective:Studies have shown that pathogenic E.coli-related virulence factor Cif(cycle inhibiting factor)may affect the occurrence and development of tumors.In this study,we investigated the effect of Cif effector protein on the biological behavior of human colon cancer cell lines SW620 and HCT116 from different technical perspectives,and further explored its possible mechanism.Methods:Wild type Cif(HA-cifWT)and mutant Cif(HA-cifC/A)were induced by tetracycline-induced expression system(Tet-on system).The expression of HA-cifWT and HA-cifC/A effect protein was regulated by Tet-on system,and the empty vector was used as a blank control.Stable cell lines were induced by doxycycline(Dox),and the expression of Cif effect protein was detected by Western blot.The effect of Cif effector protein on the cell activity and clonogenic ability of colorectal cancer cells was analyzed by cloning assay and MTT.GFPdgn protein was used as an indicator system to analyze the expression level of GFPdgn protein by fluorescence microscopy to evaluate the effect of Cif effector protein on Cullin-RING Ligase(CRL)activity in tumor cells.Western blot was used to analyze the accumulation of key CRL substrates in tumor cells involved in the regulation of DNA replication/damage,cell cycle progression,apoptosis and senescence.Results:Western blot has identified colon cancer cell line HCT116/SW620-HA-cifWT and HCT116/SW620-HA-cifC/A,which can induce cifWT and cifC/A^ effect proteins,respectively.The results of clonal formation and MTT confirmed that the cell activity and the ability of clonal formation of HA-cifWT group was significantly lower than that of HA-cifC/A group and blank control group(p<0.05).The results of fluorescence microscopy showed that GFPdgn protein was high in the cell line HCT116/SW620-HA-cifWT and low in HA-cifC/A and blank control group.Western blot analysis showed that Cif effector protein could promote the accumulation of p21 and p27 protein in CRL substrate.Conclusion:The above results indicate that the Cif effector protein can specifically inhibit the activity of CRL,thus blocking the ubiquitination of its substrate such as p21 and p27 protein,and finally achieve the goal of inhibiting the proliferation of colorectal cancer cells.