| BackgroundEpithelial ovarian cancer(EOC)seriously threatens the health of women.Owing to a lack of reliable early detection methods,about 70%patients are initially diagnosed as stage III or IV.At present,the standard treatment plan for patients with EOC is aggressive surgical debulking followed by platinum-based chemotherapy.Although the surgery and chemotherapy have been improved,there is still an 80%risk of recurrence in patients with advanced ovarian cancer,which is mainly due to acquired chemotherapy resistance.Therefore,exploring the mechanism of drug resistance in EOC and formulating effective prevention and treatment measures become the primary task to improve the prognosis of ovarian cancer patientsO-GlcNAc modification is a post-translational modification of proteins,which participates in the modification of many proteins in cells.It has been found that the abnormality of O-GlcNAc modification is related to the progression and prognosis of various tumors,but the study of O-GlcNAc modification in chemotherapy resistance of ovarian cancer is rare.As a self-protection mechanism,autophagy can help cells response to harsh environment.It is this self-protection ability that increases the tolerance of cancer cells to chemotherapy drugs.However,the mechanism of autophagy enhancement in drug resistance is still unclear.Studies have found that the SNAP-29 protein involved in the regulation of autophagic lysosome formation can be modified by O-GlcNAc.SNAP-29 mainly mediates the fusion process of autophagosome and lysosome by combining with Stxl7 and VAMP8.However,it is not clear whether the O-GlcNAc modification level of SNAP-29 affects the autophagy of ovarian cancer cells.In conclusion,we hypothesis that during the formation of EOC chemoresistance,the O-GlcNAc modification of SNAP-29 is reduced,promoting its binding to Stxl7 and VAMP8 to form a SNARE complex,mediating the formation of autophagosomes,and subsequently enhances the level of autophagy and increases the chemoresistance of ovarian cancer cells.ObjectiveThe expression of O-GlcNAc,OGT and OGA in epithelial ovarian cancer was analyzed,and the relationship between the expression of them and chemotherapy resistance was clarified.The effect of O-GlcNAc modification on chemoresistance of ovarian cancer cells was explored.In addition,the role of autophagy in chemoresistance of ovarian cancer induced by O-GlcNAc modification and its molecular mechanisms were investigated.Methods1.The expression of O-GlcNAc in clinical specimens.Clinical specimens of epithelial ovarian cancer were collected and divided into chemoresistant and chemosensitive group according to the response to chemotherapy.The age,clinical stage and histology of the two groups were compared.Immunohistochemistry was used to detect the expression of O-GlcNAc,OGT and OGA.Statistical analysis was used to determine whether there was any difference in the expression of them between the two groups.2.The effect of down-regulation of OGT on chemosensitivity of ovarian cancer cells.OGT-specific shRNA was used to generate OGT-deficient ovarian cancer cell lines.The effects of down-regulation of OGT on proliferation and apoptosis of ovarian cancer cells were detected,and the sensitivity of ovarian cancer cells to chemotherapy drugs were examined both in vivo and in vitro.3.The effect of down-regulation of OGT on autophagy in ovarian cancer cells.Western blot was used to detect the expression of autophagy-related proteins after down-regulation of OGT;immunofluorescence was used to observe the expression and distribution of LC3 after down-regulation of OGT;mRFP-GFP-LC3 adenovirus was used to observe the autophagy flux after down-regulation of OGT.Morphological changes of autophagy structure of ovarian cancer cells after OGT down-regulation were observed by transmission electron microscopy4.The effect of OGT knockdown on autolysosome formation.Autophagy was inhibited by Baf or 3-MA respectively.The sensitivity of cisplatin in OGT knockdown ovarian cancer cells was observed and the difference between the two groups was compared.The number of RFP-LC3 signals that co-localized with Lamp1,a late endosome-lysosome marker,was detected by immunofluorescence.5.OGT regulates autophagy through SNAP-29.The expression of SNAP-29 in SKOV3 cells was down-regulated by siRNA and verified by Western blot.After SNAP-29 was down-regulation,the expression of autophagy-related protein was tested by Western blot and the autophagy flux was detected by mRFP-GFP-LC3.After SNAP-29-GFP and RFP-LC3 plasmid was transfected,the co-localization of SNAP-29 and LC3 was detected by confocal microscope.6.The O-GlcNAc modification level of SNAP-29 affects the formation of SNARE complex.Western blot was used to detect the effect of OGT knockdown on the expression of SNAP-29 and Co-IP was used to detect the effect of OGT knockdown on the O-GlcNAc modification of SNAP-29.The O-GlcNAc modification site mutation plasmid of SNAP-29 was constructed and transfected into SKOV3 cells.Co-IP was used to detect the changes of O-GlcNAc modification of SNAP-29 and Western blot was used to detect autophagy-related proteins.The co-localization of SNAP-29 and LC3 was detected by confocal microscope.Results1.Low expression of O-GlcNAc was found in drug-resistant group.There was no significant difference in age,tumor stage and pathological type between chemoresistant and chemosensitive group.Compared with the chemosensitive group,the expression levels of O-GlcNAc and OGT in chemoresistant group decreased significantly,while there was no significant difference in the expression of OGA between the two groups.2.Down-regulation of OGT enhances cisplatin resistance in ovarian cancer cells.OGT-deficient ovarian cancer cell lines were generated by OGT-specific shRNA.The results of CCK8 assay showed that down-regulation of OGT had no effect on the proliferation of ovarian cancer cells,but the down-regulation of OGT after cisplatin treatment could partially rescue the inhibitory effect of cisplatin on the proliferation of ovarian cancer cells.Flow cytometry showed that down-regulation of OGT had no effect on the apoptotic rate of ovarian cancer cells.When treated with cisplatin,the apoptotic rate of ovarian cancer cells decreased significantly compared with the control group.Both the results of CCK8 assay and flow cytometry showed that down-regulation of OGT did not affect the sensitivity of ovarian cancer cells to paclitaxel.3.Down-regulation of OGT enhances the level of autophagy in ovarian cancer cells.Western blot showed that the level of p62 decreased significantly after OGT knockdown,while the ratio of LC3-?/? increased significantly.Immunofluorescence showed that the number of LC3 signals increased significantly after OGT knockdown.In addition,the RFP fluorescence increased significantly in OGT-deficient ovarian cancer cells as detected by autophagy flux assay.Transmission electron microscopy showed that the number of autophagic vacuoles per cell increased significantly in OGT-deficient cells after cisplatin treatment.4.Down-regulation of OGT promotes autoplysosome formation in ovarian cancer cells.After the treatment with Baf or 3-MA,cisplatin resistance induced by OGT knockdown was partially reversed,especially Baf.The co-localization of LC3 and Lamp 1 increased significantly after down-regulation of OGT.5.OGT regulates autophagy through SNAP-29.The expression of SNAP-29 in SKOV3 cells was down-regulated by siRNA technology.Western blot analysis showed that the expression of p62,LC3-II and LC3-I increased after down-regulation of SNAP-29.The addition of si-SNAP-29 reduced the number of red puncta induced by the down-regulation of OGT but increased the yellow puncta.The co-localization of SNAP-29 and LC3 increased significantly after OGT was down-regulated.6.The O-GlcNAc modification level of SNAP-29 affects the formation of SNARE complex.Western blot analysis showed that the down-regulation of OGT did not affect the expression of SNAP-29,but the O-GlcNAc modification of SNAP-29 was decreased.After the mutantion of O-GlcNAc modification site of SNAP-29,the autophagy level increased,and the binding of SNAP-29 to Stx17 and VAMP8 increased.Conclusion1.The expression of O-GlcNAc and OGT were correlated with chemoresistance in EOC.Down-regulation of OGT enhanced cisplatin resistance in ovarian cancer cells.2.Down-regulation of OGT enhanced cisplatin resistance of ovarian cancer cells by promoting autolysosome formation and inducing autophagy.3.Down-regulation of OGT reduced the O-GlcNAc modification level of SNAP-29 and promoted its combination with Stx17 and VAMP8 to form SNAP-29-Stx17-VAMP8 complex,thus mediating the binding of autophagosomes and lysosomes and promoting autophagy. |
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