| Objective: 1)Construct the DNA methyltransferase 1(DNMT1)high expression model of Kazakh esophageal epithelial cells,to provide a impotent research tool for studying the mechanism of esophageal cancer;2)Explore the role of DNMT1 high expression on N-methyl-N’-nitro-N-nitrosoguanidine(MNNG)to induce malignant transformation of Kazakh esophageal epithelial cell,and construct the malignant transformation model of Kazakh esophageal epithelial cells with high levels of DNMT1 expression,to provide a stable,simple experiment methods and research tool for future studying the mechanism of esophageal cancer;3)Explore the intervention of folic acid of MNNG in Kazakh esophageal epithelial cells,epigenetics mechanism and the regulatory mechanism of Wnt/β-catenin signaling transduction pathway,to provide theoretical basis for the prevention and treatment of esophageal cancer.Methods: 1)The constructed plasmids of pXanthoTMV.basic.puro-WV0133,p XanthoTMV.basic.puro-WV0132 and pXanthoTMV.basic.puro-WV072 were transformed into esophageal epithelial cells by TALE.The DNMT1 high expression cell were selected by puromycin.Transfection of the cells was observed under the inverted fluorescence microscope.The mRNA and protein expression of DNMT1 were determined by Real-Time PCR and Western blot;2)The dosage of MNNG malignant transformation was determined by MTT and the clone forming assay;The Kazakh esophageal epithelial cells with high levels of DNMT1 expression were treated by exposure to the effective concentration of MNNG for lh each time and digest by trypsin.The colony formation conditions was observed in different exposure frequency and passage number cells by soft AGAR colony forming experiment,and clon amplification culture of Kazakh esophageal epithelial cells-zhz(malignant transformed cells);24 nude mice(BALB/c-nu)were divided into 3 groups in nude mouse transplantation tumor experiment,namely,the control group(saline group),the DNMT1 high expression of Kazakh esophageal epithelial cells group and the malignant transformation model of Kazakh esophageal epithelial cells with high levels of DNMT1 expression group(malignant transformed cells).There was 8 mice in each group.The dosage of 0.2mL per mouse were injected into the near right side on the back of the nude mice.After 4 weeks,the malignant degree of Kazakh esophageal epithelial cells with high levels of DNMT1 expression and its transformed cells(Kazakh esophageal epithelial cells-zhz)were observed by histopathology.3)The change of total DNA methylation level was detected by HPLC;The methylation of MTHFR、CBS、MGMT、P16、RASSF1A and FHIT were detected by MSP;The mRNA and protein level of MTHFR、 CBS、 MGMT、 P16、 RASSF1 A and FHIT were tested by RT-PCR and Western-Blot.4)The m RNA and protein expression levels of key factors(β-catenin、APC and TCF)in Wnt/β-catenin signaling transduction pathway were tested by RT-PCR and Western blot,respectively.Results: 1)Compared with normal cell group,the mRNA expression levels of DNMT1 in the transfected pXanthoTMV.basic.puro-WV0133 cell and pXanthoTMV.basic.puro-WV0132 cell were 1.786 times and 2.721 times respectively,and their protein expression levels of DNMT1 were 2.734 times and 3.100 times respectively.They were significantly higher than in normal cell group(P<0.01).The mRNA and protein expression levels of DNMT1 in transfected pXanthoTMV.basic.puro-WV0132 cell were the highest.2)With the increase of the exposure frequency and passage number cells,the cell morphology tended to be irregular,the cell contacted inhibition was gradually disappeared,the tolerance was gradually increased,and the growth rate increased gradually.The 27 th generation of Kazakh esophageal epithelial cells with high levels of DNMT1 expression showed cell colonys,central bulge,and composed of multilayer cells.The Kazakh esophageal epithelial cells had no any positive results,and cell pyknosis and death were observed.The lumps were abserved when we injected the Kazakh esophageal epithelial cells-zhz to the nude mice.The lump were considered to be squamous cell carcinoma by histopathology.3)Under the long-term exposure of MNNG which final concentration was 1.500 x 10-5mol/L,the following results appeared:(1)With the increase of the concentration of folic acid,the overall methylation level of Kazakh esophageal epithelial cells and the DNMT1 high expression cells was significantly increased(P<0.01),the enzymatic activity of DNMT1 was significantly reduced(P<0.01);With the extension of time,the overall methylation level of Kazakh esophageal epithelial cells and the DNMT1 high expression cells was significantly reduced(P<0.01),the enzymatic activity of DNMT1 was significantly increased(P<0.01);At the same condition,the overall methylation level of Kazakh esophageal epithelial cells was higher than in the DNMT1 high expression cells at the late stage(tearly stage=0.696,P=0.494;tmedium stage=0.605,P=0.551;tlate stage=2.746,P=0.012);(2)The increase of the concentrations of folic acid had some effection on methylation condition of MTHFR,RASSF1 A and FHIT(except the Kazakh esophageal epithelial cells)in Kazakh esophageal epithelial cells and the DNMT1 high expression cells.The methylation condition of MTHFR,RASSF1 A and FHIT was reversed in high-dose folic acid.There was no effection on methylation condition of CBS,MGMT and P16;(3)With the increase of the concentration of folic acid,the expression of mRNA and protein of MTHFR,CBS,MGMT,P16,RASSF1 A and FHIT in Kazakh esophageal epithelial cells and the DNMT1 high expression cells were significantly increased(P<0.01);With the extension of time,the expression of mRNA and protein of MTHFR,CBS,MGMT,P16,RASSF1 A and FHIT were significantly reduced(P<0.01);At the same condition,the expression of mRNA and protein of MTHFR,CBS,MGMT,P16,RASSF1 A and FHIT in Kazakh esophageal epithelial cells was higher than in the DNMT1 high expression cells(P<0.01).4)With the increase of the concentration of folic acid,the expression of mRNA and protein of key factors(β-catenin and TCF)in Wnt/β-catenin signal transduction pathway of Kazakh esophageal epithelial cells and the DNMT1 high expression cells were significantly reduced,the expression of mRNA and protein of APC was significantly increased;With the extension of time,the expression of mRNA and protein of key factors(β-catenin and TCF)in Wnt/β-catenin signal transduction pathway of Kazakh esophageal epithelial cells and the DNMT1 high expression cells were significantly increased,the expression of mRNA and protein of APC were significantly reduced;At the same condition,the expression of mRNA and protein of key factors(β-catenin and TCF)in Wnt/β-catenin signal transduction pathway of Kazakh esophageal epithelial cells were lower than in the DNMT1 high expression cells,the expression of mRNA and protein of APC were higher than in the DNMT1 high expression cells.Conclusion: 1)The model of Kazakh esophageal epithelial cells with high levels of DNMT1 expression was successfully constructed,which can provide a research tool to study on esophageal cancer mechanism.2)The malignant transformation model of Kazakh esophageal epithelial cells with high levels of DNMT1 expression was successfully constructed.DNMT1 high expression had effection of promote cell malignant transformation and could shorten the time of malignant transformation.3)Compared with the esophageal epithelial cells,the DNMT1 high expression of Kazakh esophageal epithelial cells became sensitive to MNNG and low-dose folic acid.It suggested that the high-dose folic acid could reduce the damage of Kazak normal esophageal epithelial cells which caused by MNNG,and reverse the changes of epigenetics indicators.Adequate folic acid plays a antagonistic role in cells adverse reactionn.4)Sufficient folic acid could inhibit activation of Wnt/β-catenin signaling transduction pathway with MNNG which played a protective role in the damage caused by MNNG. |
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