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目的:运用RNA干扰(RNA interference,RNAi)技术抑制同源盒基因A7(homeobox geneA7,HOXA7)表达,并探讨其对白血病U937细胞多药耐药的逆转作用。方法:将靶向HOXA7基因的特异性真核表达载体(pGPU6/GFP/Neo-shHOXA7)及阴性对照载体(pGPU6/GFP/Neo-shNC)分别转染U937细胞,G418稳定筛选。实验分3组:实验组(稳定表达pGPU6/GFP/Neo-shHOXA7的U937细胞)、阴性对照组(稳定表达pGPU6/GFP/Neo-shNC的U937细胞)和空白对照组(U937细胞)。RT-PCR和蛋白质印迹法分别在mRNA和蛋白质水平检测各组细胞中HOXA7的表达情况。MTT法检测各组细胞对阿糖胞苷(cytarabine,Ara-C)和高三尖杉酯碱(homo harringtonine,HHT)的敏感性。FCM法分别检测Ara-C(10μg/mL)和HHT(0.5μg/mL)作用下各组细胞的凋亡情况。结果:实验组HOXA7在mRNA和蛋白质水平的相对表达量明显低于阴性对照组和空白对照组(P<0.05)。实验组Ara-C和HHT的半数抑制浓度(half-inhibitory concentration,IC50)较阴性对照组和空白对照组的IC50分别降低了3.5倍和8.5倍(P<0.05),提示实验组细胞对Ara-C和HHT的敏感性增加。实验组细胞分别经Ara-C(10μg/mL)和HHT(0.5μg/mL)诱导后,细胞凋亡率明显高于相同药物作用下阴性对照组和空白对照组的细胞凋亡率(P<0.05)。结论:RNAi抑制HOXA7表达能增强Ara-C和HHT对U937细胞的增殖抑制及凋亡诱导作用,在一定程度上可逆转白血病细胞的多药耐药性。
OBJECTIVE: To inhibit the expression of homeobox gene A7 (HOXA7) by RNA interference (RNAi) technology and to investigate its reversal of multidrug resistance in leukemic U937 cells. Methods: The specific eukaryotic expression vector targeting HOXA7 gene (pGPU6 / GFP / Neo-shHOXA7) and negative control vector (pGPU6 / GFP / Neo-shNC) were transfected into U937 cells respectively. The experiment was divided into three groups: experimental group (U937 cells stably expressing pGPU6 / GFP / Neo-shHOXA7), negative control group (U937 cells stably expressing pGPU6 / GFP / Neo-shNC) and blank control group (U937 cells). RT-PCR and Western blotting were used to detect the expression of HOXA7 in each group of cells at mRNA and protein levels. The sensitivity of cells to cytarabine (Ara-C) and homoharringtonine (HHT) was determined by MTT assay. FCM was used to detect the apoptosis of cells in each group under Ara-C (10μg / mL) and HHT (0.5μg / mL). Results: The relative expression level of HOXA7 mRNA and protein in experimental group was significantly lower than that in negative control group and blank control group (P <0.05). The IC50 of Ara-C and HHT in experimental group were reduced by 3.5 times and 8.5 times (P <0.05), respectively, compared with negative control group and blank control group, Sensitivity of C and HHT increased. After induced by Ara-C (10μg / mL) and HHT (0.5μg / mL), the apoptotic rate of the experimental group was significantly higher than that of the negative control group and the blank control group (P < 0.05). CONCLUSION: Inhibition of HOXA7 expression by RNAi can enhance the proliferation and apoptosis-inducing effect of Ara-C and HHT on U937 cells, which can reverse the multidrug resistance of leukemia cells to a certain extent.