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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 investigate the effects of 5-iodotubercidin (ITU) on proliferation and apoptosis of human colon cancer cell line HT-29 and the expression of DLC-1 gene. METHODS: ITU-T cells were treated with ITU at different concentrations (0.1, 0.5, 1, 2, 4, 6, 8, 10μmol / L) for 48 hours. HT- (0,2,4,6μmol / L) for 48 h, the apoptosis rate of HT-29 cells was detected by flow cytometry; the effect of ITU (0,2,4,6μmol / L) -29 cells for 48 h, the methylation of CpG island in promoter region of DLC-1 gene was detected by bisulfite cloning sequencing (BSP) method; the same as the methods of cell processing, reverse transcription polymerase chain reaction ) Was used to detect the expression of DLC-1 mRNA in DLC-1. The protein expression of DLC-1 was detected by Western blot. Results 1ITU could significantly inhibit the viability of HT-29 cells. The inhibitory effect of 1ITU on HT-29 cells was stronger than that of HT-29 cells in a certain range (P <0.05) ± 0.62) μmol / L. 2 were treated with 0, 2, 4 and 6μmol / L ITU respectively. The apoptotic rates of HT-29 cells were detected by flow cytometry (FCM), and were respectively 9.56 ± 2.21%, 22.18 ± 3.16% ± 3.16)%, (40.50 ± 3.16)%, indicating that as the concentration of ITU increased, the stronger the pro-apoptotic effect on HT-29 cells. 3 The methylation rates of CpG island in promoter region of DLC-1 gene after treated with 0, 2, 4 and 6μmol / L of ITU were 91.67%, 75.00%, 61.67% and 43.33%, respectively, indicating that ITU can cause DLC-1 gene Promoter methylation reduced. 4 A small amount of DLC-1 mRNA and protein could be detected in HT-29 cells. After the same conditions were treated with 0, 2, 4, and 6 μmol / L ITU, the expression of DLC-1 mRNA and protein increased With increasing concentration, mRNA and protein expression increased accordingly. Conclusions ITU can inhibit the proliferation of human colon cancer HT-29 cells and promote the apoptosis of HT-29 cells, and this inhibitory effect may be related to the decrease of methylation level of DLC-1 promoter and the up-regulation of DLC-1 gene by ITU .