| PART1 THE EXPRESSION OF PROTEASOME SUBUNIT PSMD2 IN BREAST CANCERObjective: To investigate the expression patterns of ubiquitin-proteasome system(UPS)-associated members in breast cancer(BC).PSMD2 with significant expression difference was selected for further analysis.Correlations between PSMD2 and clinicopathological features,as well as the prognostic value of PSMD2,were investigated.Methods: The expression patterns of 797 UPS-related genes were determined using BC mRNA HiSeq expression data from The Cancer Genome Atlas(TCGA)database.RT-qPCR and immunohistochemistry(IHC)were performed to confirm the expression difference of PSMD2 between BC and normal tissues.The correlation between PSMD2 expression and clinicopathological features of BC patients was determined by Chi-square tests.Kaplan–Meier analysis was used to plot survival curves.Univariate and multivariate Cox regression analyses were used to analyze the prognostic significance of PSMD2 in BC.Results: The expression pattern analysis showed that 208 UPS-related genes were upregulated and 239 genes were downregulated in BC.Among the significantly changed subunits,the mRNA expression level of PSMD2 was the highest in breast cancer tissues.With 32 paired BC and normal tissues,we confirmed that the mRNA expression level was significantlyhigher in BC tissues.PSMD2 protein expression was also examined with immunohistochemistry,and the results showed that PSMD2 immunoreactivity was remarkably higher in tumor tissues.High PSMD2 expression was significantly associated with age(p=0.031),tumor size(p=0.006),lymph node metastasis(p=0.023),and advanced TNM stage(p=0.016).Patients with high level of PSMD2 associated with shorter overall survival(OS)and distant metastasis free survival(DMFS).However,there was no clear correlation between PSMD2 and relapse-free survival(RFS).Multivariate Cox regression analysis showed that PSMD2 was an independent predictor for OS(HR=3.15,95% CI: 1.08–9.23,p=0.036)and DMFS(HR=3.16,95% CI: 1.29–7.72,p=0.012).Conclusion: Compared with normal tissues,PSMD2 was significantly unregulated in BC.The expression of PSMD2 was significantly associated with clinical outcomes of BC.PSMD2 was demonstrated to be an independent prognostic factor for BC.PART2 THE BIOLOGICAL ROLE OF PSMD2 IN BREAST CANCER AND PRELIMINARY MECHANISM INVESTIGATION Objective: To investigate the biological roles and related mechanisms of PSMD2 in breast cancer(BC).Methods: GSEA was performed to explore potential biological functions of PSMD2 in BC.The impact of PSMD2 knockdown on cell proliferation was detected by CCK-8,colony formation,and xenograft tumor assays.Flow cytometry was used to determine the cell cycle distribution and apoptosis of BC cells treated with PSMD2 silencing.Cell cycle associated factors CDK4,CDK6,cyclin D1,E2F1,Rb,p Rb,p21,and p27,and apoptosis associated proteins caspase-7,cleaved caspase-7,and cleaved caspase-9 were detected by western blot.Immunofluorescence was used to determine the localization of upregulated p21 and p27.Knockdown PSMD2 combined with p21 and/or p27 silencing to determine effects of p21 and p27 on cell cycle arrest caused by PSMD2 silencing.Results: The results of GSEA showed that PSMD2 was significantly associated with cell proliferation,cell cycle,and apoptosis.Silencing PSMD2 obviously attenuated BC cell proliferation,colony formation abilities,and tumor growth.Flow cytometry showed that PSMD2 silencing significantly increased the G0/G1 phase ratio and reduced the S phase ratio.The expression of CDK4,CDK6,cyclin D1,E2F1,and p Rb were decreased following PSMD2 knockdown,while p21,p27,cleaved caspase-7 and cleaved caspase-9 were increased.Immunofluorescence and nuclear and cytoplasmic proteins separation assays showed that the upregulated p21 and p27 were mainly localized in the nucleus.Knockdown p21 and/or p27 could reverse the cell cycle arrest caused by PSMD2 silencing.Conclusion: PSMD2 knockdown significantly inhibited cell proliferation,promoted apoptosis,suppressed tumor growth,and induced cell cycle arrested at G0/G1.PSMD2 silencing disturbed cell cycle progression partially through p21 and/or p27.PART3 THE MOLECULAR MECHANISMS OF PSMD2 REGULATING THE EXPRESSION OF P21 AND P27Objective: To further explore the molecular mechanisms of the regulation of PSMD2 to p21 and p27.Methods: Proteasome activity kit was used to detect the impact of PSMD2 silencing on proteasome activity.Western blot was used to determine the impact of PSMD2 silencing on half-life of p21 and p27.The effect of PSMD2 silencing on the expression level of ubiquitinated p21 and p27 was detected by immunoprecipitation.IHC was performed to determine the expression correlation between PSMD2 and p21 and p27 in BC tissues.Immunofluorescence assay was used to detect the co-localization of PSMD2 with p21 and p27.The combination of PSMD2 and USP14 was detected by co-immunoprecipitation and GST pull-down assays.The expression of ubiquitinated p21 and p27 following USP14 knockdown was measured by western blot.Results: No clear correlation was observed between PSMD2 and p21 or p27 at the m RNA level.Proteasome activity was decreased by PSMD2 silencing in BC cells.Meanwhile,levels of total ubiquitinated proteins were modestly elevated.The half-life of p21 and p27 was significantly extended by PSMD2 silencing.IHC assay with 50 cases of breast cancer tissues showed that PSMD2 protein expression was negatively associated with p21 and p27.Co-transfection and co-immunoprecipitation assays demonstrated that PSMD2 could interact with p21 and p27.Co-localization analysis showed that PSMD2 was mainly co-localized with p21 and p27 in the nucleus.The interaction of PSMD2 with USP14 was confirmed by co-immunoprecipitation and GST pull-down.After knockdown USP14,the expression of p21 and p27 was significantly upregulated,while PSMD2 was unaffected.Moreover,the ubiquitinated p21 and p27 levels were significantly enhanced by USP14 knockdown.Conclusion: PSMD2 could interact with p21 and p27.USP14 may be required for PSMD2-mediated p21 and p27 degradation. |
PDF Full Text Request