Effect Of SENP2 On The Stability Of NDRG2 In Grstric Cancer Cells And Its Regulatory Mechanism

Background: Gastric cancer is one of the most common malignancies worldwide,it has the second highest incidence and mortality rate of all cancers,second only to lung cancer.Although early gastric cancer patients undergo radical surgery followed by chemotherapy,and the postoperative5-year survival rate is 90%,However,the detection rate is low because of the lack of specific diagnosis markers of early gastric cancer,and therefore,most patients(>70%)develop advanced-stage gastric cancer and,need to be treated with molecule-targeted drugs.So to investigate the pathogenesis gastric cancer can provide specific diagnosis markers for gastric cancer and provide molecular targets for the research on targeted drug,which have great significance for the prevention and treatment on gastric cancer.The SUMO-specific proteases(SENPs)family has six members,including SENP1~3 and SENP5~7,are involved in tumorigenesis.,SENP2 has been reported to be a tumor suppressor in various cancers,and demonstrates the role of tumor suppression by de-SUMOization to maintain protein stability and inhibit activation of tumor-associated signaling pathways.N-Myc downstream regulated gene 2 also belongs to the tumor suppressor,and is inactivated or degraded in the tumor tissue by methylation and SUMOylation,thereby losing the tumor suppressing effect.However,the expression characteristics of SENP2 in GC tissues and themechanism of action on the stability of NDRG2 is unkown.Objective: In this study,we aim to investigate the expression characteristics of SENP2 in gastric cancer tissues and its effect on the proliferation of GC cells.Further to investigate the mechanism of action on the stability of NDRG2 in GC cells.Methods:1.The mRNA and protein expression levels of SENP2 in gastric cancer tissues were tested by reverse transcription-real-time quantitative polymerase chain reaction and western blot.2.The mRNA and protein expression levels of SENP2 in gastric cancer cell lines(AGS,BGC823,MGC803,NCI-N87 and SGC7901)were tested by reverse transcription-real-time quantitative polymerase chain reaction and Western Blot.The cell line with highest expression of SENP2 was selected to observe the effect on cell proliferation after silencing the SENP2.3.The interaction of SENP2 and NDRG2 were tested by co-immunoprecipitation.4.Overexpressing the SENP1~3 and SENP5~7 vector in AGS cells,respectively,and,to investigate the role on the stability of NDRG2 by Western blot.5.To investigate effects on NDRG2 stability and ubiquitination after silencing the SENP2 in AGS cells by Western blot or co-immunoprecipitation.6.Overexpressing the HA-NDRG2+Myc-Sumo1 plasmid,HA-NDRG2+Flag-SENP2 WT+Myc-Sumo1 plasmid,HA-NDRG2+Flag-SENP2 CA+Myc-Sumo1 plasmid in 293 T cells,respectively.Meanwhile,using the MG132 treatment the AGS(Vector)and AGS(SENP2-shRNA)cell.andthen,to investigate the role of SENP2 on the deSUMOylates of NDRG2 by co-immunoprecipitation.Results:1.The mRNA and protein expression levels of SENP2 in GC tissues were significantly lower than those in adjacent normal tissues(P<0.001).2.Compared with the control group,SENP2 in silencing AGS cells significantly promoted the proliferation of AGS cells(P<0.01).3.The results of co-immunoprecipitation showed that SENP2 and NDRG2 could directly interact.4.The western blot results showed that the stability of NDRG2 in AGS cell transfected with SENP2 overexpression vector was significantly increased than the AGS cell transfected with vector,SENP1,SENP3,SENP5,SENP6 and SENP7 overexpression vector.5.Co-immunoprecipitation assay showed that SENP2 in silencing AGS cells significantly increased the ubiquitination level of NDRG2.6.Mutation or silencing of SENP2 increased the SUMOylation level of NDRG2.Conclusion:1.The expression levels of SENP2 significantly decreased in gastric cancer tissues and promote the proliferation of gastric cancer cell.2.SENP2 is the only member of the SENPs family that enhances the stabilization of NDRG2.3.One of the mechanisms by which SENP2 maintains NDRG2 stability in GC cells is inhibition of SUMOylation and ubiquitination of NDRG2.