Ring1B/BZH2-Mediated Histone Modification And TWIST-Mediated Transcriptional Repression In Pancreatic Cancer

Background: Polycomb group(Pc G)proteins Ring1 B and EZH2,which have been characterized as catalyzing the two epigenetic modifications H2AK119 monoubiquitination(H2AK119Ub1)and H3K27 trimethylation(H3K27Me3),are well-known epigenetic silencers implicated in embryonic development and tumorigenesis.Transcriptional repressor Twist is a master regulator of epithelialmesenchymal transition,yet the epigenetic mechanism governing Twist to induce EMT is poorly understood.Here we studied the epigenetic mechanism that Ring1B/EZH2-mediated H2AK119Ub1 and H3K27me3 involves in the Twist-induced transcriptional repression and tumor metastasis.Methods: First immunohistochemistry was performed on tissue microarrays of human resected pancreatic duct adenocarcinoma to detect Ring1 B,H2AK119Ub1,EZH2,and H3K27Me3 expressions in tumor tissues,and the correlations between these proteins and clinical prognosis were analyzed.Then we silence Ring1 B and EZH2 via sh RNA to detect the level of H2AK119Ub1 and H3K27Me3 in the pancreatic cancer cells,and observe the tumor cell growth in vitro and in tumor xenograft models.Next,we performed immunohistochemistry on tissue microarrays of human distal pancreatic cancer specimens to assess the expression levels of Twist,then performed co-immunoprecipitation to verify the interaction between Ring1B/EZH2 and Twist.Twist,Ring1 B and EZH2 were simultaneously knocked down via sh RNA in hypoxic pancreatic cancer cells,and the Twist target gene E-cadherin and P16 was checked by real-time PCR and Western blotting,and cell growth and migration ability were tested thereafter by transwell assay and mice model.Finally,Chromatin-immunoprecipitation was employed in the above cells to detect Ring1 B,H2AK119Ub1,EZH2,and H3K27Me3 enriching at the promoter region of Twist target locus.Results: Here we demonstrated that both Ring1 B and EZH2 were elevated in pancreatic ductal adenocarcinoma(PDAC),and H2AK119Ub1 and H3K27Me3 cooperate in tumors and are associated with the clinical prognosis in combinatorial patterns.Simultaneous silencing of Ring1 B and EZH2 via sh RNA depleted H2AK119Ub1 and H3K27Me3 in the pancreatic cancer cells,enhanced HOX gene derepression.TWIST was overexpressed in distal pancreatic cancer invading splenic artery and was associated with the worst prognosis,which might be activated by hypoxic microenvironment.We identified Ring1 B and EZH2 as Twist interacting proteins.Ring1B/EZH2 knocking down simultaneously in pancreatic cancer cells resulted in derepression of Twist target gene E-cadherin and P16,as well as impairement of Twist-induced cell growth and migration ability.Finally,Ch IP on the above stable cells demonstrated that Twist recruited Ring1B/EZH2 to E-cadherin and P16 gene promoter region to catalyze H2AK119Ub1 and H3K27Me3.Conclusion: This study shows that Ring1B/EZH2-mediated H2AK119Ub1 and H3K27me3 may serve as discriminatory biomarkers for molecular staging of pancreatic cancer,and Ring1B/EZH2 involving in hypoxic Twist-mediated transcriptional repression.