| Objective: To investigate the relationship between methylation of promoter region of mismatch repair gene and 5-FU chemosensitivity of colon cancer.Methods:(1)Different concentrations of 5-azacytidine(0,1,5,10,20 ?g/mL)were used to induce the demethylation of promoter regions of mismatch repair genes in HT-29 and SW480 cell lines.Methylation-specific PCR was used to detect the methylation levels of the promoter regions of hMSH2 and hMLH1,and to screen the optimal concentration which could change the methylation levels of the promoter regions of hMSH2 and hMLH1.(2)Cell lines HT-29 and SW480 were treated with 5-FU at different concentrations(0,10,20,50,100,200 ?g/mL).Cell survival rate was measured by CCK8 to determine the most sensitive concentration of 5-FU.(3)Cell lines were divided into three groups: control group(without 5-Aza and 5-FU),5-FU group(with 100 ?g/mL,without 5-Aza),5-Aza+5-FU group(with 10 ?g/mL 5-Aza and 100 ?g /mL 5-FU).The mRNA level of hMLH1 and hMSH2 in two colon cancer cell lines were detected by PCR,and the expression of hMLH1 and hMSH2 in two colon cancer cell lines were detected by Western Blot.Flow cytometry was used to detect the apoptotic level of two colon cancer cell lines.(4)To establish orthotopic transplantation model of colon cancer in mice;(5)Orthotopic transplantation of colon cancer model mice were divided into three groups: control group(without 5-Aza and 5-FU),5-FU group(2.5 mg kg-1 d-1,twice a week for 4 weeks),5-Aza+5-FU group(5-Aza and 5-FU,25 mg kg-1 d-1,twice a week for 4 weeks),10 mice in each group.The size of orthotopic transplanted colon cancer in mice was observed,and mRNA level of hMLH1 and hMSH2 in transplanted tumor was detected by PCR,and expression of hMLH1 and hMSH2 and the apoptotic level in transplanted tumor were detected by Western Blot.Results:(1)The methylation level of promoter regions of hMLH1 and hMSH2 in colon cancer cell HT-29 and SW480 decreased with increase of 5-Aza concentration.And the optimum concentration of 5-Aza was 10 ?g/mL.(2)The survival rate of HT-29 and SW480 cells was not significantly affected by 5-Aza at different concentrations.But the survival rate of HT-29 and SW48 0 cell lines decreased with increase of 5-FU concentration,and the optimal concentration of 5-FU was 100 ?g/mL.When the concentration of 5-FU was from 20 to 200 ?g/mL,the survival rate of demethylated cell lines was significantly lower than that of methylated cell lines(p< 0.05).(3)There was no significant difference in expression of hMLH1 and hMSH2 between 5-FU group and control group in HT-29 and SW480 cell lines(p>0.05).The expression levels of hMLH1 and hMSH2 genes in 5-Aza+5-FU group were significantly higher than those in 5-FU group and control group(p< 0.05).(4)The number of apoptotic HT-29 cells in 5-FU group(51.9%)and 5-Aza+5-FU group(65.5%)were significantly higher than that in control group(3%)(p>0.05).And the number ofapoptotic HT-29 cells in 5-Aza+5-FU group was significantly higher than that in 5-FU group(p<0.05).The number of apoptotic SW480 cells in 5-FU group(40.2%)and 5-Aza+5-FU group(53%)were significantly higher than that in control group(1.8%)(p>0.05).The number of apoptotic SW480 cells in 5-Aza+5-FU group was significantly higher than that in 5-FU group(p< 0.05).(5)The volume of transplanted colon cancer in 5-FU group was significantly smaller than that in control group(p<0.05),and the average volume of transplanted colon cancer in 5-FU + 5-Aza group was significantly smaller than that in 5-FU group(p<0.05).(6)The expression of hMLH1 and hMSH2 in 5-FU group were not significantly different from those in control group(p>0.05).The expression of hMLH1 and hMSH2 in 5-Aza+5-FU group were significantly higher than those in 5-FU group and control group(p<0.05).The results were basically consistent with those in vitro.(7)The expression of Bcl-2 in 5-FU group and 5-Aza+5-FU group was significantly lower than that in control group(p<0.05),and Bcl-2 in 5-Aza+5-FU group was significantly lower than that in 5-FU group(p<0.05).The expression of Bax in 5-FU group and 5-Aza+5-FU group was significantly higher than that in control group(p<0.05),and Bcl-2 in 5-Aza+5-FU group was significantly higher than that in 5-FU group(p<0.05).Conclusions:The methylation of promoter regions of hMLH1 and hMSH2 may reduce the sensitivity of colon cancer to 5-FU in HT-29 and SW480 cell lines and orthotopic transplantation tumors of colon cancer.It was suggested that methylation of promoter regions of hMLH1 and hMSH2 may down-regulateor silence the expression of hMLH1 and hMSH2.And then,the function of mismatch repair of cells decreased or lost,the ability to recognize damaged DNA and induce apoptosis reduced,resulting in mutants resistant to 5-FU,leading to decreased chemosensitivity of colon cancer cells. |
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