Research On The O-GlcNAcylation Of C-Jun To Promote The Proliferation Of Ovarian Cancer Through Regulating The Expression Of MUC1

Objective:1.Bioinformatics was used to analyze the expression of MUC1 between ovarian cancer and normal ovarian tissues,to analyze the relationship between the expression of MUC1 and the prognosis of patients with ovarian cancer,and to verify the expressions of MUC1 at tissue and cell levels.2.To investigate the relationship between the expression of MUC1 and the proliferation and apoptosis of ovarian cancer cells,and to verify the expression level of MUC1 and its O-GlcNAcylation level after treated with high glucose.3.To investigate the mechanism that O-GlcNAcylation of c-Jun can promote the proliferation of ovarian cancer through regulating the expression of MUC1.Methods:1.Bioinformatics analyzed that the expressions of MUC1 in ovarian cancer tissues and normal ovarian tissues,the expressions of MUC1 in different stages of ovarian cancer,and its relationship with the prognosis of ovarian cancer patients.2.qRT-PCR and western blot were used to detect the m RNA and protein expressions of MUC1 in ovarian cancer tissues and normal ovarian tissues,respectively.3.The m RNA and protein expressions of MUC1 between five human ovarian cancer cells(SKOV3,OVCAR3,UWB1.289,CAOV-3,HO-8910)and normal ovarian epithelial cells(IOSE80)were detected by qRT-PCR and western blot,respectively.4.Cell transfection technology was used to transfect the ovarian cancer cells with MUC1 interference sequences and overexpression sequences.Cell proliferation was detected by CCK8;The cell colony formation experiment was used to detect the colony formation.Apoptosis was detected by flow cytometry(Annexin V/PI staining).5.The ovarian cancer cells were treated with high glucose,while the control group was the cells without high glucose treatment.The m RNA and protein expressions of MUC1 were detected by qRT-PCR and western blot respectively.The O-GlcNAcylation of MUC1 was detected by enzymatic labeling of O-GlcNAc site experiments and Co-immunoprecipitation(Co-IP).6.The MUC1 transcription factor c-Jun was predicted by bioinformatics;Chromatin immunoprecipitation(Chip)assay and double luciferase reporter assay verified that the c-Jun could bind to the promoter regions of MUC1 gene;c-Jun interference sequences and overexpression sequences were transfected into ovarian cancer cells,and the m RNA and protein expressions of MUC1 was detected by qRT-PCR and western blot,respectively.7.The ovarian cancer cells were treated with high glucose,and the control group without high glucose treatment.The m RNA and protein expressions of c-Jun were detected by qRT-PCR and western blot respectively;The ovarian cancer cells were treated with O-GlcNAcylation accelerant PUGNAc and GlcNAc,while the control group without the accelerant.The m RNA and protein expressions of c-Jun were detected by qRT-PCR and western blot respectively;The protein stability of c-Jun was detected by Cycloheximide(CHX)assay.The O-GlcNAcylation level of c-Jun was detected by enzymatic labeling of O-GlcNAc site experiments and Co-IP;The ovarian cancer cells were treated with O-GlcNAcylation accelerant PUGNAc and GlcNAc and meanwhile knockdown the c-Jun.CCK8 was used to detect cell proliferation;the cell colony formation experiment was used to detect colony formation;the apoptosis was detected by flow cytometry assay(Annexin V/PI staining);c-Jun and MUC1 were overexpressed,and the level of O-GlcNAcylation were detected by western blot.8.The c-Jun was upregulated by the overexpression sequences.The O-GlcNAcylation related proteins(OGT,GUCY1A3,UAP1,and SGLT1)were detected by western blot.Overexpressing the c-Jun,and meanwhile knocking down the SGLT1,the O-GlcNAcylation of c-Jun was detected by the Co-IP;qRT-PCR and western blot were used to detect the m RNA and protein expressions of MUC1 in ovarian cancer cells after SGLT1 was knocked down.Results:1.Bioinformatics analyzed that the expressions of MUC1 in ovarian cancer tissues and normal ovarian tissues,the expressions of MUC1 in different stages of ovarian cancer,and its relationship with the prognosis of ovarian cancer patients(1)The m RNA and protein expressions of MUC1 in ovarian cancer tissues increased significantly compared with normal ovarian tissues.(2)The expressions of MUC1 in ovarian cancer tissues was highest in stage IV tumors.(3)The survival rate of patients with high expression of the MUC1 gene was significantly lower than that of patients with low expression.2.Expressions of MUC1 in ovarian cancer tissuesThe expressions of MUC1 increased in ovarian cancer tissues compared with normal ovarian tissues.3.Expressions of MUC1 in ovarian cancer cellsCompared with normal ovarian epithelial cells,the m RNA and protein expressions of MUC1 in ovarian cancer cells(SKOV3,OVCAR3,UWB1.289,CAOV-3,and HO-8910)increased.4.The effects of MUC1 on proliferation and apoptosis of ovarian cancer cells(1)Compared with the control group,overexpression of MUC1 can promote cell proliferation(CCK-8)and the formation of cell colony colonies,but knocking down MUC1 can reduce cell proliferation(CCK-8)and the formation of cell colony.(2)Compared with the control group,apoptosis decreased in overexpressed MUC1 cells,and apoptosis increased in knockdown MUC1 cells.5.The effects of high glucose and MUC1 on the proliferation and apoptosis of ovarian cancer cells and detection on O-GlcNAcylation of MUC1(1)The m RNA and protein expressions of MUC1 increased in ovarian cancer cells treated with high glucose compared with the control group.(2)Compared with the control group,the proliferation of ovarian cancer cells and the ability of colony formation increased and apoptosis decreased after treatment with high glucose.compared with the high-glucose group,the proliferation of ovarian cancer cells and the ability of colony formation decreased and apoptosis increased after treatment with high-glucose + sh-MUC1.(3)The protein of MUC1 was not modified by O-GlcNAcylated in the ovarian cancer cells.6.The molecular mechanism research on the MUC1 regulated by c-Jun(1)According to the bioinformatics website,the upstream transcription factor c-Jun of MUC1 was found.(2)Chromatin immunoprecipitation(Chip)assay and double luciferase reporter assay confirmed that transcription factor c-Jun can bind to the promoter region of MUC1.(3)Compared with the control group,m RNA and protein expressions of MUC1 increased when c-Jun was overexpressed,and expressions of MUC1 decreased when c-Jun was knocked down.7.The roles of c-Jun and O-GlcNAcylation in promoting proliferation and inhibiting apoptosis of ovarian cancer cells,and the effect of c-Jun on O-GlcNAcylation(1)The expressions of c-Jun in ovarian cancer cells treated with high glucose.After treated with high-glucose,the m RNA of c-Jun were not significantly increased;The protein of c-Jun increased significantly.(2)The expression of c-Jun in ovarian cancer cells treated with PUGNAc and GlcNAc.The m RNA and protein levels of c-Jun were both significantly increased after treated with PUGNAc and GlcNAc.(3)The protein stability of c-Jun in ovarian cancer cells treated with PUGNAc and GlcNAc was detected by CHX assay.The protein stability of the c-Jun increased after being treated with PUGNAc and GlcNAc.(4)The O-GlcNAcylation of c-Jun was detected in ovarian cancer cells.The protein of c-Jun was O-GlcNAcylated in ovarian cancer cells.(5)The role of c-Jun and O-GlcNAcylation in promoting proliferation and inhibiting apoptosis of ovarian cancer cellsCompared with the control group,the proliferation of ovarian cancer cells and the ability of colony formation increased and apoptosis decreased after being treated with PUGNAc and GlcNAc.compared with the PUGNAc and GlcNAc treatments group,the proliferation of ovarian cancer cells and the ability of colony formation decreased and apoptosis increased after being treated with PUGNAc and GlcNAc + sh-c-Jun.(6)The effect of c-Jun on O-GlcNAcylationThe up-regulation of c-Jun increased the level of O-GlcNAcylation,while the up-regulation of MUC1 showed no effect on O-GlcNAcylation.8.The detection of the O-GlcNAcylation-related proteins.(1)Compared with the control group,after overexpressing of the c-Jun gene,O-GlcNAcylation related protein SGLT1 significantly increased.(2)Compared with the control group,after overexpressing of the c-Jun gene,the level of O-GlcNAcylation increased,and after the overexpressing of the c-Jun gene+ si-SGLT1,the level of O-GlcNAcylation decreased.(3)In ovarian cancer cells,the m RNA and protein expressions of MUC1 decreased after si-SGLT1.Conclusion:1.The expression of MUC1 is higher in ovarian cancer tissues than normal ovarian tissues and the high expression of MUC1 is associated with poor prognosis.MUC1 can promote proliferation and inhibit the apoptosis of ovarian cancer cells.2.The O-GlcNAcylation promotes the expression of MUC1 through increasing the O-GlcNAcylation of c-Jun protein.Meanwhile,the increased protein expression of c-Jun also promotes the level of O-GlcNAcylation.O-GlcNAcylation /c-Jun can form a positive feedback loop,and promote proliferation and inhibit the apoptosis of ovarian cancer cells.3.The O-GlcNAcylation/c-Jun axis regulates the O-GlcNAcylation through SGLT1.In turn,the down-regulation of SGLT1 can reduce the O-GlcNAcylation and the m RNA and protein expressions of MUC1.