The Role Of EZH2 And Its Mediated STAT3 Methylation In Autosomal Dominant Polycystic Kidney Disease

Background and Objective Autosomal dominant polycystic kidney disease(ADPKD)is one of the most common genetic disorders caused by a single gene mutation.It’s mostly caused by mutations in either PKD1 or PKD2 and is characterized by formation of renal cysts along with the enlargement of kidneys,development of mμltiple bilateral renal cysts that replace normal kidney tissue and deterioration of their function,eventually leading to renal insufficiency.The disease affects about 1 in 400 to every 1 in 1000 live births,it usually manifests itself at the age of 30–40 years and accounts for 8–10% of cases of end-stage renal disease(ESRD).Despite of all the progress made these years,ADPKD is still regarded as an untreatable,progressing relentlessly towards end-stage renal disease.Studys showed that proliferation and dedifferentiation of cyst lining epithelial cells derived from normal renal tubular epithelial cells were the key factor in the development and progression of polycystic kidney disease,the processing is very similar to benign tumors.EZH2 is a histone-lysine N-methyltransferase enzyme,which can regulate the methylation of H3K27 and plays an important role in pathological/physiological processes.Studies showed that EZH2 was overexpressed in many solid tumors,such as breast,bladder,ovarian,etc.Yet it has become a hot spots and new targets for cancer therapy.EZH2 mediated non-histone protein methylation is an important ost-translational modification involved in many life processes,such as cell cycle,DNA repair,cell senescence,differentiation,apoptosis and tumorigenesis.The microarray data analysis in our laboratory showed that EZH2 was over-expressed in the rat model of polycystic kidney disease,and was gradually increased with the increase of age,which sμggest there EZH2 might play a role in ADPKD.Until now,there is no report about the relationship between ADPKD and EZH2 worldwide.This research is aimed to explore value of EZH2 to be a new therapeutic of ADPKD,to study the effect and role of EZH2 mediated non-histone protein methylation in the mechanism of ADPKD,so as to provide theoretical and experimental basis for ADPKD.This study will explore the role of EZH2 from cells,tissues and the overall level,the contents is divided into the following 4 parts:Part I: EZH2 expression and enzymatic activity in PKD tissureObjective: To observe the expression and enzymatic activity of EZH2 in PKD tissuesMethods: Polycystic kidney tissues were deprived from Pkd1 knockout mice and ADPKD patients.PCR and western blot were used to detect the expression of EZH2,the activity of EZH2 were shown by the expression of its specific substract,using Western blot to detect the methylation of histione 3 lysine 27.Result: 1.The expresson of EZH2 in kidney tissues of PKD patients was significantly higher than in control group,and the enzymatic activity of EZH2 was significantly higer,too.2.The express and enzymatic activity of EZH2 in the kidney tissues of pkd1 knockout mice were both up-regulated in comparison to control,and gradually increased with age increase.Conclusion: EZH2 was overexpressioned in ADPKD.Part II: Effect and mechanism of EZH2 inhibitor on cell proliferation of ADPKD cell lines and cyst growth of Pkd1 knockout miceObjective: To explore the effect and mechanism of EZH2 inhibitor on the cell proliferation of ADPKD cell lines and cyst growth of Pkd1 knockout mice.Methods: Using selective EZH2 inhibitor GSK126 to explore the effect on ADPKD cell(WT9-12)proliferation,cell proliferation of WT9-12 was examined by MTT assay,using the LDH assay to evaluate the toxicity of GSK126 on WT9-12 cells.Using selective EZH2 inhibitor GSK126 to explore the effect on the cyst growth and development of pkd1 knockout mice.Using the HE staining and Ki67 staining to evaluate the kidney cyst growth and cell proliferation of Pkd1 knockout mice.Using EZH2 plasmid and si RNA to overexpress and reduce the expression of EZH2,then we can observe the activity of STAT3 through Western blotting and verify the effect of EZH2 inhibition on STAT3 phosphorylation.Results: EZH2 selective inhibitor GSK126 inhibited the cell proliferation of ADPKD cell lines and the inhibition concentration wasn’t toxic.EZH2 selective inhibitor GSK126 delayed cyst growth,reduced KW/BW ratio,BUN level and Scr level and improved kidney function of Pkd1 knockout mice.Overexpress of EZH2 through plasimid transfection lead to increase of STAT3 activity,inhibition of EZH2 through si-EZH2 or GSK126 resulted in concentration-dependent decrease of STAT3 phosphorylation.GSK126 also inhibited STAT3 phosphorylation of the kidney of Pkd1 knockout mice.Conclusion: EZH2 inhibitor inhibited cell proliferation of ADPKD cell lines,delayed cysts growth and improved kidney function of Pkd1 knockout mice,EZH2 might be anew target for ADPKD treatment.EZH2 regulated the phosphorylation of STAT3.Part III: EZH2 overexpression induced renal cyst formation in zebrafishesObjective: To study if EZH2 overexpression can induce renal cyst formation in zebrafishes.Methods: Using p CS2-EGFP as vector and Bam HI/Xho I as restriction enzymes to construct p CS2-EZH2-EGFP plasmid for zebrafishes,using the Not I linearization assay to transcribe the plasmid and synthesis full-length EZH2 m RNA.Overexpress EZH2 in zebrafish through microinjection the EZH2 m RNA and observe if EZH2 can induce renal cyst formation in zebrafishes.Results: Part of the zebrafishes with EZH2 overexpression developed renal cyst,while there is no cyst formation in zebrafishes from control group.The proportion of the EZH2 overexpressed zebrafishes developed cyst was about 17.1%±9.8%.Conclusion: EZH2 overexpression induced renal cyst formation in zebrafishes.Part IV: the mechanism of EZH2 overexpression and the role of EZH2 mediated STAT3 methylation in polycystic kidney diseasObjective: To explore the mechanism of EZH2 overexpression in polycystic kidney disease and to study the role of EZH2 mediated STAT3 methylation in the mechanism of polycystic kidney disease.Methods: Using c AMP activator Forskolin,c AMP analogue 8-Br-camp,c AMP inhibitor Rp-camp and PKA inhibitor H89 to explore the impact of c AMP/PKA signaling pathway on the expression of EZH2 in ADPKD cell lines;Using immunoprecipitation and Western blotting to explore whether EZH2 directly bind to and methylate STAT3 and if GSK126 regulated STAT3 ativity through regulating its methylation.Results: The c AMP activator Forsklin and c AMP analogues 8-Br-c AMP increased EZH2 expression and activity in WT9-12 cells,while c AMP competitive inhibitors Rp-c AMP and PKA inhibitor H89 decreased EZH2 expression and activity in WT9-12 cells.EZH2 bind to STAT3 directly in WT9-12 cells and kidney tissue of ADPKD patients.STAT3 methylation was increased in the kidney tissue of ADPKD patients.GSK126 reduced STAT3 methylation in WT9-12 cells.Conclusion: The expression of EZH2 was regulated by c AMP/PKA signal pathway in ADPKD,EZH2 medicated methylation of STAT3 plays important role in ADPKD by regμlating the phosphorylation of STAT3.