Mutant P53 Induces EZH2 Expression And Promotes Epithelial-mesenchymal Transition By Attenuating MiR-26a Yield In Endometrial Carcinoma

Objective: To explore the molecular mechanism of mutant type p53 promoting epithelial-mesenchymal transition(EMT)in endometrial carcinoma(EC).Contents:Mutant type p53 and EZH2 protein expression were examined in(EC)tissue specimens.We analyze the relationship between mutant type p53 and EZH2 expression level and its association with clinical parameters in EC.We explore mutual regulatory relationship between EZH2 and mutp53 and its underlying molecular mechanism.We also investigate the effects of EZH2 on biological behaviors of EC cells.Plasma miR-26 a expression was detected and analyzed its association with prognosis in EC patients.Methods: Immunohistochemistry(IHC),Western blot,and cell immunofluorescence were used to detect protein expression.QRT-PCR was performed to examine mRNA and miRNA expression.SiRNA and shRNA were carried out to silencing gene expression.Overexpression plasmids and lentivirus were used to up-regulate gene expression.ChIP-PCR and RNA-ChIP were performed to check the interaction between protein and nucleic acid.Co-immunoprecipitation(CO-IP)was used to check the interaction between protein and protein.Luciferase reporter plasmid was constructed to demonstrate the direct target of miRNA.Transwell assay was carried out to analyze the migration and invasion ability changes.Results: Both EZH2 and mutant p53(mutp53)are highly expressed in type ? EC specimens compared with type ?.EZH2 expression was positively correlated with p53 expression,and it associated with high FIGO stage,deep myometrial invasion,higher tumor grade,and lymph node metastasis.EZH2 protein expression was significantly decreased with mutp53 knockdown,but its mRNA expression remained unchanged in HEC-1B cells.Mutant type p53 up-regulate EZH2 by inhibiting miR-26 a expression at post-transcriptional level.P53 mutations not only impair its ability to bind the promoter of miR-26a-1,but also impair p68-Drosha complex assembly and attenuate miR-26a-1 processing.In addition,EZH2 is a direct downstream target of miR-26 a.Re-expression of miR-26 a decreases the migration and invasion ability and reverses mesenchymalepithelial transition(MET)of HEC-1B cells with TP53 mutation.MiR-26 a significantly decreased the tumor formation of HEC-1B cells in vivo.Rescuing miR-26 a expression also inhibits EZH2,N-cadherin,Vimentin,and Snail expression and induces E-cadherin expression both in vitro and in vivo.Expression of miR-26 a was significantly reduced in type ? EC serum compared to type ? EC.Patients with higher serum miR-26 a levels have a better survival rate.Conclusion: mutant-type p53 promotes EC cells EMT through p53/miR-26a/EZH2 regulation axis,leading to increase of its invasion ability.MiR-26 a not only may be a molecular target in EC therapy but also a diagnostic tool for EC patients.Part ? The significance of EZH2 and mutant p53 expression in endometrial carcinoma tissuesObjective: To analyze EZH2 expression level in endometrial carcinoma(EC)tissue specimens and its association with clinical parameters and p53 expression level.Methods: Immunohistochemistry(IHC)was performed to examine EZH2 and p53 protein expression in EC tissue specimens.Results:(1)Both EZH2 and p53 are highly expressed in type ? EC tissue specimens compared with type ?(58.8% vs 10% and 67.6% vs 6.7% respectively).(2)EZH2 expression was positively correlated with p53 expression in type ? EC(Spearman correlation r = 0.739).(3)EZH2 expression was higher in cases with a high FIGO stage(P < 0.001),a deep myometrial invasion(P = 0.023),a high histological grade(P < 0.001),and lymph node metastasis(P = 0.002).Conclusion: Both EZH2 and mutant p53(mutp53)are highly expressed in type ? EC specimens compared with type ?.EZH2 expression was positively correlated with p53 expression,and it associated with high FIGO stage,deep myometrial invasion,higher tumor grade,and lymph node metastasis.Part ? Mutant p53 induces EZH2 expression by inhibiting miR-26 a expressionObjective: To explore mutual regulatory relationship between EZH2 and mutp53 using EC cell lines with wild type TP53 or mutant type TP53.Methods: Western blot was performed to detect p53 and EZH2 protein expression in EC cell lines.HEC-1B and KLE cells were transiently transfected with EZH2 siRNA?TP53 siRNA or counterpart negative control repectively.QRT-PCR and Western Blot were carried out to examine EZH2 and p53 mRNA and protein expression after manipulating EZH2 and p53 protein expression in HEC-1B and KLE cells.To determine which miRNAs are regulated by mutp53 and regulate EZH2 expression,we used qRT-PCR and the biological predicition software,Target Scan.HEC-1B and Ishikawa cells were transiently transfected with miR-26 a mimics?miR-26 a inhibitors(anti-miR-26a)or counterpart negative control(Ctrl)repectively.QRT-PCR and Western blot were performed to examine EZH2 mRNA and protein expression after miR-26 a expression change in HEC-1B and Ishikawa cells.Results:(1)EZH2 was overexpressed in HEC-1B,KLE,and AN3 CA cells compared with SPEC-2 and Ishikawa cells(P < 0.001).(2)Mutp53 mRNA and protein expression were not changed obviously with EZH2 knockdown in HEC-1B cells(P > 0.05).(3)EZH2 protein expression was decreased with mutp53 knockdown(P < 0.05),but its mRNA expression remained unchanged in HEC-1B cells(P > 0.05).(4)Down-regulation of mutp53 by siRNA increased miR-26 a expression in HEC-1B and KLE cells(P < 0.05).(5)Compared with controls,transfection of miR-26 a mimics decreased EZH2 protein expression in HEC-1B cells.In contrast,EZH2 protein expression was increased in Ishikawa cells transfected with anti-miR-26 a.Neither treatment altered EZH2 mRNA expression.Conclusion: Mutp53 possibly induces EZH2 expression by inhibiting miR-26 a expressionPart ? The molecular mechanism about the effects of mutp53 on miR-26 a expression and the effects of miR-26 a on EZH2 expressionObjective: To explore the complex molecular mechanism about the effects of mutp53 on miR-26 a expression and the effects of miR-26 a on EZH2 expression.Methods: HEC-1B cells were transiently transfected with TP53 siRNA or counterpart negative control repectively.The expression of pre-miR-26a-1 and pre-miR-26a-2 expression were examined after manipulating mutp53 protein expression in HEC-1B cells using qRT-PCR.Ch IP-PCR was performed to evaluate the binding ability of wild type p53 and mutant type p53 on miR-26a-1 promoter region.Co-immunoprecipitation(CO-IP)assay was used to detect binding between mutp53 and p68,Drosha and p68 respectively.QRT-PCR was performed to examine miR-26 a expression after stable transfection with p68 sh RNA and p68 overexpression plasmid in HEC-1B cells.Western blot and qRT-PCR were performed to examine EZH2 protein and miR-26 a expression after stable transfection with different mutant type p53 and wild type p53 expression plasmid in p53-null HEC-50 cells respectively?RNA-Chi P was used to detect binding between pri-miR-26a-1 and Drosha/p68.Luciferase reporter plasmid was constructed to demonstrate the direct target of miR-26 a using dual luciferase reporter assay system.Results:(1)After knockdown of mutp53 both pre-miR-26a-1 and pre-miR-26a-2 were increased about 2-fold over the control group(P < 0.01).However,the baseline expression of pre-miR-26a-1 was nearly 150-fold that of pre-miR-26a-2 in HEC-1B cells(P < 0.0001).(2)The binding ability between wild type p53 and miR-26a-1 promoter region is significantly stronger than that between mutp53 and miR-26a-1 promoter region.(3)Knockdown of p68 decreased miR-26 a expression(P < 0.001),while re-expression of p68 rescued miR-26 a expression in HEC-1B cells.(4)Wild type p53 induced miR-26 a expression and suppressed EZH2 protein expression(P<0.01),while mutp53 suppressed miR-26 a expression(P<0.01)and increased EZH2 protein expression in p53-null HEC-50 cells after stable transfection with wild type p53 and mutant type p53 expression plasmids respectively.(5)TP53 mutant cells had reduced pre-miR-26a-1 expression but no change in pri-miR-26a-1 expression in p53-null HEC-50 cells after stable transfection with mutant type p53 expression plasmids.In contrast,wild-type p53 transfection increased pre-miR-26a-1 expression in p53-null HEC-50 cells.(6)TP53-null HEC-50 cells transfected with mutp53 had decreased interactions between Drosha and p68,while cells with wild-type p53 showed a modest increase in the Drosha–p68 association.(7)TP53-null HEC-50 cells with mutp53 transfection had decreased associations between pri-miR-26a-1 and p68/Drosha.In contrast,wild-type p53 had increased associations between pri-miR-26a-1 and p68/Drosha.(8)miR-26 a decreased the relative luciferase activity of the wild-type EZH2 3'UTR vector by more than 65%(P<0.001),whereas the reduction in luciferase activity of the mutant EZH2 3'UTR vector was not as dramatic(P = 0.0122).Conclusion: p53 mutations not only impair its ability to bind the promoter of miR-26a-1,but also impair p68-Drosha complex assembly and attenuate miR-26a-1 processing.In addition,EZH2 is a direct downstream target of miR-26 a.Together,this results in a reduction in miR-26 a expression and upregulation of EZH2 expression in EC.Part ? The effects of EZH2 on biological behaviors of endometrial carcinoma cellsObjective: To investigate the effects of EZH2 on migration,invasion and the epithelial-mesenchymal transition(EMT)of EC cells.Methods: HEC-1B and AN3 CA cells were transiently transfected with miR-26 a mimics and counterpart negative control respectively.Ishikawa cells were transiently transfected with miR-26 a inhibitors(anti-miR-26a)and counterpart negative control respectively.The effects of miR-26 a on EC cells migration,invasion and epithelial-mesenchymal transition(EMT)were evaluated using transwell migration assay,transwell invasion assay.EMT-related marker E-cadherin,N-cadherin,Vimentin and Snail protein expression were detected by western blot assay and cell immunofluorescence.Results:(1)Re-expression of miR-26 a decreases the migration and invasion ability of HEC-1B with TP53 mutation(P < 0.001).Re-expression of miR-26 a also decreases invasion ability of AN3 CA cells with EZH2 high expression(P < 0.001)?(2)Inhibiting miR-26 a increases the invasion ability of Ishikawa cells with EZH2 low expression(P < 0.001).(3)Re-expression of miR-26 a induced E-cadherin expression and inhibited Vimentin,N-cadherin,and Snail expression in HEC-1B cells with TP53 mutation.Conclusions: miR-26 a promotes mesenchymal-epithelial transition(MET)of EC cells by inhibiting EZH2 protein expression.Part ? miR-26 a acts as a tumor suppressor and inhibits EZH2 protein expression in vivoObjective: To investigate the effects of miR-26 a on tumorigenicity of EC cells with TP53 mutation and its direct downstream target,EZH2,in vivo.Methods: miR-26 a expression lentivirus(LV-miR-26a)and counterpart negative control were constructed and packed.Subcutaneous tumor-formation assay in adult female nude mice was performed using HEC-1B cells with stable transfection of miR-26 a expression lentivirus or its control respectively.We drawed the tumor gowth volume curve and weighted the weight of the tumor mass.E-cadherin,Vimentin,Ki67 and EZH2 protein expression were detected using immunohistochemistry(IHC).Results:(1)The size and weights of tumors were smaller in LV-miR-26 a group ainimals than controls.(2)The expression of epithelial cell marker E-cadherin was lower in LV-miR-26 a group tumor tissue samples than controls.While the expression of mesenchymal cell marker Vimentin and proliferation marker Ki67 was lower in LV-miR-26 a group tumor tissue samples than controls.(3)The downstream target of miR-26 a,EZH2,was lower expression in LV-miR-26 a group tumor tissue samples than controls.Conclusions: miR-26 a acts as a tumor suppressor and EZH2 is the direct downstream target of miR-26 a in vivo.Part ? Plasma miR-26 a expression predicts prognosisObjective: To investigate plasma miR-26 a expression in EC patients and analyze its association with prognosis.Methods: qRT-PCR was carried out to examine plasma miR-26 a expression from EC patients.Results:(1)Expression of miR-26 a was significantly reduced in type ? EC serum compared to type ? EC(P = 0.0319).(2)Patients with higher serum miR-26 a levels have a better survival rate(Log-rank P = 0.0136).Conclusions: Plasma miR-26 a expression may be a diagnostic tool for EC patients.