Preliminary Studyon The Molecular Mechanisms Of SETDB1 Regulating Colorectal Carcinogenesis Through Wnt/?-catenin Signaling Pathway

Objective: Colorectal cancer,which ranking the fifth in the incidence and mortality of cancerin China,is one of the most common malignant tumors.A series of biological changes such as activation of proto-oncogenes,inactivation of tumor suppressor genes,changes in mismatch repair genes,and disturbances in apoptosis regulation occur during the development of colorectal cancer.Currently,there are no efficient molecular markers that can monitor or reflect these biological changes.Therefore,the study of colorectal cancer related genes and their functions will be not only beneficial to elucidate the molecular mechanism of colorectal cancer,but also may provide new molecular markers for clinical diagnosis,treatment,drug screening and prognosis.SETDB1(SET domain bifurcated 1)protein is a histone methyltransferase,which is highly expressed in a variety of human solid tumors,and its high expression is closely related to tumor growth and metastasis.Recent studies have shown that SETDB1 is probably an oncogenic gene,but the function and molecular mechanism of SETDB1 in colorectal cancer remain unclear.This project intends to study the function of SETDB1 in the development of colorectal cancer in clinical specimens,in vitroexperiments,and in vivo animal models,and to study the molecular mechanism of SETDB1 in regulating Wnt/?-catenin signaling pathway in colorectal cancer through in vitro experiments,so as to provide a new theoretical basis for the occurrence of colorectal cancer.Methods: 1.The expression of SETDB1 in colorectal cancer tissues was detected by immunohistochemical techniques,and the relationship between the expression of SETDB1 in cancer tissues and various clinical pathological parameters was analyzed.2.The plasmid transfection technique was used to establish HCT116 and SW480 cell lines with stable expression/knockdown SETDB1.The effects of in vitro proliferation abilities,colony cloning abilities,migration abilities,invasion abilities,cell cycle progress and in vivo tumorigenesis abilities in HCT116 and SW480 cells with stable expression/knockdown SETDB1 were detected by CCK8 experiment,EdU cell proliferation experiment,soft agar colony formation experiment,scratch experiment,Transwell invasion experiment,flow cytometry and subcutaneous tumor formation experiment.3.The effects of overexpression / knockdown of SETDB1 in HCT116 and SW480 cells on EMT(epithelial-mesenchymal transition)markers E-cadherin and Vimentin,Wnt/?-catenin signaling pathway key factors?-catenin,APC,GSK-3?,and Wnt/?-catenin signaling pathway target factors c-Myc and CyclinD1 were detected by RT-qPCR,Western Blot and immunofluorescence experiments.Results: 1.The expression of SETDB1 in colorectal cancer tissues:in colorectal cancer,the expression of SETDB1 in cancer tissues was significantly higher than that in adjacent tissues(P <0.001).The positive expression and differentiation degree of SETDB1,tumor size,preoperative serum CEA levels were statistically correlated(P <0.05),but not statistically correlated with age,gender,tumor site,pTNM stage,and lymph node metastasis(P> 0.05).2.The effects of SETDB1 on biological behaviors of HCT116 and SW480 cells in vivo and in vitro: the overexpression of SETDB1 enhanced the abilities of proliferation,colony cloning formation,migration,invasion in vitro and the abilities of tumorigenesis in vivo of HCT116 and SW480 cells,and promoted cell cycle differentiation from G0/G1 to S and G2/GM;knocking down SETDB1 reduced the abilities of proliferation,colony cloning formation,migration,invasion in vitro and the abilities of tumorigenesis in vivo of HCT116 and SW480 cells,and led to cell cycle arrest in the G0/G1 phase.3.Molecular mechanism of SETDB1 regulating Wnt/?-catenin of EMT pathway in colorectal cancer cells: after the overexpression of SETDB1,E-cadherin significantly decreased in mRNA and protein levels,Vimentin,c-Myc and CyclinD1 significantly increased in mRNA and protein levels.The levels of ?-catenin protein in the nucleus were significantly increased,and the protein levels of APC and GSK-3? were significantly reduced.After knocking down SETDB1,E-cadherin significantly increased in mRNA and protein levels.Vimentin,c-Myc and CyclinD1 in mRNA and protein levels were significantly reduced,?-catenin protein levels in thenucleus were significantly reduced,and APC and GSK-3? protein levels were significantly increased.Conclusions: 1.The expression of SETDB1 is up-regulated in colorectal cancer tissues,and its high expression is related to the degree of differentiation,tumor size,and serum CEA level before surgery.2.SETDB1 promotes the proliferation,migration,invasion and cell cycle of colorectal cancer cells in vitro,and enhances the tumorigenic capacity of cancer cells in vivo.3.SETDB1 may induce colorectal cancer EMT by activating Wnt/?-catenin signaling pathway,and promote the occurrence of colorectal cancer.