摘要目的 通过构建靶向同源盒A10(HOXA10)的真核表达载体,探讨利用RNA干扰沉默HOXA10基因对人慢性髓系白血病细胞株K562增殖和凋亡的影响.方法 根据筛选的针对HOXA10的特异性有效小干扰RNA(siRNA)序列设计合成短发夹RNA(shRNA)寡核苷酸链,构建pGPHI-GFP-Neo-HOXA10真核表达载体并测序,应用阳离子脂质体转染K562细胞.实验分为细胞对照组(仅加等量细胞及培养基),阴性对照组(脂质体转染阴性对照质粒)、实验组(脂质体转染pGPHI-GFP-Neo-HOXA10).转染载体24 h后利用RT-PCR检测各组HOXA10 mRNA表达；转染载体24、48、72 h后应用MTT法检测各组细胞增殖并计算细胞抑制率；转染载体48 h后应用流式细胞术检测各组细胞凋亡.结果 成功构建pGPHI-GFP-Neo-HOXA10载体并转染K562细胞.与细胞对照组和阴性对照组比较,实验组转染载体能有效降低HOXA10 mRNA的表达水平[(38.86±4.49)％比(88.52±9.24)％、(86.75±7.38)％,P＜0.05],而阴性对照组与细胞对照组比较差异则无统计学意义(P＞0.05).与阴性对照组比较,实验组载体作用于K562细胞24、48、72 h后,细胞增殖能力均明显下降,细胞抑制率明显升高[(39.92±0.74)％比(7.98±5.52)％;(55.62±1.18)％比(8.27±3.45)％;(66.30±1.26)％比(8.63±3.58)％;均P＜0.05].实验组细胞凋亡率较细胞对照组、阴性对照组也显著升高[(22.29±1.67)％比(9.82±0.69)％、(10.14±0.96)％,P＜0.05],而阴性对照组与细胞对照组比较差异则没有统计学意义(P＞0.05).结论 构建的真核表达载体pGPHI-GFP-Neo-HOXA10可有效沉默K562细胞中HOXA10基因的表达,能明显抑制K562细胞增殖并诱导其凋亡.

abstractsObjective To investigate the impact of RNA interference silencing HOXA10 gene on proliferation and apoptosis of K562 cell strain of human chronic myeloid leukemia (CML) by construction of eukaryotic expression vector targeting HOXA10.Methods Based on short hairpin RNA(shRNA) oligo that was designed and compounded by the selected specific small interference RNA (siRNA) targeting HOXA 10,the eukaryotic expression vector of pGPHI-GFP-Neo-HOXA10 was successfully constructed and sequenced,and the cationic liposome was then used to transfect K562 cells.There were cell control group (equivalent cells and culture medium only),negative control group (negative control plasmid transfected by liposome)and experimental group (pGPHI- GFP- Neo- HOXA10 transfected by liposome).The HOXA10 mRNA expression was detected by RT-PCR at 24 h after transfection,cell proliferation by MTT at 24,48 and 72 h for cell inhibition ratio,and the apoptosis by flow cytometry at 48 h in all the groups.Results The vector pGPHI-GFP-Neo-HOXA10 was successfully constructed and used to transfect K562 cells.The transfection vector in experimental group could effectively decrease the expression level of HOXA10 mRNA [ (38.86±4.49)％ vs (88.52±9.24)％,(86.75±7.38)％,P＜0.05] as compared with that in cell control and negative control group,with no difference between negative control and cell control group (P＞0.05).Compared with negative control group,the experimental group had a significant decrease in cell proliferation capacity but an increase in cell inhibition ratio at 24,48 and 72 h after transfection [ (39.92±0.74)％ vs (7.98±5.52)％;(55.62±1.18)％ vs (8.27±3.45)％; (66.30±1.26)％ vs (8.63±3.58)％; all P＜0.05].Moreover,the apoptosis rate was significantly increased in experimental group as compared with that in control and negative control groups [ (22.29± 1.67)％ vs (9.82±0.69)％,(10.14±0.96)％,P＜0.05],however,no difference was found between negative control and cell control group (P＞0.05).Conclusion Since eukaryotic expression vector pGPHI-GFP-Neo-HOXA10 can effectively silence the expression of HOXA10 in K562 cells,pGPHI-GFP-Neo-HOXA10 may significantly inhibit proliferation of K562 cell and induce its apoptosis.