Mechanisms And Clinical Significance Of Targeting THBS1 By Pinocembrin In Inhibiting Invasion And Metastasis Of Ovarian Cancer

Part I: Screening the Functions and Targets of Pinocembrin in Ovarian Cancer Cells Objective: The objective of this study is to elucidate the role of pinocembrin in ovarian cancer cells and to screen and validate its targets.Methods: We conducted CCK-8 and plate clone formation assays to evaluate the impact of pinocembrin on the proliferation of ovarian cancer cells(ES-2 and SK-OV-3).The IC50 value of pinocembrin in ovarian cancer cells was calculated to determine the optimal concentration for subsequent experiments.Scratch and transwell assays were performed to assess the effect of pinocembrin on the migration and invasion abilities of ES-2 and SK-OV-3 cells.Additionally,Western blot assays were conducted to examine the influence of pinocembrin on the expression levels of MMP2,MMP9,FAK,p FAK,E-cadherin,N-cadherin,and Vimentin in ovarian cancer cells.Network pharmacology technology was employed to retrieve and predict potential targets of pinocembrin.Correlation analysis was then used to screen these targets,which were further validated using Western blot assays.Results: Pinocembrin demonstrated a time-and dose-dependent inhibition of ovarian cancer cell proliferation.It also suppressed the migration and invasion abilities of ovarian cancer cells,while reducing the expression levels of MMP2,MMP9,p FAK,N-cadherin,and Vimentin,and upregulating the expression of E-cadherin.Through network pharmacology analysis,the top ten potential target genes of pinocembrin were identified as AR,ESR1,DICER1,CDKN1 B,HDAC7,USP7,THBS1,HCK,NCBP1,and EIF4A1.Among these,THBS1 was found to be closely associated with migration and invasion,and pinocembrin was able to dose-dependently downregulate its expression.Conclusion: Pinocembrin exhibits inhibitory effects on cell viability,proliferation,migration,and invasion in ovarian cancer cells,with THBS1 identified as one of its key targets.Part II: The Role and Clinical Significance of THBS1 in Ovarian Cancer Objective: The objective of this study is to elucidate the role of THBS1 in the migration and invasion of ovarian cancer cells and to explore the expression and clinical significance of THBS1 in ovarian cancer.Methods: THBS1 was knocked down and overexpressed in ovarian cancer cells.The changes in migratory and invasive abilities of tumor cells and the additional effects of pinocembrin on these cells were evaluated using scratch and transwell assays.The Western blot assay was employed to examine the expression levels of migration and invasion-related proteins including MMP2,MMP9,FAK,p FAK,E-cadherin,Ncadherin,and Vimentin in ovarian cancer cells.The expression levels of MMP2,MMP9,FAK,p FAK,E-cadherin,N-cadherin,and Vimentin were also assessed through Western blot analysis.Results: Knocking down THBS1 significantly inhibited the migratory and invasive abilities of ovarian cancer cells.Furthermore,pinocembrin was able to further suppress the migratory and invasive characteristics of these tumor cells.The expression levels of MMP2,MMP9,p FAK,N-cadherin,and Vimentin proteins decreased,while the expression of E-cadherin was upregulated.On the other hand,overexpressing THBS1 significantly increased the migratory and invasive abilities of ovarian cancer cells.Western blot analysis showed that the expression levels of MMP2,MMP9,p FAK,N-cadherin,and Vimentin proteins increased,while Ecadherin expression was upregulated.Immunohistochemistry(IHC)results demonstrated higher expression of THBS1 in ovarian cancer compared to normal ovarian tissues.In the tumor group,THBS1 expression positively correlated with lymph node metastasis and poor prognosis of ovarian cancer.ROC analysis indicated that THBS1 expression levels were better predictors of 3-year survival compared to 5-year survival in patients.Conclusions: THBS1 promotes the migration and invasion of ovarian cancer cells and is overexpressed in ovarian cancer compared to normal ovarian tissue.Furthermore,its expression is positively associated with poor prognosis in ovarian cancer patients.Part III: Mechanism of Pinocembrin in Down-regulating THBS1Objective: The objective of this study is to investigate the mechanism by which pinocembrin down-regulates THBS1 and to provide a theoretical basis for the clinical application of pinocembrin.Methods: RT-q PCR was used to assess the effect of pinocembrin on the expression level of THBS1 m RNA in ovarian cancer cells.Western blot analysis was performed to examine the impact of pinocembrin on the expression of TGF-β1,a transcriptional regulatory protein of THBS1.Molecular docking and cellular thermal shift assay(CETSA)were conducted to validate the interaction between pinocembrin and THBS1.Protein synthesis in ovarian cancer cells was inhibited to observe the effect of pinocembrin on THBS1 degradation using molecular docking and CETSA assays.CHL and MG132 were used to block lysosomal and proteasomal pathways of protein degradation,respectively,in order to determine the specific pathways affected by pinocembrin on THBS1 degradation.Co-immunoprecipitation(Co-IP)technique was employed to examine the co-expression and ubiquitination of THBS1 and Ubiquitin.Co-IP experiments were also conducted to investigate the interaction between THBS1 and USP7.Western blot analysis was performed to investigate the effect of pinocembrin on USP7 protein expression,and molecular docking and CETSA assays were employed to validate the direct binding ability of pinocembrin to USP7.Results: RT-q PCR revealed that pinocembrin significantly inhibited THBS1 m RNA at a concentration of 40μM,but THBS1 m RNA levels increased at a concentration of80μM.Western blot analysis demonstrated that pinocembrin had a significant effect on the transcription-inducing factor TGF-β1 of THBS1 at both 40μM concentrations.Molecular docking and CETSA assays provided evidence for the direct binding of pinocembrin to THBS1.Pinocembrin reduced THBS1 expression even after protein synthesis was inhibited in ovarian cancer cells.Inhibition of THBS1 by pinocembrin was reduced after proteasome inhibition with MG132,while inhibition of lysosomal enzymes by CHL had no effect on the degradation of THBS1 promoted by pinocembrin.Co-IP experiments revealed the ubiquitination of THBS1 in ES-2 and SK-OV-3 cells,and pinocembrin was found to promote its ubiquitination.An interaction between USP7 and THBS1 was also observed,and further Western blot assays demonstrated that pinocembrin had no effect on the expression of the deubiquitinating protein USP7.However,molecular docking and CETSA assays showed that pinocembrin could bind directly to USP7.Conclusion: Pinocembrin can directly bind to THBS1,promoting its ubiquitination,and it also inhibits the deubiquitination ability by binding to USP7.These findings shed light on the mechanism of action of pinocembrin in down-regulating THBS1 and provide insights for its potential clinical application.Overall Conclusion:1.Pinocembrin inhibits the proliferation and migration invasion of ovarian cancer cells.2.THBS1 promotes the migration and invasion of ovarian cancer cells,and its expression is higher in ovarian cancer compared to normal ovarian tissue,correlating with poor prognosis.3.Pinocembrin down-regulates the expression of THBS1 by promoting its ubiquitination,thus suppressing the migration and invasion of ovarian cancer.