摘要
目的探讨FLT3靶向RNA干扰对HL-60细胞增殖、凋亡及cyclinD1、cyclinA表达的影响。方法体外构建FLT3靶向短发夹状干扰RNA(FLT3-shRNA),转染HL-60细胞,RT-PCR、流式细胞术(FCM)鉴定FLT3的表达;CCK-8法检测细胞增殖活力;FCM检测细胞周期;AnnexinV染色法检测凋亡;RT-PCR及Western blot检测cyclinD1,cyclinA在mRNA、蛋白水平的表达。结果FLT3-shRNA转染可下调FLT3的表达,它对FLT3 mRNA和蛋白的抑制率分别达(81.66±10.25)%,(76.76±11.23)%。FLT3表达下降后细胞增殖活力受到抑制,转染48h抑制率达(31.66±2.97)%,72h达(33.10±3.43)%;细胞生长曲线低平,缺乏指数增殖特征。与对照组比,转染48h细胞周期重新分布,G0/G1期(58.48±6.17)%的明显上升,S期(27.72±5.10)%下降;细胞早期凋亡率(8.95±0.88)%上升;细胞内cyclinD1的mRNA、蛋白表达下降,cyclinA的表达无明显改变。结论FLT3靶向RNA干扰通过下调cyclinD1表达有效地抑制细胞增殖,具有潜在的临床应用价值。
Aim To explore the effects of FLT3-targeted RNAi on proliferation, apoptosis and expression of cyclin D1, cyclin A in HL-60 cell line. Method FLT3-targeted small hairpin interfering RNA （FLT3- shRNA） was constructed by in vitro transcription system and transfected into HL-60 cells. FLT3 mRNA was detected by semi-quantitative RT-PCR, FLT3 protein was detected by Flow Cytometry （FCM）, cell growth viability was measured by CCK-8, the distribution of cell cycle was assayed by FCM, apoptosis was analyzed by Annexin V staining, and the mRNA, protein expression of cyclin D1, cyclin A were detected by RTPCR or Westerning Blotting. Results FLT3 expression was downregulated by FLT3-shRNA which the inhibiting rate to mRNA was （81.66 ± 10. 25 ）%, and to protein was （76. 76 ± 11.23 ）%. The suppression of FLT3 resulted in markedly inhibition of cell growth with percentages of inhibition were （31.66 ± 2. 97 ） % for48 h, （33. 10±3.43）% for72 h, the growth curve was low, lacking of typical character of exponential growth as controls ; Compared with controls, at 48 h following transfection cell cycle was redistributed again which the percentage of sub-G0/G1 phase （55.48 ± 6. 17）% increased while that of sub-S phase （25.11 ± 2. 70） % decreased, and the percentage of early apoptosis （ 8. 95 ± 0. 88 ） % increased ; Furthermore the expression of cyclin D1 mRNA and protein decreased significantly, but the expression of cyclin A didn＇t change. Conclusion Through downregulating the expression of cyclin D1, FLT3-targeted RNAi can inhibit proliferation in HL-60 cells, which illustrates the potential benefit of therapeutic approaches.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2007年第5期645-650,共6页
Chinese Pharmacological Bulletin
基金
河南省自然科学基础研究计划项目
