Ubiquitin E3 Ligase UBE3C Mediates Estrogen-induced Invasion And EMT In Endometrioid Adenocarcinoma

Objective:To investigate the expression of UBE3 C in endometrioid adenocarcinoma;to examine whether estrogen plays a role on the expression of UBE3 C in ERα-positive endometrial cancner cells;to investigate the roles and related mechanism of UBE3 C on estrogen-induced migration,invasion and EMT in endometrial cancer cell line Ishikawa.Methods : We investigated the UBE3 C protein expression in endometrioid adenocarcinoma tissues and benign endometrial tissues using IHC;Western Blotting was used to examine the protein level of UBE3 C in different endometrial cancer cell lines.We also used immunofluorescence to pinpoint the intracellular distribution of UBE3 C.We treated ERα-positive endometrial cancer cell line Ishikawa with E₂,q RT-PCR and Western Blotting were used to investigate the influence of E2 on the expression levels of m RNA and protein of UBE3 C.We transfected Ishikawa with si RNA to suppress the expression of UBE3 C,si UBE3 C and control groups were treated with E2 or not,Wound healing assay and cell invasion assay were used to investigate the migration and invasion abilities of cells in four groups.We transfected Ishikawa with si UBE3 C,Western Blotting was used to find out the changes of protein levels of EMT-and Wnt/β-catenin pathway-related molecules.The same was examined after DKK1 treatment（inhibitor of Wnt pathway）.Results:（1）UBE3C was overexpressed in endometrioid adenocarcinoma tissues,and the protein level tend to show higher in endometrial cancer cell lines compared to normal endometrial epithelial cells and ESC.UBE3 C protein was localized in both cytoplasm and nucleus.（2）E₂ upregulates the expression of UBE3 C in a dose-and time-dependent manner in ERα positive EC cells.（3）Wound-healing assay and Transwell assay confirmed that estrogen stimulation promoted the abilities of migation and invasion,decreased UBE3 C reduced the abilities of migration and invasion in E2-treated Ishikawa cells.（4）The protein levels of mesenchymal marker N-cadherin and transcription factors snail,slug were upregulated,whereas the epithelial marker E-cadherin decreased in E2-treated cells.After knockdown of UBE3 C,the epithelial marker E-cadherin level enhanced,the mesenchymal marker N-cadherin and transcription factors snail,slug levels were reduced.Downregulation of UBE3 C significantly reversed E₂-induced EMT.（5）We also discovered that ablation of UBE3 C downregulated the expression of β-catenin and p-GSK3β（ser9）while total GSK3β protein level remained unchanged.After treatment with DKK1,the inhibitor of Wnt pathway,the protein level of epithelial marker E-cadherin further inceased,the mesenchymal marker N-cadherin and transcription factors snail,slug levels were further reduced.In conclusion,UBE3 C facilitates EMT through Wnt/β-catenin pathway in Ishikawa cells.Conclusions:UBE3C was overexpressed in endometrioid adenocarcinoma tissues.E₂ upregulates the expression of UBE3 C in ERα-positive endometrial cancer cells.UBE3 C mediates estrogen-induced migration,invasion and EMT in endometrial cancer cells.UBE3 C facilitates epithelial-mesenchymal transition through Wnt/β-catenin pathway in EC cells.