载体介导的RNA干扰技术对人类髓系白血病HL-60细胞端粒酶反转录酶基因、蛋白表达和凋亡的影响

Effect of Vector-Based RNA Interference Technology on Telomerase Reverse Transcriptase Gene,Protein Expressions and Apoptosis in HL-60 Cell Line

目的探讨由载体介导的靶向端粒酶反转录酶(hTERT)基因的RNA干扰(RNAi)技术对白血病HL-60细胞hTERT基因、蛋白表达及细胞凋亡的影响。方法用已经构建好的表达针对hTERT基因siRNA的质粒载体经转染试剂RNAi-Mate转染HL-60细胞;以转染后24 h、72 h、120 h细胞作为实验组,以空质粒、转染试剂及空白组作对照,采用反转录-PCR(RT-PCR)检测细胞hTERT基因mRNA表达,Western blot检测细胞hTERT蛋白表达,膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/PI双染法流式细胞仪检测细胞凋亡。结果转染后24 h、72 h、120 h实验组HL-60细胞hTERT基因mRNA相对表达水平分别为0.31±0.08、0.28±0.05、0.32±0.06,质粒组、转染试剂组、空白对照组分别为0.55±0.13、0.49±0.08、0.58±0.19;24 h、72 h、120 h实验组蛋白相对表达水平分别为0.47±0.09、0.32±0.07、0.51±0.08,质粒组、转染试剂组、空白对照组分别为0.75±0.11、0.76±0.15、0.73±0.14;24 h、72 h、120 h实验组,质粒组,转染试剂组,空白对照组凋亡率分别为(11.95±3.61)%、(14.61±2.97)%、(12.82±3.01)%、(4.25±2.16)%、(5.09±1.97)%、(3.76±1.84)%。24 h、72 h、120 h实验组hTERT mRNA、蛋白表达水平均低于各对照组(Pa<0.05),细胞凋亡率高于各对照组(Pa<0.05),hTERT mRNA、蛋白表达水平、细胞凋亡率实验组组间比较差异均无统计学意义(Pa>0.05);质粒组、转染试剂组hTERT mRNA、蛋白表达水平、细胞凋亡率与空白对照组比较差异均无统计学意义(Pa>0.05)。结论载体介导的靶向hTERT基因的RNAi技术能在体外抑制HL-60细胞hTERT基因和蛋白的表达,增加细胞凋亡。

Objective To explore the effect of RNA interference（RNAi） technology targeting telomerase reverse transcriptase（hTERT） by vector on hTERT mRNA,protein expressions and apoptosis of leukemia HL-60 cell.Methods hTERT-siRNA plasmid which had been structured was transferred into HL-60 cells by RNAi-Mate,while the empty plasmid,transferring reagent and blank cells were used as controls.Cells were collected at 24 h,72 h,120 h after transferred,and inverse transcription polymerase chain reaction was used to detect the expression of hTERT mRNA,while Western blot was used to detect the expression of hTERT protein,and flow cytometry was used to observe the apoptosis of cells.Results hTERT mRNA expression of test groups at 24 h,72 h,120 h after transferred were 0.31±0.08,0.28±0.05,and 0.32±0.06,respectively,while expression level of empty plasmid,transferring reagent and blank groups were 0.55±0.13,0.49±0.08,and 0.58±0.19,respectively.Compared with control groups,hTERT mRNA expression in test groups were significantly lower（Pa0.05）;compared with blank group,hTERT mRNA in the empty plasmid and transferring reagent groups didn′t change significantly（Pa0.05）.hTERT protein expression of test groups at 24 h,72 h,120 h after transferred were 0.47±0.09,0.32±0.07,and 0.51±0.08,respectively,expression level of empty plasmid,transfering reagent and blank groups were 0.75±0.11,0.76±0.15,0.73±0.14,respectively.Compared with control groups,hTERT protein in test groups was significantly lower,too（Pa0.05）;but compared with blank group,hTERT protein expression in empty plasmid and transferring reagent groups had no significant change（Pa0.05）.The apoptosis rate of 24 h,72 h,120 h test groups and control groups were（11.95±3.61）%,（14.61±2.97）%,（12.82±3.01）%,（4.25±2.16）%,（5.09±1.97）%,and（3.76±1.84）%,respectively.They were higher in test groups than those in control groups（Pa0.05）;but compared among the test groups,there was no significant change（Pa0.05）;and compared with blank group,the apoptosis rate in the empty plasmid and transferring reagent groups didn′t change significantly（Pa0.05）.Conclusions RNAi technology targeting hTERT by vector can inhibit the expression of hTERT gene,protein,and increase the apoptosis in HL-60 cell in vitro.