5'-氮杂-2'-脱氧胞苷对HT-29和LoVo结直肠癌细胞株中Wif-1基因甲基化状态、mRNA表达及蛋白表达的影响

Effect of 5-Aza-dC on mRNA expression, protein expression and methylation of Wif-1 gene status in HT-29 and LoVo Colorectal cancer

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 effects of 5-Aza-2-deoxycytidine （5-Aza-dC）, a methylation inhibitor, on the mRNA expression, protein expression of W if-1 gene in HT-29 and LoVo Colorectal cancer cell lines. Methods HT-29 and LoVo Colorectal cancer cell lines was treated with different dosages of 5-Aza-dC. W if-1 gene DNA, mRNA and protein were determined by Methylight, SYBR Green PCR and Western blot respectively. HT-29 and LoVo Colorectal cancer cell lines were treated with different dosages of 5-Aza-dC （0.5, 1.0, 1.5μmol/L）. W if-1 gene DNA, mRNA and protein were determined by Methylight, SYBR Green PCR and Western blot respectively. Results Methylight detection showed that the W if-1 gene methylation had effectively been reverserd by 5-Aza-dC. Moreover, the expression levels of W if-1 gene mRNA treated with 5-Aza-dC increased respectively in HT-29 Colorectal cell line [（1.000±0.000）, （1.207±0.052）, （1.790±0.033）, （2.016±0.123）], and the expression levels of W if-1 gene mRNA treated with 5-Aza-Dc increased respectively in LoVo Colorectal cell line [（1.000±0.000）, （1.294±0.048）, （1.893±0.061）, （2.204±0.041）]. West-ern blot indicated that 5-Aza-dC could recover the Wif-1 protein expression respectively in HT-29 Colorectal cell line [（0.456±0.040）, （0.511±0.025）, （0.857±0.031）, （0.934±0.047）], and the protein expression was （0.842±0.032）, （0.844±0.044）, （0.854±0.037）, （0.856±0.034）, respectively in LoVo Colorectal cell line. The W if-1 gene mRNA effects within certain extent dose and time dependent with statistical significance in HT-29 Colorectal cancer line （F=144.823, P=0.000） and LoVo Colorectal cancer line （F=476.195, P=0.000）, and the Wif-1 protein effects within certain extent dose and time dependent with statistical significance in HT-29 Colorectal cancer line （F=129.674, P=0.000）, but without statistical significance in LoVo Colorectal cancer line （F=0.117, P=0.948）. Conclusion The methylation of pro-moter region is a main cause for transcriptional inactivation of W if-1 gene in HT-29 and LoVo Colorectal cancer cell lines. 5-Aza-dC may effectively reactivate the gene transcription through a demethylation role.