载体介导RNAi技术对HL-60细胞hTERT基因和细胞生长影响

Effect of hTERT gene expression and cell growth in HL-60 cell line by vector-based RNAi technology

目的探究靶向人端粒酶逆转录酶(hTERT)的核糖核酸干扰(RNAi)对白血病HL-60细胞生长及hTERT基因的作用。方法用已经构建好的表达针对pSilencer1.0-U6/hTERT siRNA质粒载体,经RNAi-Mate转染试剂转染HL-60细胞。将转染细胞分为对照组(3亚组)和实验组(3亚组),即pSilencer1.0-U6空质粒组、RNAi-Mate转染试剂组及生理盐水空白对照组为3个对照组,pSilencer1.0-U6/hTERT质粒转染后24 h组、pSilencer1.0-U6/hTERT质粒转染后72 h组、pSilencer1.0-U6/hTERT质粒转染后120 h组为3个实验组。用反转录聚合酶链反应(RT-PCR)法检测细胞hTERT基因mRNA的表达,Western blot法检测细胞hTERT蛋白的表达,Annexin V-FITC/PI流式细胞仪双染法检测细胞凋亡情况,用四甲基偶氮唑盐(MTT)比色法检测细胞增殖抑制率。结果实验组(3亚组)HL-60细胞hTERT蛋白表达水平(0.47±0.09、0.32±0.07、0.51±0.08)、mRNA(0.31±0.08、0.28±0.05、0.32±0.06)低于对照组(3亚组)(0.73±0.14、0.76±0.15、0.75±0.11,0.55±0.13、0.49±0.08、0.58±0.19);实验组(3亚组)细胞增殖抑制率[(23.7±4.0)%、(35.1±5.9)%、(29.6±3.8)%]及凋亡率[(11.95±3.61)%、(14.61±2.97)%、(12.82±3.01)%]均高于对照组(3亚组)(P<0.05),但实验组各亚组间、对照组各亚组间差异无统计学意义(P>0.05)。结论载体介导的靶向hTERT基因的RNAi技术在体外能抑制HL-60细胞hTERT蛋白和基因的表达,能抑制细胞增殖,增加细胞凋亡。

Objective To explore the effect of RNA interference（RNAi） of targeting human telomerase reverse transcriptase（hTERT） by vector on hTERT gene expression and cell growth of leukemia HL-60 cell. Methods pSilencer1.0-U6/hTERTsiRNA plasmid was constructed to transfer into HL-60 cell by RNAi-Mate. The transferred cells were divided into control group（3 subgroups which were pSilencer 1.0-U6 null plasmid group, RNAi-Mate transfection reagent group and Saline control group）and experimental group（3 subgroups which were pSilencer1.0-U6/hTERT plasmid transfection after-24-hour group, pSilencer1.0-U6/hTERT plasmid transfection after-72-hour group and pSilencer1.0-U6/hTERT plasmid transfection after-120-hour group）. The gene mRNA expression of hTERT was detected by reverse transcription-polymerase chain reaction（RT-PCR）,protein expression of hTERT by Western blot, cell apoptosis by Annexin V-FITC/PI flow cytometry double staining method and inhibition rate of cell proliferation measured by MTT. Results The levels of hTERT protein of HL-60 cell and mRNA expression in experiment group（protein 0.47 ± 0.09, 0.32 ± 0.07, 0.51 ± 0.08; mRNA 0.31 ± 0.08, 0.28 ± 0.05, 0.32 ± 0.06）were lower than that of control group（protein 0.73 ± 0.14, 0.76 ± 0.15, 0.75 ± 0.11; mRNA 0.55 ± 0.13, 0.49 ± 0.08, 0.58 ±0.19）. The inhibition rate of cell proliferation[（23.7 ± 4.0） %,（35.1 ± 5.9） %,（29.6 ± 3.8） %] and apoptosis[（11.95 ± 3.61） %,（14.61 ± 2.97） %,（12.82 ± 3.01） %] of experimental group were higher than that of control group（P〈 0.05）, while there was no significant difference between subgroups of experimental group and control group（P 〉0.05）. Conclusion It is demonstrated that RNAi technology targeting hTERT by vector could inhibit the expression of hTERT gene, protein and proliferation activity,which increase apoptosis level of HL-60 cell in vitro.