5-碘代杀结核菌素对HT-29细胞DLC-1基因的去甲基化作用

Effect of 5-iodotubercidin on proliferation and apoptosis in human colon cancer HT-29 cells and demethylation of deleted in liver cancer-1 gene

目的探讨5-碘代杀结核菌素(5-iodotubercidin,ITU)对人结肠癌细胞HT-29株增殖凋亡及DLC-1基因表达的影响。方法不同浓度(0.1、0.5、1、2、4、6、8、10μmol/L)ITU作用于HT-29细胞48 h,四甲基偶氮唑盐(MTT)比色法检测HT-29细胞的增殖活性;ITU(0、2、4、6μmol/L)作用于HT-29细胞48 h,流式细胞仪检测其凋亡率;ITU(0、2、4、6μmol/L)作用于HT-29细胞48 h,亚硫酸氢盐克隆测序法(BSP)检测DLC-1基因启动子区Cp G岛甲基化情况;同上的处理细胞的方法,逆转录聚合酶链式反应(RT-PCR)检测肝癌缺失基因-1(deleted in liver cancer-1,DLC-1)mRNA的表达;Western blot检测DLC-1基因蛋白表达。结果 1ITU可明显抑制HT-29细胞活力,在一定范围内随着药物浓度增加抑制细胞活力的作用越强(P<0.05),以6μmol/L组对HT-29细胞影响最大,IC50=(5.92±0.62)μmol/L。2分别以0、2、4、6μmol/L ITU作用于HT-29细胞后,流式细胞仪检测各组凋亡率,分别为(9.56±2.21)%、(22.18±3.16)%、(34.23±3.16)%、(40.50±3.16)%,表明随着ITU浓度增加,对HT-29细胞的促凋亡作用越强。3经0、2、4、6μmol/L ITU处理后DLC-1基因启动子区Cp G岛甲基化率分别为91.67%、75.00%、61.67%、43.33%,表明ITU可以引起DLC-1基因启动子区甲基化程度降低。4在HT-29细胞中可检测到少量DLC-1 mRNA和蛋白的表达;相同条件经0、2、4、6μmol/L ITU处理细胞后检测显示DLC-1基因mRNA和蛋白的表达增加,并随浓度增长,mRNA及蛋白表达随之增加。结论 ITU可抑制人结肠癌HT-29细胞增殖,促进HT-29细胞凋亡,而这种抑制增殖作用可能和ITU降低DLC-1基因启动子区甲基化水平而诱导DLC-1基因上调有关。

Objective To explore the effect of 5-iodotubercidin （ITU） on the proliferation and apoptosis in human colon cancer HT-29 cells and on the expression of deleted in liver cancer-1 （ DLC-1 ） gene. Methods HT-29 cells were treated with different concentrations of ITU （0.1, 0.5, 1, 2, 4, 6, 8, and 10 μmol/L） for 48 h, and the cell viability was studied by MTT assay. HT-29 cells were co-cultured with different concentrations of ITU （0, 2, 4, and 6 μmol/L） for 48 h, and the apoptosis rate was detected by flow cytometry. Meanwhile, the methylation levels of CpG islands at promoter region of DLC-1 gene were determined by bisulfite sequencing PCR （BSP）. The DLC-1 mRNA expression levels were detected by reverse transcription-polymerase chain reaction （RT-PCR） and the DLC-1 protein expression levels were detected by Western blotting. Results ITU distinctly inhibited the proliferation of HT-29 cells in a concentration-dependent manner in a certain range, and the 6 ixmol/L group had the greatest effect, with ICs0 of 5.92 ± 0. 62μmol/L. The apoptotic rates of HT-29 cells treated with ITU （0, 2, 4, and 6 I^mol/L） were （9.56 ± 2.21）%, （22.18 ±3. 16）%, （34.23 ±3. 16）%, and （40.5 ±3. 16）%, respectively, which were increased in a concentration-dependent manner. The methylation rates of CpG islands at the promoter region of DLC-1 gene were 91.67%, 75.00%, 61.67%, and 43.33%, respectively, after treatment with ITU （0, 2, 4, and 6 μmol/L）. The results showed that ITU could decrease the methylation of DLC-1 gene promoter region. Little of DLC-1 mRNA and protein expression was detected in HT-29 cells, and ITU treatment increased the expression in a concentration-dependent manner. Conclusion ITU can inhibit the proliferation of HT-29 cells and induce apoptosis through decreasing the methylation level of DLC-1 gene promoter region and increasing the DLC-1 gene expression.