| Objective: Endometrial cancer is one of the three major malignant tumors in the female reproductive system,with a rising incidence worldwide.Treatment for endometrial cancer includes surgery,radiotherapy,chemotherapy,and hormone therapy.Most ECs are diagnosed as early-stage,low-grade endometrioid endometrial cancers,and possessed a satisfying 5-year overall survival in excess of 90%.However,20% of advanced-stage(IIIIV)patients will experience metastasis and recurrence after primary treatment.Endometrial cancer stem cells(ECSCs)comprise a small portion of endometrial cancer cells,distinguished by unlimited self-renewal and pluripotency.ECSCs have been regarded as the source of metastasis,chemotherapy resistance,and disease relapse,which can be a cause for treatment failure of EC.Up to now,the underlying biologic mechanism of ECSCs in EC is yet to be fully characterized.Therefore,information about stemness regulation of ECSCs may be useful in the development of specific targeted therapies.Upframeshift mutant 1(UPF1)is the core protein of the nonsense-mediated m RNA degradation pathway,which is tightly associated with the tumorigenesis and progression of many cancers.Previous studies demonstrated that UPF1 was crucial to dictate whether a neural cell proliferates or differentiates.UPF1 destabilized the NMD substrate encoding the TGF-b inhibitor SMAD7 and was downregulated to permit neural differentiation.UPF1 can also act as the RNA-binding protein(RBPs),playing an important role in posttranscriptional gene regulation.RBPs are involved in various aspects of RNA metabolism by affecting stability,splicing,RNA folding,base modification,transport,localization,and translation.RNAs and RBPs bind to each other,forming complexes that influence the development and progression of many diseases including cancer.Long non-codingRNAs(lncRNAs)are a group ofRNAs with lengths exceeding 200 nucleotides and have no protein-coding capacity.LncRNAs play important regulatory roles in many biological processes,such as cell proliferation,invasion,differentiation,stemness maintenance,and oncogenesis.Plasmacytoma Variant Translocation 1(PVT1),a lnc RNA,is located at 8q24,which is upregulated in ECSCs and promotes malignant biological behaviors of ECSCs.PVT1 can interact with microRNAs(miRNAs)and influence the expression of mRNAs,subsequently affecting cancer risk.This study aimed to determine whether RBP UPF1 and lnc RNA PVT1 influenced the stemness in ECSCs and to further explore the potential mechanism.We further aimed to evaluate whether UPF1 could enhance the level of SOX2 by negatively regulating miR-508-5p via stabilizing PVT1,subsequently regulating selfrenewal and other biological behavior in ECSCs.Methods: First,the expression of UPF1 in endometrial carcinoma and normal endometrium was assessed via qRT-PCR and Western blot,and the association between the expression of UPF1 and the clinicopathological characteristics was evaluated.The expression of miR-508-5p in endometrial carcinoma and normal endometrium was assessed by qRT-PCR,and the relationship between the expression of miR-508-5p and the clinicopathological parameters was also analyzed.The human endometrial carcinoma cell line Ishikawa was cultured and Ishikawa-ECSCs were extracted from serum-free suspension cultivation and flow cytometry.Next,fluorescence in situ hybridization combined with immunofluorescence was applied to observe the expression and distribution of UPF1 and PVT1 in parental cells and ECSCs via laser confocal microscopy.RIP-seq and RIP were conducted to verify the binding effect of UPF1 and PVT1.qRTPCR was used to detect the expression of UPF1 and PVT1 in endometrial cancer parent cells and ECSCs.The overexpression and knockdown of UPF1/PVT1 were induced by transfection in parent cells and ECSCs,and the transfection efficiency was analyzed by qRT-PCR and Western blot.In parental cells and ECSCs transfected with UPF1 and cotransfected with UPF1 and PVT1,RNA stability measurement was used to detect the changes in the half-life of PVT1;Western blot was used to detect changes in the expression of stemness-related genes SOX2,OCT4,and NANOG;Sphere-forming assay was used to detect changes in self-renewal capacity;CCK-8 assay was used to detect changes in proliferation capacity;Transwell assay was used to detect changes in migration and invasion capacity;Flow cytometry was used to detect changes in cell cycle progression and apoptosis capacity.Finally,the regulation of miR-508-5p targeting PVT1 and the regulation of miR-508-5p targeting SOX2 were evaluated with bioinformatics software and the luciferase reporter assay.The overexpression and knockdown of miR-508-5p were achieved via transfection in parent cells and ECSCs,and the transfection efficiency was detected by qRT-PCR and Western blot.In parental cells and ECSCs transfected with miR-508-5p and co-transfected with miR-508-5p and PVT1,Western blot,sphere-forming assay,CCK-8 assay,Transwell assay,and flow cytometry were applied to detect changes in stemness-related genes,self-renewal,proliferation,migration and invasion,cell cycle,and apoptosis.Results: In this study,UPF1 was highly expressed in endometrial cancer tissues and was significantly correlated with myometrial invasion,and advanced FIGO stage(P<0.001).The CCK-8 assay showed that downregulation of UPF1 resulted in inhibition of proliferation in ECSCs.The Transwell assay showed that downregulation of UPF1 inhibited the abilities of migration and invasion in ECSCs.Cell cycle assay showed that the cells in the G2-M phase were significantly decreased after depleting UPF1.The overall apoptotic rate of ECSCs(early apoptosis + late apoptosis)increased(all P<0.05).A total of 322 lncRNAs interacting with UPF1 were screened by RIP-seq.Due to the peak with higher enrichment,lnc RNA PVT1 was selected for further study.The RIP assay verified that there were binding sites between UPF1 and PVT1.The results of RNA stability measurement,Western blot,sphere-forming assay,CCK-8 assay,Transwell assay,and flow cytometry showed that UPF1 regulates cell self-renewal,proliferation,migration,invasion,cell cycle progression,and apoptosis via stabilizing PVT1(all P<0.05).Both bioinformatics software and dual-luciferase assay confirmed that PVT1 and miR-508-5p bind to each other specifically.In endometrial cancer tissues,miR-508-5p was highly expressed and was significantly related to myometrial invasion,lymph node metastasis as well as advanced FIGO stage(P<0.001).The results of Western blot,sphere-forming assay,CCK-8 assay,Transwell assay,and flow cytometry confirmed that overexpression of miR-508-5p significantly inhibited cell self-renewal,proliferation,migration,and invasion ability in ECSCs.The ECSCs in G2-M phase were significantly decreased,and the overall apoptotic rate of ECSCs(early apoptosis + late apoptosis)increased(all P<0.05).Through bioinformatics software and dual-luciferase gene reporter system,we confirmed that miR-508-5p could bind to the 3’UTR of SOX2.The results of Western blot,sphere-forming assay,CCK-8 assay,Transwell assay,and flow cytometry verified that downregulation of PVT1 reduced expression of SOX2 by negatively regulating miR-508-5p,thus inhibiting the self-renewal,proliferation,migration,invasion,and cell cycle progression,and promoting cell apoptosis(all P<0.05).Conclusion:1.UPF1 is highly expressed in endometrial cancer tissues.miR-508-5p is under-expressed in endometrial cancer tissues.The high expression of UPF1 and low expression of miR-508-5p were related to clinicopathological parameters such as myometrial invasion and advanced FIGO stage.2.UPF1 is highly expressed in ECSCs.Knockdown of UPF1 significantly down-regulated the expression of PVT1,inhibited the self-renewal,proliferation,migration,invasion,and cell cycle progression,and promoted cell apoptosis in ECSCs.UPF1 bound to and increased the stability of PVT1,affecting the biological behavior of ECSCs.3.PVT1 competitively combined with miR-508-5p as a ce RNA to reduce the suppressive effect of miR-508-5p on SOX2.Silencing UPF1 reduced the stability of PVT1,lowered PVT1 binding to miR-508-5p,and reduced SOX2 expression,thereby inhibiting the selfrenewal,proliferation,migration,invasion,and cell cycle progression,and promote cell apoptosis in ECSCs.UPF1/PVT1/miR-508-5p axis provides insights and new targets for the regulatory mechanism of stemness in ECSCs. |
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