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目的采用小干扰RNA(siRNA)沉默HOXA9基因,探讨siRNA转染后人白血病细胞株U937能否逆转化疗药物的耐药性。方法设计合成针对HOXA9的特异性siRNA寡核苷酸链,应用阳离子脂质体介导瞬时转染U937细胞。实验分为3组:实验组(脂质体转染靶向HOXA9的siRNA)、细胞对照组(仅加等量细胞及培养基)和阴性对照组(脂质体转染阴性对照siRNA)。利用反转录(RT)-PCR法、蛋白免疫印迹法分别检测各组细胞HOXA9 mRNA、蛋白的表达;四甲基偶氮唑蓝法(MTT)、流式细胞术检测U937细胞转染前后对长春新碱(VCR)、依托泊苷(VP-16)的敏感性及凋亡率变化。结果靶向HOXA9的siRNA可有效沉默HOXA9的表达,HOXA9 mRNA及蛋白相对水平明显下降。siRNA转染后实验组U937细胞可明显增加对VCR、VP-16的敏感性,实验组细胞的半数抑制浓度值显著低于细胞对照组及阴性对照组(Pa<0.05)。流式细胞术检测结果表明,siRNA干扰后实验组细胞联合化疗药物较细胞对照组及阴性对照组细胞凋亡率显著增高(Pa<0.05)。而阴性对照组与细胞对照组之间差异均无统计学意义(Pa>0.05)。结论靶向HOXA9的siRNA可有效沉默HOXA9基因表达,增加其对VCR、VP-16的敏感性,能逆转白血病细胞对化疗药物的耐药性。
Objective To silence the HOXA9 gene with small interfering RNA (siRNA), and to explore whether the transfection of human leukemia cell line U937 with siRNA could reverse the chemoresistance of chemotherapeutic drugs. Methods Specific siRNA oligonucleotides targeting HOXA9 were designed and synthesized. U937 cells were transiently transfected with cationic liposomes. The experiment was divided into three groups: experimental group (liposome transfection siRNA targeted to HOXA9), cell control group (only plus equal cells and medium) and negative control group (liposome transfected negative control siRNA). The mRNA and protein expression of HOXA9 in each group were detected by RT-PCR and Western blotting respectively. The expressions of HOXA9 mRNA and protein were detected by MTT and flow cytometry VCR, Etoposide (VP-16) sensitivity and apoptosis rate changes. Results siRNA targeting HOXA9 effectively silenced the expression of HOXA9, and the relative levels of HOXA9 mRNA and protein were significantly decreased. After transfected with siRNA, U937 cells could significantly increase the sensitivity to VCR and VP-16. The median inhibitory concentration of cells in the experimental group was significantly lower than that in the cell control group and the negative control group (Pa <0.05). The results of flow cytometry showed that the apoptosis rate of cells in experimental group and chemotherapy group was significantly higher than that of cell control group and negative control group (P <0.05). However, there was no significant difference between the negative control group and the cell control group (Pa> 0.05). Conclusion siRNA targeting HOXA9 can effectively silence HOXA9 gene expression and increase its sensitivity to VCR and VP-16, and can reverse the drug resistance of leukemia cells to chemotherapeutic drugs.