The Mechanism Of UBP43 Promotes The Progression Of Epithelial Ovarian Cancer By Activating The β-catenin Signaling Pathway

Background:Ovarian cancer has the highest mortality rate among all gynecological malignancies,and its 5-year survival rate is less than 50%.so it poses a serious threat to women’s health.Among ovarian cancer,epithelial ovarian cancer is the most common type,accounting for up to 90%.Because of its high incidence rate and mortality,the research on its related mechanisms has attracted much attention in the medical field.Therefore,in-depth research on the occurrence and development mechanism of EOC,as well as the search for new drug targets and treatment strategies,is of great research significance.UBP43 is a member of the deubiquitinating enzyme family and known for its role in stabilizing downstream protein substrates.Its imbalance is associated with many human diseases,including cancer.However,the promoting or inhibitory effects of UBP43 vary among different types of tumors.Therefore,it is necessary to clarify the role of UBP43 in specific cancer types through relevant research.At present,the function of UBP43 in the occurrence and development of epithelial ovarian cancer is still unknown.Previously,by searching the databases,the research team found that the expression level of UBP43 in OC was much higher than that in more than 20 other types of tumors such as lung cancer and liver cancer,and there was an important correlation between UBP43 and β-catenin.It is worth nothing that β-catenin is a key pathway promoting the malignant phenotype of tumors.Meanwhile,it has not been reported whether UBP43 can play a role by affecting the β-catenin signaling pathway.Therefore,this research takes this as the breakthrough point and proposes a research hypothesis: "That UBP43 regulates the β-catenin signaling pathway may be a new mechanism affecting EOC development,and the regulatory effects on theβ-catenin may be related to the deubiquitination activity and the functional characteristics of stabilizing downstream protein substrates of UBP43."Objective:The aim of this study is to explore the impact of UBP43 on the biological behavior of epithelial ovarian cancer cells and revealing the new mechanism of UBP43 affects EOC progression through the β-catenin signal pathway.so as to provide potential new therapeutic targets for ovarian cancer,and to provide a theoretical basis for the exploration and development of UBP43 specific inhibitors in the future.Methods:1.Firstly,this study analyzed the clinical correlation between UBP43 and EOC.The expression of UBP43 in EOC was confirmed by searching the TNMplot database and the q RT-PCR and Western blot analysis of 10 pairs of EOC clinical samples(cancer and adjacent).The Kaplan-Meier-Plotter database was used to analyze the correlation between the expression of UBP43 and the clinical prognosis of ovarian cancer.2.Explore the impact of UBP43 on the biological behavior of EOC cells.The expression of UBP43 in normal ovarian epithelial cell lines(HOSEpi C)and five EOC cell lines(OVCAR-3,Caov-3,TOV-112 D,A2780,SK-OV-3)was detected by Western blot assay.Then constructed UBP43 knockdown and overexpression stable transfection cell lines of A2780 and TOV-112 D using lentivirus.The effects of UBP43 knockdown and overexpression on the proliferation,migration,and invasion ability of EOC cells were detected through CCK-8,plate cloning,flow cytometry,and Transwell experiments.And Western blot was used to detect the expression of Cyclin B1 and CDK1 proteins related to G2/M phase.In addition,genes related to epithelial mesenchymal transition(EMT),migration,and invasion,such as mature-MMP2,mature-MMP9,N-cadherin,and E-cadherin,also were detected by Western blot.3.Clarified the carcinogenic mechanism of UBP43.Part 1: The research on the regulatory effects and mechanism of UBP43 on β-catenin.Western blot assay were used to detect the effects of UBP43 knockdown and overexpression on the expression levels of β-catenin and Cyclin D1 in EOC cells,which was the downstream target genes of β-catenin.Immunofluorescence assay were used to detect the effects of UBP43 on the intracellular distribution of β-catenin.Detect the effects of UBP43 on β-catenin activity through dual luciferase experiment.The q RT-PCR was used to detect the m RNA levels of β-catenin and Cyclin D1 of the EOC clinical sample tissues.Double staining immunofluorescence and immunoprecipitation assay were used to detect the interaction between UBP43 and Xβ-catenin.Cells were pretreated with 50 μg/m L cycloheximide,and the effect of UBP43 on the stability of β-catenin protein was detected by Western blot assay at 0h,0.5h,1h,and 2h after treatment.Finally,the effect of UBP43 on the ubiquitination level of β-catenin was detected through immunoprecipitation experiments.Part 2:This study verified whether UBP43 exerted its cancer promoting effect by regulating the β-catenin signaling pathway through response experiments.The successfully constructed UBP43 knockdown stable transfection cell line(Lv-sh UBP43)was transfected with β-catenin(S33Y)overexpression plasmid.Western blot,CCK-8,and Transwell experiments were used to detect whether the activation of β-catenin signaling can reverse the downregulation of Cyclin D1,mature-MMP2,and mature-MMP9 mediated by UBP43 knockdown,as well as the negative impact on cells proliferation,migration,and invasion ability.4.The effect of UBP43 on the proliferation ability of epithelial ovarian cancer cells were studied in vivo experiments.The nude mouses subcutaneous xenograft tumor models were constructed.After tumor cells inoculation,the longest and shortest diameters of the tumors were measured every 4 days.On the 28 th day,the tumors were harvested,and the weight and volume of the tumors were recorded and photographed.A tumor growth curve was drawn to examine the effect of UBP43 on the growth rate of xenograft tumors in vivo.Ki67 and PAX8 immunohistochemistry assays were used to detect the effects of UBP43 on the proliferation activity of tumor cells in vivo.Immunofluorescence experiment was used to detect the regulatory effects of UBP43 on β-catenin in vivo,including the expression and colocalization of UBP43 and β-catenin in tumor tissues.At the same time,it also showed the nuclear expression of β-catenin.The effects of UBP43 on the expression levels of β-catenin,Cyclin D1,mature-MMP2 and mature-MMP9 in tumor tissue were detected through Western blot experiment.Results:1.The results of searching the TNMplot online database,the q RT-PCR and Western blot experiments on 10 pairs of clinical samples showed that the expression of UBP43 was significantly upregulated in EOC tissues compared to normal tissues.Compared with other tumor tissues,UBP43 was particularly highly expressed in ovarian tumor tissues.Moreover,the high expression of UBP43 was associated with poor clinical prognosis,and ovarian cancer patients with higher levels of UBP43 had significantly poorer survival rate.2.Compared with HOSEpi C,UBP43 expression was also significantly upregulated in five EOC cell lines.This study successfully constructed UBP43 knockdown and overexpression stable transfection cell lines of A2780 and TOV-112 D.In the UBP43 knockdown groups,the cell proliferation ability and the colony formation rate were significantly inhibited,and the percentage of S phase cells was significantly reduced,and the percentage of G2/M phase cells was significantly increased;the cell migration and invasion ability were significantly reduced.At the same time,the protein levels of mature-MMP2,mature-MMP9,and N-cadherin in UBP43 knockdown groups were significantly reduced,while E-cadherin level was significantly upregulated,the progression in the Epithelia XII Mesenchymal Transition was inhibited.The detection results of UBP43 overexpression group were opposite.3.Part 1:The overexpression of UBP43 resulted in the upregulation of the expression of β-catenin and its downstream target gene Cyclin D1;Meanwhile,the overexpression of UBP43 caused the changes in intracellular distribution and abnormal nuclear accumulation of β-catenin.The results of the double luciferase experiment showed that the overexpression of UBP43 significantly increasedβ-catenin activity.The results of double staining immunofluorescence and immunoprecipitation experiments indicated that the subcellular positions of UBP43 and β-catenin significantly overlaped,and there was an interaction between them.Meanwhile,the upregulation of UBP43 increased the deubiquitination of β-catenin,weakened β-catenin degradation,and enhanced its protein stability.In the EOC clinical tissue samples,Cyclin D1 was significantly overexpressed in cancer tissues compared to adjacent tissues,but there was no significant upregulation of β-catenin.Part 2: The response experiments results showed that the upregulation of β-catenin significantly reversed the downregulation of Cyclin D1,mature-MMP2,and mature-MMP9 expression levels in cells caused by UBP43 knockdown,as well as the negative impact on cell proliferation,migration,and invasion ability.Meanwhile,the overexpression of β-catenin significantly eliminated the inhibitory effect of UBP43 knockdown on EMT,inhibited the expression of E-cadherin,and induced N-cadherin expression.4.Consistent with the results of vitro cell experiments: The tumor size,tumor weight and the tumor growth rates in the UBP43 knockdown group were significantly reduced;The expression of Ki67 in the tumor tissue of the UBP43 knockdown group was significantly reduced,indicating that the UBP43 knockdown inhibited tumor cells proliferation activity.The low magnification image of the tumor area showed that PAX8 existed in all tumor tissues,and the expression of PAX8 in the tumor tissue of the UBP43 knockdown group was significantly reduced,the results of the overexpression group were opposite;The protein levels of UBP43 and β-catenin in tumor tissue were significantly reduced in the UBP43 knockdown group,and the nuclear expression of β-catenin was reduced.The results were opposite in the UBP43 overexpression group.In addition,fluorescence overlap between UBP43 and β-catenin and abnormal nuclear accumulation of β-catenin were observed.Indicating there was an interaction between UBP43 and β-catenin in vivo.Similarly,the expression levels of β-catenin,Cyclin D1,mature-MMP2,and mature-MMP9 in tumor tissue of the UBP43 knockdown group were significantly lower than the control group,while the results of the UBP43 overexpression group were opposite.Conclusions:1.UBP43 is highly expressed in EOC and is associated with poor patient prognosis.2.UBP43 plays a carcinogenic role in EOC,promoting EMT,proliferation,migration,and invasion of tumor cells.3.UBP43 can promote the progression of EOC by activating β-catenin signaling,upregulating β-catenin protein level and promoting β-catenin entry into the nucleus.4.UBP43 may improve the stability of β-catenin protein by removing ubiquitination modifications,which promotes the activation of the β-catenin signaling pathway and promotes the malignant progression of EOC thereby.