A Study Of The Effect And Mechanism Of SHCBP1 On Proliferation And Metastasis In Prostate Cancer

PART 1: THE EXPRESSION AND CLINICAL SIGNIFICANCE OF SHCBP1 IN PROSTATE CANCERObjectives: To determine the role of SHCBP1 in PCa and explore the relationship between the expression of SHCBP1 and clinicopathological parameters in PCa.Methods: We analyzed the expression of SHCBP1 in PCa tissues and adjacent normal tissues using bioinformatics methods.Tissues were collected from the First Affiliated Hospital of Fujian Medical University.After constructing the TMA,IHC staining was performed to compare the expression of SHCBP1 between PCa tissue and BPH tissue.The correlation of SHCBP1 expression with clinicopathological parameters and survival outcomes was explored based on the clinicopathological data from TCGA database and our center.Results: The analysis of the TCGA database and IHC results showed that the expression of SHCBP1 in PCa tissues was significantly higher than that in BPH tissue and adjacent normal tissues(P<0.05).The results from the analysis of the TCGA database revealed that the expression level of SHCBP1 in PCa patients was closely correlated with T stage and N stage(P<0.05).The overall survival(OS)and prostate cancer-free survival (PCFS)were significantly increased in patients with low SHCBP1expression(P<0.05).The correlation analysis demonstrated that preoperative PSA level,Gleason score,and p T stage were higher in PCa patients with higher expression of SHCBP1 and that patients with higher expression of SHCBP1 were more likely to have prostate extracapsular invasion and seminal vesicle infiltration(P<0.05).Multivariate Cox regression analysis showed that high Gleason score,high p T staging,and high SHCBP1 expression were independent predictors of BCR after radical prostatectomy.The biochemical recurrence-free survival(BFS)in PCa patients with high Gleason score,high p T staging,and high expression of SHCBP1 were significantly lower than those with low Gleason score,low p T staging,and low SHCBP1 expression(P<0.05).Conclusion: The expression of SHCBP1 was elevated in PCa and closely correlated with T stage,extracapsular invasion,seminal vesicle infiltration,lymph node metastasis,Gleason score,and p T stage.The OS,PCFS,and BFS in PCa patients with high SHCBP1 expression were significantly decreased.The high expression of SHCBP1 was an independent predictor of biochemical recurrence after radical prostatectomy.PART 2: THE EFFECT OF SHCBP1 ON PROLIFERATION AND METASTASIS OF PROSTATE CANCER CELLSObjectives: To investigate the effect of the SHCBP1 knowdown on the proliferation,cell cycle,migration,invasion,and metastasis of PCa cells.Methods: The Lentivirus transfection method was used to silence the SHCBP1 in PC-3 and DU145 cell lines.Western blot and q RT-PCR were used to detect the knockdown efficiency of SHCBP1.The MTT assay,flow cytometry assay,and Transwell assay were used to determine the proliferation,cell cycle,migration and invasion ability of PCa after the knockdown of SHCBP1.The effect of silencing the SHCBP1 on the growth and metastasis of PC-3 cell was validated by utilizing subcutaneous tumorigenesis and tail vein injection in nude mice,respectively.Results: The results of Western blot and q RT-PCR indicated that the m RNA and protein expression of SHCBP1 was significantly decreased.The proliferation ability of PC-3 cells and DU145 cells treated with sh SHCBP1 were significantly decreased(P<0.05).The proportion of G2/M-phase cells was significantly increased after SHCBP1 knockdown(P<0.05).Transwell assay demonstrated that the migration and invasion ability of PC-3 and DU145 cells after SHCBP1 knockdown were significantly decreased(P<0.05).In vivo experiments confirmed that the growth and metastasis of PC-3 cells after SHCBP1 knockdown were significantly decreased(P<0.05).Conclusion: SHCBP1 can promote the proliferation,invasion,and metastasis of PCa cells in vitro and in vivo.PART 3: EXPLORATION OF THE MECHANISM OF SHCBP1 ON PROMOTING PROSTATE CANCER PROLIFERATION AND METASTASISObjectives: To explore the mechanism of SHCBP1 on promoting the proliferation and metastasis of PCa.Methods: Gene chips and bioinformatics analysis were used to determine the expression profile changes of the downstream gene after SHCBP1 knockdown in PC-3 cells.q RT-PCR,Western blot,and rescue assays were used to validate the predicated signaling pathways.CHX and MG-132 assays were used to explore the mechanism of SHCBP1 on regulation of TP53.Results: 1.A total of 1820 differentially expressed genes were identified,including 633 up-regulated genes and 1187 down-regulated genes.The results of bioinformatics analyses revealed that the Hippo signaling pathway and MDM2-TP53 signaling pathway were significantly activated after SHCBP1 knockdown.2.The results of western blot indicated that there was no significant difference in the m RNA expression,the protein expression,and the protein phosphorylation levels of YAP1 and TAZ in Hippo pathway after SHCBP1 knockdown.3.The results of Western blot indicated that the protein expression level of MDM2 was significantly downregulated,and the protein expression of LATS1 and TP53 was significantly upregulated(P<0.05).The rescue assays indicated that LATS1 and TP53 knockdown can rescue the proliferation and migration of PC-3 cells,respectively(P<0.05).The results of CHX and MG-132 assays indicated that SHCBP1 knockdown can attenuate the degradation of TP53 by proteasome,and prolong the half-life of TP53.Conclusion: These results indicated that SHCBP1 might promote the proliferation and metastasis of prostate cancer through the LATS1-MDM2-TP53 signaling pathway by decreasing the stabilization of TP53.PART 4: CONSTRUCTION AND ANALYSIS OF COMPETING ENDOGENOUS RNAS REGULATORY NETWORK AND MRNA PROGNOSTIC SIGNATURE IN PROSTATE CANCERObjectives: To explore the regulatory mechanisms and prognostic values of differentially expressed RNAs using competing endogenous RNAs(ce RNA)and construct an m RNA prognostic signature that predicts survival outcome in prostate cancer(PCa).Methods: The expression profiles data of PCa were obtained from the TCGA database.The RNA expression of 499 PCa tissues and 52non? PCa tissues from TCGA were analyzed.The differentially expressed RNAs were examined using the edge R package.Survival was analyzed by the Kaplan–Meier method.micro RNA(mi RNA),messenger RNA(m RNA),and long non? coding RNA(lnc RNA)ce RNA regulatory networks were constructed based on the differentially expressed RNAs between non? PCa and PCa tissues.Results: A total of 773 lnc RNAs,1417 mRNAs,and 58 mi RNAs were differentially expressed between non? PCa and PCa samples(P<0.05).The newly constructed ce RNA network comprised 63 PCa-specific lnc RNAs,13 mi RNAs,and 18 m RNAs.Three of 63 differentially expressed lnc RNAs and one of 18 differentially expressed m RNAs were significantly associated with overall survival(OS)in PCa(P<0.05).4m RNAs(HOXB5,GPC2,PGA5,and AMBN)were screened by utilizing univariate and multivariate Cox regression methods and used to establish a predictive model for the OS of patients.Our ROC curve analysis revealed that the 4? m RNA signature performed well.Conclusion: The lnc RNA-mi RNA-m RNA ce RNA regulatory network of prostate cancer constructed in this study may play a critical role in the progression and metastasis of PCa and are thus the potential therapeutic targets and prognostic biomarkers.A novel model that incorporated these candidates was established and might provide more powerful prognostic information in predicting survival in PCa.