二氯二苯基三氯乙烷诱导人结直肠癌增殖、侵袭与miR-129/CDK-14表达的关系

Relationship between proliferation and invasion of human colorectal cancer induced by dichlorodiphenyltrichloroethane and expression of miR-129/CDK-14

目的探讨二氯二苯基三氯乙烷(dichlorodiphenyltrichloroethane,DDT)诱导人结直肠癌增殖、侵袭与微小RNA-129(microRNA-129,miR-129)/细胞周期蛋白依赖性激酶-14(cyclin-dependent kinase-14,CDK-14)表达的关系。方法5×106个/m L密度的人结直肠癌细胞分别用终浓度为0、100、250和500 nmol/L的DDT处理72 h,运用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]法及甲基紫染色测定细胞活力的和细胞细胞集落,流式细胞仪测定细胞凋亡水平,Transwell小室(transwell chamber,Transwell)侵袭室及划痕试验测定细胞侵袭迁移水平,实时荧光逆转录(real-time fluorescent reverse transcription,RT-PCR)法及蛋白印迹法测定细胞miR-129、CDK-14、卷曲螺旋结构域-34(coiled-coil domain-34,CCDC34)、半乳糖激酶1(Galactokinase 1,GALK1)水平。结果100、250和500 nmol/L DDT组细胞增殖水平[(70.59±7.72、76.63±8.32、84.25±9.04 vs 62.58±6.83)%]、细胞集落[(99.95±9.58、215.63±19.85、398.96±35.26 vs 62.63±6.52)个]、侵袭数目[(76.25±9.58、154.62±19.51、298.96±38.57 vs 49.65±6.65)个]、迁移水平(75.96±14.58、119.69±20.59、165.63±32.68 vs 11.59±2.28)、CDK-14、CCDC34、GALK1 m RNA蛋白水平明显高于对照组(P<0.01),凋亡率、miR-129水平明显低于对照组(P<0.01);且500 nmol/L DDT组细胞增殖水平、细胞集落、侵袭数目、迁移水平、CDK-14 mRNA蛋白[(1.60±0.28、2.14±0.33、2.99±0.51 vs 0.92±0.16),(0.42±0.07、0.85±0.14、1.20±0.20 vs 0.21±0.03)]、CCDC34(0.48±0.08、0.87±0.15、1.18±0.18 vs 0.28±0.07)、GALK1(0.50±0.09、0.84±0.14、1.22±0.19 vs 0.30±0.08)蛋白水平均高于250 nmol/L DDT组,差异均有统计学意义(均P<0.01),凋亡率(1.70±0.31、1.00±0.16、0.60±0.11 vs 3.00±0.51)、miR-129(1.42±0.24、0.92±0.15、0.64±0.10 vs 1.78±0.28)水平均低于250 nmol/L DDT组,差异均有统计学意义(均P<0.01);250nmol/L DDT组细胞增殖水平、细胞集落、侵袭数目、迁移水平、CDK-14、CCDC34、GALK1 mRNA蛋白水平均高于100 nmol/L DDT组,差异均有统计学意义(均P<0.01),250 nmol/L DDT组细胞凋亡率、miR-129水平均低于100 nmol/L DDT组,差异均有统计学意义(均P<0.01);DDT浓度与细胞增殖水平、细胞集落、侵袭数目、迁移水平、凋亡率、miR-129水平、CDK-14、CCDC34、GALK1 mRNA蛋白水平有明显剂量-反应关系(P<0.01)。结论DDT下调miR-129表达靶向上调调控CDK-14以及CCDC34、GALK1的表达,从而促进结直肠癌细胞的增殖和侵袭,并抑制其凋亡。

Objective To investigate the relationship between the proliferation and invasion of human colorectal cancer induced by dichlorodiphenyltrichloroethane(DDT)and the expression of microRNA-129(miR-129)/cyclin-dependent kinase-14(CDK-14).Methods The human colorectal cancer cells with a density of 5×106/mL were treated with DDT at final concentrations of 0.0,100,250,and 500 nmol/L for 72 hours,respectively.The cell viability and cell colony were determined by the 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)method and methyl violet staining.The apoptosis level was measured by flow cytometry.The invasion and migration levels of cells were measured by transwell chamber(Transwell)and scratch assay.The levels of miR-129,CDK-14,coiled-coil domain-34(CCDC34)and Galactokinase 1(GALK1)were measured by real-time fluorescent reverse transcription(RT-PCR)and Western blotting method.Results The cell proliferation level[(70.59±7.72,76.63±8.32,84.25±9.04 vs 62.58±6.83)%],cell colony(99.95±9.58,215.63±19.85,398.96±35.26 vs 62.63±6.52),invasion number(76.25±9.58,154.62±19.51,298.96±38.57 vs 49.65±6.65),migration level(75.96±14.58,119.69±20.59,165.63±32.68 vs 11.59±2.28),as well as CDK-14,CCDC34,GALK1 mRNA protein levels in 100,250,500 nmol/L DDT groups were significantly higher than those in the control group(P<0.01),and the apoptosis rate and miR-129 level were significantly lower than those in the control group(P<0.01).The cell proliferation level,cell colony,invasion number,migration level,CDK-14 mRNA protein[(1.60±0.28,2.14±0.33,2.99±0.51 vs 0.92±0.16),(0.42±0.07,0.85±0.14,1.20±0.20 vs 0.21±0.03)],CCDC34(0.48±0.08,0.87±0.15,1.18±0.18 vs 0.28±0.07),GALK1(0.50±0.09,0.84±0.14,1.22±0.19 vs 0.30±0.08)protein levels in 500 nmol/LDDT group were higher than those in 250 nmol/L DDT group,and the differences were statistically significant(all P<0.01).The apoptosis rate(1.70±0.31,1.00±0.16,0.60±0.11 vs 3.00±0.51)and miR-129(1.42±0.24,0.92±0.15,0.64±0.10 vs 1.78±0.28)level were lower than those in 250 nmol/L DDT group,and the differences were statistically significant(all P<0.01).The cell proliferation level,cell colony,invasion number,migration level,CDK-14,CCDC34,and GALK1 mRNA protein levels in 250 nmol/L DDT group were higher than those in 100 nmol/L DDT group,and the differences were statistically significant(all P<0.01).The apoptosis rateand miR-129 level in 250 nmol/L DDT group were lower than those in 100 nmol/L DDT group,and the differences were statistically significant(all P<0.01).There was a significant dose-response relationship between DDT concentration and cell proliferation level,cell colony,invasion number,migration level,apoptosis rate,miR-129 level,CDK-14,CCDC34,GALK1 mRNA protein level(P<0.01).Conclusion DDT down-regulates the expression of miR-129 and targets to up-regulate the expression of CDK-14,CCDC34 and GALK1,thereby promoting the proliferation and invasion of colorectal cancer cells and inhibiting their apoptosis.