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目的探讨甲基化酶抑制剂5’-氮杂-2’-脱氧胞苷(5’-Aza-2’-deoxycytidine,5’-Aza-CdR)对结直肠癌细胞株HT-29和LoVo中Wif-1基因甲基化水平及蛋白表达的影响。方法用不同浓度(0.5、1.0、1.5μmol/L)5’-Aza-CdR处理结直肠癌细胞株HT-29和LoVo。应用TaqMan探针为基础的实时定量PCR(Methylight)方法、SYBR Green PCR方法及蛋白印迹实验(Western blot)检测药物处理前后HT-29和LoVo细胞中Wif-1基因的甲基化状态、mRNA和蛋白表达。结果 Methylight检测HT-29和LoVo细胞中Wif-1蛋白在药物作用后异常甲基化得到逆转;实时荧光定量PCR和Western Blot检测到0.5、1.0、1.5μmol/L 5’-Aza-CdR处理后Wif-1基因mRNA和蛋白重新表达,实时荧光定量PCR检测DMSO对照组和不同浓度5’-Aza-CdR(0.5、1.0、1.5μmol/L)在HT-29细胞株相对表达量分别为(1.000±0.000)、(1.207±0.052)、(1.790±0.033)和(2.016±0.123);在LoVo细胞株相对表达量分别为(1.000±0.000)、(1.294±0.048)、(1.893±0.061)和(2.204±0.041)。Western blot检测Wif-1蛋白检测DMSO对照组和不同浓度5’-Aza-CdR在HT-29细胞株相对表达量分别为(0.456±0.040)、(0.511±0.025)、(0.857±0.031)和(0.934±0.047);LoVo细胞株相对表达量分别为(0.842±0.032)、(0.844±0.044)、(0.854±0.037)和(0.856±0.034)。以上作用均呈时间、剂量依赖性,Wif-1基因mRNA在HT-29细胞株表达和LoVo细胞株表达差异均有高度统计学意义(F=144.823,P=0.000;F=476.195,P=0.000)。Wif-1蛋白表达在HT-29细胞株表达差异有高度统计学意义(F=129.674,P=0.000),但Wif-1蛋白表达在LoVo细胞株表达差异无统计学意义(F=0.117,P=0.948)。结论结直肠癌细胞株HT-29和LoVo中Wif-1启动子甲基化可能是导致该基因表达下调甚至失活的主要原因。5’-Aza-CdR能够较成功地逆转结直肠癌细胞株HT-29和LoVo中Wif-1基因的甲基化状态,并能恢复mRNA及蛋白重新表达。
Objective To investigate the effect of 5’-Aza-2’-deoxycytidine (5’-Aza-CdR), a methylase inhibitor, on colorectal cancer cell lines HT-29 and LoVo Effects of Wif-1 Gene Methylation and Protein Expression. Methods Colorectal cancer cell lines HT-29 and LoVo were treated with different concentrations (0.5, 1.0 and 1.5 μmol / L) of 5’-Aza-CdR. The methylation status of Wif-1 gene in HT-29 and LoVo cells before and after drug treatment was detected by TaqMan probe-based real-time quantitative PCR (SYBR Green) method and SYBR Green PCR method and Western blot. MRNA and Protein. Results Methylight reversed the abnormal methylation of Wif-1 protein in HT-29 and LoVo cells after treatment with 0.5,1.0,1.5μmol / L 5’-Aza-CdR by real-time fluorescence quantitative PCR and Western Blot The mRNA and protein expressions of Wif-1 gene were re-expressed in real-time fluorescent quantitative PCR (RT-PCR). The relative expression levels of HT-29 cells in DMSO control group and different concentration of 5’-Aza- ± 0.000), (1.207 ± 0.052), (1.790 ± 0.033) and (2.016 ± 0.123) respectively. The relative expression levels in LoVo cells were (1.000 ± 0.000), (1.294 ± 0.048), 2.204 ± 0.041). The relative expression of Wif-1 protein detected by Western blot in DMSO control group and different concentrations of 5’-Aza-CdR in HT-29 cell line were (0.456 ± 0.040), (0.511 ± 0.025), (0.857 ± 0.031) and 0.934 ± 0.047). The relative expression levels of LoVo cells were (0.842 ± 0.032), (0.844 ± 0.044), (0.854 ± 0.037) and (0.856 ± 0.034), respectively. The above effects were time-and dose-dependent. The Wif-1 gene mRNA expression in HT-29 cells and LoVo cell lines were highly statistically significant (F = 144.823, P = 0.000; F = 476.195, P = 0.000 ). The expression of Wif-1 protein in HT-29 cell line was highly significant (F = 129.674, P = 0.000), but there was no significant difference in Wif-1 protein expression in LoVo cell line (F = 0.117, P = 0.948). Conclusion Methylation of Wif-1 promoter in colorectal cancer cell lines HT-29 and LoVo may be the main reason for the down-regulation or inactivation of this gene. 5’-Aza-CdR can reverse the methylation status of Wif-1 gene in colorectal cancer cell lines HT-29 and LoVo more successfully and restore mRNA and protein re-expression.