UBE2C-mediated Autophagy Inhibition Via Ubiquitination Of SIRT1 Contributes To Endometrial Cancer Progression

Part I.Effect of UBE2 C on autophagy and malignant biological behavior of endometrial cancerObjectives: To investigate the effect of UBE2 C on autophagy and related malignant biological behavior of endometrial cancer cells.Material and methods: UBE2 C short hairpin RNAs(shRNAs)and overexpression lentivirus were constructed and transfected with Ishikawa cells and HEC-1B cells,and endometrial cancer cell lines were stably transfected by puromycin screening.protein expression of autophagy-related genes LC3 B,P62,BECN-1,ATG5 and ATG7 were detected by Western-blot.The effect of overexpression of UBE2 C on cellular autophagy was observed by transmission electron microscopy.chloroquine(CQ)and bafilomycin(Baf A1)were introduced to block autophagy,and the expression of LC3 B was verified by Western-blot and immunofluorescence.m RFP-GFP-LC3 adenovirus assay was used to track the changes in autophagic flow.CCK8 and Clone formation assay were used to detect cell proliferation.flow cytometry 7-AAD/PE Annexin V staining was used to detect apoptosis.Results:1.Ishikawa cells and HEC-1B cells after knockdown of UBE2 C showed reduced proliferation and increased apoptosis.LC3 B,BECN-1,ATG5,and ATG7 protein expression increased and P62 expression decreased in endothelial cancer cells.UBE2 C overexpression resulted in decreased LC3 B,BECN-1,ATG5,and ATG7 protein levels and increased P62 expression.2.The accumulation of LC3 B in endometrial cancer cells after UBE2 C knockdown was significantly increased compared to the control group after the addition of the autophagy inhibitor CQ with Baf A1,whereas the accumulation of LC3 B was decreased after UBE2 C overexpression.3.The number of autophagic vacuoles was significantly increased in all endometrial cancer cells after UBE2 C knockdown by fluoroscopic electron microscopy.Correspondingly,the number of autophagic vacuoles was significantly reduced in cells with stable UBE2 C overexpression.4.Immunofluorescence and m RFP-GFP-LC3 adenovirus assays showed that the knockdown of UBE2 C promoted autophagic flow.in contrast,overexpression of UBE2 C significantly inhibited autophagic flow.Conclusions: UBE2 C inhibited autophagy,promoted proliferation,and inhibited apoptosis of endometrial cancer cells.Part II Mechanisms of UBE2 C regulation of autophagy in endometrial cancerObjectives: Exploring the specific molecular mechanism of UBE2 C regulating autophagy in endometrial cancer cellsMaterial and methods: TMT quantitative proteomics to detect differentially expressed proteins after knockdown of UBE2 C in endometrial cancer cells.Predicting the manner and mechanism by which UBE2 C affects autophagy in endometrial cancer cells by KEGG and GO enrichment analysis.To detect UBE2 C and the effect of UBE2 C via SIRT1 on the acetylation level of histone H4 lysine 16(H4K16)using Western-blot.Detection of changes in SIRT1 expression in endometrial cancer cells after knockdown or overexpression of UBE2 C by Western-blot.Western-blot and q RT-PCR were used to detect the protein and m RNA expression of LC3 B,BECN-1,ATG5,and ATG7 in the rescue expression of combined UBE2C-overexpression and SIRT1-overexpression(UBE2C-knockdown and SIRT1-knockdown).Examine autophagy based on the transfection of adenovirus with an expression of m Cherry-GFP-LC3 fusion protein.Results:1.Analysis of quantitative proteomics data from endometrial cancer cells after knockdown of UBE2 C showed that tumor cells differentially expressed proteins contained a large number of histones,and KEGG and GO enrichment analysis predicted that UBE2 C played a regulatory epigenetic modification function.2.The protein level of SIRT1 was increased in endometrial cancer cells after the knockdown of UBE2 C,and the protein expression of SIRT1 was decreased in cells after the overexpression of UBE2 C.In addition,the protein amount of H4K16 ac was significantly reduced in UBE2 C knockdown cells,while the expression of H4K16 ac was increased in the rescue expression of combined UBE2 C knockdown and SIRT1-knockdown.Although H4K16 ac protein expression was increased after UBE2 C overexpression,H4K16 ac was reduced in the combined rescue expression of UBE2C-overexpression and SIRT1-overexpression.3.the inhibitory effects of UBE2 C overexpression on m RNA and protein expression of autophagy-related genes LC3,BECN-1,ATG5,ATG7 and ATG12 could be partially rescinded by SIRT1 overexpression.the m RNA and protein expression-promoting effects of UBE2 C knockdown on autophagy-related genes LC3,BECN-1,ATG5,ATG7 and ATG12 could be partially offset by SIRT1 knockdown.4.The m RFP-GFP-LC3 adenovirus assay showed that the pro-autophagy effect could be significantly reversed by SIRT1 knockdown after the knockdown of UBE2 C.After overexpression of UBE2 C,the inhibited autophagy could be partially reverted by SIRT1 overexpression.CCK8 and Ed U staining and clone formation assays were performed to detect the proliferative capacity of UBE2 C knockdown or overexpressed endometrial cancer cells and to observe the changes in cell proliferation capacity in the rescue assay combining UBE2 C knockdown and SIRT1 knockdown(UBE2C-overexpression and SIRT1-overexpression).Flow cytometry 7-AAD/PE Annexin V staining was performed to detect apoptosis in UBE2 C knockdown or overexpressed endometrial cancer cells,as well as to observe changes in apoptosis in rescue assays combining UBE2 C knockdown and SIRT1 knockdown(UBE2C-overexpression and SIRT1-overexpression).Conclusions: UBE2 C epigenetically suppresses the expression of autophagy-related genes in endometrial cancer through histone acetylation modification.Part III UBE2 C regulates the proliferation and apoptosis of endometrial cancer cells through down-regulation of SIRT1Objectives: To investigate the regulatory mechanism of UBE2 C on SIRT1 and the role of SIRT1 in the regulation of proliferation and apoptosis of endometrial cancer cells by UBE2 C.Material and methods: Detection of the protein degradation rate of SIRT1 in endometrial cancer cells with UBE2 C knockdown or overexpression by Western-blot after blocking protein synthesis using Cycloheximide(CHX).Based on this rescue experiment,the effect of UBE2C-overexpression on SIRT1 degradation was assessed through the treatment of CHX,or together with protease inhibitor MG132.Immunoprecipitation assay and Westernblot assay were used to observe the effect of UBE2 C knockdown or overexpression on the ubiquitination level of SIRT1.Transfection of expression vector plasmids with ubiquitin wild-type WT-Ub or K48 ubiquitin mutation type 48R-Ub,immunoprecipitation,and Western-blot assays to detect the level and type of UBE2C-mediated ubiquitination of SIRT1.Results:1.The ubiquitination level of SIRT1 decreased after the knockdown of UBE2 C,and the rate of SIRT1 protein degradation slowed down and protein stability increased;the ubiquitination level of SIRT1 increased after overexpression of UBE2 C,and the rate of SIRT1 protein degradation increased and protein stability decreased,but this process could be reversed by MG132.2.The increase in ubiquitination level of SIRT1 induced by UBE2 C overexpression was partially reversed after transfection of plasmid with HA-Ub-K48 R.3.UBE2 C knockdown greatly reduced proliferation and favored apoptosis,while SIRT1 effectively reversed the proliferative effects of UBE2 C overexpression.Likewise,SIRT1 knockdown partially counteracted the inhibitory effects of UBE2 C knockdown on proliferation.Conclusions: UBE2 C induces K48-linked SIRT1 ubiquitination and promotes ubiquitination-dependent degradation of SIRT1,and UBE2 C regulates the proliferation and apoptosis of endometrial cancer cells through SIRT1.Part IV UBE2C-mediated autophagy regulates the malignant progression of endometrial cancerObjectives: To investigate the role of UBE2 C on the malignant biological behavior of endometrial cancer cells through the regulation of autophagy.Material and methods: In a combined UBE2 C knockdown and BECN-1 knockdown(UBE2C-overexpression and BECN-1-overexpression)rescue assay,proliferation was detected by CCK8,clone formation assay,and Ed U cell staining assay,and apoptotic changes in XIB were detected by flow cytometry 7-AAD/PE Annexin V staining.Hoechst staining to detect apoptosis in endometrial cancer cells with knockdown of UBE2 C along with knockdown of BECN-1.Immunofluorescence staining was performed to detect the expression levels of LC3 and caspase-3 in endometrial cancer cells with knockdown of UBE2 C along with knockdown of BECN-1.Protein expression of BECN-1,P62,LC3 B,bax,Bcl2,cleaved-caspase 3,and cleaved-caspase 9 was detected by Western-blot in a combined UBE2 C knockdown and BECN-1 knockdown(UBE2C-overexpression and BECN-1-overexpression)cell rescue assay.Results: 1.UBE2 C knockdown greatly reduced proliferation and favored apoptosis,while BECN-1 effectively reversed the proliferative effects of UBE2 C overexpression.Likewise,BECN-1 knockdown partially counteracted the inhibitory effects of UBE2 C knockdown on proliferation.2.Nuclear condensation and DNA breaks caused by UBE2 C were attenuated by BECN1.3.Immunofluorescence assay results indicated increased endogenous LC3 puncta and Caspase-3 levels after UBE2 C was knocked down in EC cells while knocking down BECN1 effectively abolished the enhanced expression of Caspase-3 induced by UBE2 C knockdown.4.Western blotting showed that UBE2 C caused a significant increase in Bcl-2 and a decrease in Bax protein expression in EC cells,which were attenuated by BECN-1.5.3-MA effectively reversed the proliferation inhibition caused by UBE2 C knockdown,and immunoblot analysis revealed that UBE2 C knockdown-induced increases in LC3 B,cleaved-caspase 3,and cleaved-caspase 9 expressions were decreased and returned to baseline levels after 3-MA inhibited autophagy.Hoechst cell staining results suggested that 3-MA attenuated the nuclear condensation,deformation,and DNA fragmentation caused by UBE2 C knockdown.Conclusions: The pro-proliferative effect of UBE2C-mediated autophagy inhibition on endometrial cancer cells may be the result of reduced apoptotic cell death.Part V.Rapamycin attenuates the progression of UBE2C-induced endometrial cancer by restoring the autophagy programObjectives: To investigate the anti-tumor growth effect and mechanism of rapamycin on endometrial cancer.Material and methods: The proliferative effects of rapamycin on endometrial cancer cells after overexpression of UBE2 C were detected by CCK8 and Ed U assays;apoptotic changes were detected by flow cytometry 7-AAD/PE Annexin V staining.Five-week-old female nude mice were selected to be injected subcutaneously with 1.5×10^6 Ishikawa cells,followed by intraperitoneal injection of rapamycin every three days.Mice were sacrificed 28 days later.The tumors were removed,weighed,and tissues were analyzed for the expression of UBE2 C,Ki-67,Caspase-3,LC3,ATG5,ATG7,BECN-1,and P62 by immunohistochemistry.A lung metastasis model was constructed by injecting 2×10^6 Ishikawa cells in the tail vein;mice were executed after 8 weeks and the number of metastases on each lung surface was determined by H&E staining to assess lung metastasis.Results:1.UBE2 C overexpression significantly promoted cell proliferation,and Rapamycin treatment significantly inhibited the increase in cell proliferation after UBE2 C overexpression,with no significant difference in apoptosis results.2.Compared with the control group,the subcutaneous transplantation tumor volume,and weight increased in the UBE2 C overexpression group,and Rapamycin treatment significantly attenuated the increase in subcutaneous tumor volume and weight induced by UBE2 C overexpression.3.Immunohistochemical staining showed that Rapamycin significantly inhibited UBE2 Cmediated upregulation of Ki-67 and downregulation of Caspase-3 expression;compared with the control group,the expression of UBE2 C and P62 proteins was increased in the subcutaneous transplanted tumor tissues,and the levels of autophagy-related proteins such as LC3,ATG5,ATG7,and BECN-1 were reduced in the overexpressed UBE2 C group.The expression of LC3,BECN-1,ATG7,and ATG5 was increased and the expression of P62 was decreased after Rapamycin treatment.4.Histological examination showed that the number of lungs micrometastases in the UBE2 C overexpression group of mice was increased compared with the number in the control group,and rapamycin reversed the enhanced effect of lung metastasis induced by UBE2 C overexpression.Conclusions: Rapamycin attenuates the progression of UBE2C-induced endometrial cancer by restoring the autophagy program in vitro and in vivo.