RNA干扰同源盒A10基因表达可逆转白血病K562细胞多药耐药性

Reversion of multidrug resistance in leukemia K562 cells by RNA interference targeting homeobox A10 gene

目的:本研究通过建立高效干扰同源盒A10(homeobox A10,HOXA10)基因表达的短发夹RNA(short hairpin RNA,shRNA)真核表达载体,探讨RNA干扰技术逆转人慢性髓系白血病K562细胞株多药耐药的新方案。方法:构建靶向HOXA10基因的真核表达载体pGPHI/GFP/Neo-HOXA10,应用阳离子脂质体将其转染入K562细胞,G418筛选4周。RT-PCR鉴定重组载体稳定转染的细胞株。MTT法检测细胞在转染前后对长春新碱(leurocristine,VCR)、足叶乙苷(etoposide,VP-16)的敏感性变化。FCM法检测各组细胞的凋亡率。结果:经G418筛选后,反转录PCR(reverse transcriptase PCR,RT-PCR)证实成功建立了pGPHI/GFP/Neo-HOXA10稳定转染的细胞克隆。MTT结果显示,基因干扰组细胞的半数抑制浓度(half-inhibitory concentration,IC50)值明显低于对照组(P<0.05),对VCR及VP-16的敏感性明显增强。FCM检测结果表明,基因干扰组细胞联合化疗药物后细胞凋亡率显著增加(P<0.05)。而转染阴性对照组的IC50值及细胞凋亡率与正常对照组比较差异无统计学意义(P>0.05)。结论:以HOXA10基因为靶标构建的shRNA表达载体能明显增强VCR和VP-16对K562细胞的抑制增殖、诱导凋亡作用,提高白血病细胞对化疗药物的敏感性。提示HOXA10基因表达被RNA干扰可以在一定程度上逆转白血病细胞的多药耐药性。

Objective： To investigate a new strategy to reverse multidrug resistance of chronic myeloid leukemia cell line K562 by RNA interference technique through constructing an eukaryotic vector of short hairpin RNA （shRNA） targeting homeobox A10 （HOXAIO） gene. Methods： The eukaryotic vector pGPHI/ GFP/Neo-HOXA10 with shRNA targeting HOXAIO gene was constructed and then transfected into K562 cells by positive ion liposome. The stable transfectants were vertificated by reverse transcriptase PCR （RT-PCR） 4 weeks after G418 pressure selection. The changes of sensitivity of K562 cells to leurocristine （VCR） and etoposide （VP-16） after transfection with shRNA-HOXA10 were detected by MTT method, and the apoptosis rate was detected by flow cytometry. Results： After selection with G418, the cell clones stably transfected with pGPHI/GFP/Neo-HOXA10 were successfully constructed and verificated by RT-PCR. The half inhibitory concentration （/C5o） values of VCR and VP-16 for K562 cells transfected with shRNA-HOXA10 were significantly reduced and the apoptosis rate of K562 cells after HOXAIO gene interference combining with chemotherapy was increased significantly as compared with those of shRNA-negative control group and the normal control group （P〈0.05）. No differences in /Csoand apoptosis rate were observed between the shRNA-negative control group and the normal control group （P〉0.05）. Conclusion： The eukaryotic vector of short hairpin RNA （shRNA） targeting HOXAIO gene can enhance the abilities of VCR and VP-16 to inhibit the cell proliferation and induce the apoptosis of K562 cells, namely an increase of sensitivity to these chemotherapeutics in K562 cells. It is hinted that RNA interference targeting HOXAIO gene may reverse the multidrug resistance of leukemia cells in some degree.