| Objective:Speckle?type POZ protein(SPOP)is one of the substrate recognizers of E3 ubiquitin ligase.It plays an important role in many cancers.Previous experimental data indicated that SPOP plays a tumor-suppressive role in choriocarcinoma,but the specific mechanism by which SPOP regulates choriocarcinoma is still unclear.This article aims to explore the mechanism of SPOP and its ubiquitinated substrate regulating choriocarcinoma through the ubiquitination pathway.Methods:(1)The overexpression and interference lentiviral model of SPOP was transfected into choriocarcinoma JAR cells,and its overexpression efficiency was verified by q RT-PCR and western blot experiments.The combination of label-free ubiquitination modification quantitative proteomics results,SBC motifs,early SPOP research results and the highly invasive features of choriocarcinoma were used for screen out related proteins.(2)The expressions of KIF23 in extravillous trophoblast cell HTR-8/SVneo,choriocarcinoma JAR and JEG3 cells were detected by immunoblotting.The subcellular localization of KIF23 was detected by immunofluorescence assay.(3)Co-immunoprecipitation and western blot experiments were used to detect the relationship between SPOP and Kinesin family member 23(KIF23).(4)After silencing KIF23 with small interfering RNA,q RT-PCR and western blot experiments were used to select the KIF23-si RNA1 with the best interference efficiency.CCK8,colony-forming assay,western blot and transwell assay were used to investigate the effect of KIF23 on the proliferation,invasion and migration ability of JAR cells and the changes of phosphorylation levels of protein Akt and GSK3?.(5)Label-free ubiquitination modified quantitative proteomics,coimmunoprecipitation experiments,and SBC motifs were combined to screen out related protein.(6)Nuclear DNA helicase II and RNA helicase A(DHX9)expression and subcellular localization were investigated in HTR-8/SVneo cells,JAR cells and JEG3 cells using western blot and immunofluorescence assay.(7)Detection of endogenous interaction of SPOP and DHX9 in JAR cells and JEG3 cells by co-immunoprecipitation.This assay was used for explored the interaction between the SPOP and DHX9 in J AR cells over-expressing Flag-SPOP or HA-DHX9,293 T cells co-exp ressing Flag-SPOP and HA-DHX9,and the 293 T cells co-expressing Flag-SPOP and HA-DHX9-?SBC.(8)The effects of SPOP on DHX9 degradation and ubiquitination were observed by q RT-PCR,western blot and endogenous ubiquitination experiments.(9)Western blot,q RT-PCR and endogenous ubiquitination experiments were used to observe the effect of SPOP on the degradation and ubiquitination of DHX9.Western blot,q RT-PCR and transwell assay were used to observe the effect of the interaction between SPOP and DHX9 on cell invasion and migration.Western blot was used to detect the influence of the correlation between SPOP and DHX9 on the expression of EMT-related proteins.Results:(1)We constructed a JAR cell model of overexpression and knockdown and verified the efficiency from the m RNA level and protein level.Based on the results of related experiments,the kinesin KIF23 was screened out(Treat/Control > 1.50).(2)Compared with HTR-8/SVneo,KIF23 was highly expressed in choriocarcinoma cells JAR and JEG3.KIF23 is mainly located in the nucleus,which is the same as SPOP.(3)Compared with the control group,the expression of KIF23 was decreased in overexpressed SPOP group,and the expression of KIF23 was increased in the knockdown SPOP group.KIF23 was precipitated by FLAGSPOP through co-immunocoprecipitation technology,and no corresponding band of KIF23 in immunoblotting.(4)KIF23-si RNA1 could effectively silence KIF23 in JAR cells at both m RNA levels and protein levels.The KIF23-si RNA group could significantly inhibit the proliferation,the invasion and migration ability of JAR cells,total Akt and GSK3? protein levels were unchanged,p-Akt and p-GSK3? protein levels were significantly down-regulated.(5)Based on the results of related experiments,DHX9 protein was further screened.(6)DHX9 was highly expressed in JAR cells and JEG3 cells than in HTR-8/SVneo cells,it is the same localization as SPOP,DHX9 was localized in the nucleus of HTR-8/SVneo cells,JAR cells and JEG3 cells.(7)DHX9 could co-precipitate SPOP in choriocarcinoma JAR cells and JEG3 cells.Anti-Flag or anti-HA could co-precipitate DHX9/SPOP,and western blot could detect the corresponding band in 293 T cells co-expressing Flag-SPOP and HA-DHX9 and JAR cells over-expressing Flag-SPOP or HA-DHX9.However,in 293 T cells co-expressing Flag-SPOP and HADHX9-?SBC,Flag-SPOP could not pull down HA-DHX9-?SBC,and there was no DHX9 corresponding protein band in western blot.(8)DHX9 expression was significantly reduced in JAR cells overexpressing SPOP,and DHX9 expression were significantly increased in JAR cells silenced SPOP.The changes of DHX9 at the m RNA level in the cell model were not statistically significant.When MG132 was added to the JAR cells with SPOP overexpression,the expressions of DHX9 increased significantly,and the endogenous ubiquitination assays were positive.(9)Both the m RNA levels and the protein levels successfully constructed the DHX9 gene silencing JAR cell models.Compared with the sh-NC group,JAR cells knockdowning DHX9 significantly inhibited the invasion and migration of JAR cells,and the expressions of N-cadherin and vimentin were significantly reduced.However,E-Cadherin and cytokeratin 8 expressions were not statistically different.Compared with the sh-SPOP group,the group of sh-SPOP+sh-DHX9 significantly inhibited the invasion and migration ability of JAR cells,N-Cadherin and vimentin expression were significantly reduced,while E-Cadherin and cytokeratin 8 expressions were not statistically different.Conclusion:(1)There is no interaction between KIF23 and SPOP,but SPOP can promote degradation of KIF23,and KIF23 may promote the proliferation,invasion and migration of JAR cells by activating the Akt/GSK3? pathway.(2)SPOP attenuates JAR cell migration and invasion by promoting ubiquitination and degradation of DHX9. |
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