SiRNA逆转肺耐药相关蛋白表达介导的白血病细胞多药耐药
研究双链短干扰RNA(siRNA)对白血病多药耐药细胞模型(K562/NaB)肺耐药相关蛋白(LRP)表达及功能的影响. 方法: 针对LRP基因设计合成特异性siRNA,在脂质体介导下转染K562/NaB;采用半定量逆转录聚合酶链反应(RTPCR)检测K562细胞LRP mRNA的水平;用流式细胞术检测K562/NaB细胞LRP蛋白表达的变化和细胞内柔红霉素(DNR)的蓄积;MTT法检测阿霉素(ADM)对K562/NaB细胞耐药的半数抑制浓度(IC50). 结果: siRNA转染后:K562/NaB细胞的LRP mRMA水平明显降低;LRP蛋白表达由阳性转为阴性;细胞内DNR的蓄积明显增加,DNR平均荧光增强3.28倍;对ADM药物敏感相对逆转效率为78.18%. 结论: siRNA可逆转由LRP介导的白血病细胞多药耐药.
【关键词】 RNA,小分子干扰;基因,肺耐药相关蛋白;K562细胞;抗药性,多药
【Abstract】 AIM: To investigate the effect of short interfering RNA (siRNA) on expression and function of lung resistancerelated protein (LRP) in the multidrug resistant human leukemia cells (K562/NaB). METHODS: Multidrugresistant K562 cells with high level LRP expression treated with sodium butyrate (NaB), was used as an in vitro model system. LRP specific siRNA was synthesized and transfected into the K562/NaB cells. Expression of LRP mRNA was detected by reverse transcriptasepolymerase chain reaction (RTPCR), and protein level and intracellular daunorubicin (DNR) accumulation in K562/NaB cells were detected by flow cytometry. 50% inhibition concentration (IC50) of adriamycin (ADM) on K562/NaB cells was detected by MTT method. RESULTS: LRP mRNA level was decreased obviously; the protein expression was turned from positive result to negative result. Intracellular DNR accumulation was increased and the mean fluorescence of DNR was 3.28 times higher. The relative efficiency to ADM was 78.18%. CONCLUSION: The siRNA could effectively reverse the multidrug resistance of leukemia cells induced by LRP.
【Keywords】RNA, small interfering; gene, lung resistancerelated protein; K562 cells; drug resistance, multiple
0引言
肺耐药相关蛋白(lungrelated resistant protein, LRP)是一种新型的与多药耐药(multidrug resistance, MDR)相关的糖蛋白,主要导致细胞内药物聚集缺陷,而在多种肿瘤细胞内引起MDR. 在儿童白血病以及成人髓系白血病(AML)中,LRP表达是独立的预后决定因素之一,其过度表达造成患者对化疗不敏感,提示预后不良[1-2]. 双链短干扰RNA(short interfering RNA, siRNA)介导的RNA干扰(RNA interference, RNAi),是在特异性抑制哺乳动物细胞基因方面的最新突破[3-4]. 我们在建立了经丁酸钠(sodium butyrate, NaB)诱导人AML系 K562细胞,高表达LRP并介导多药耐药的细胞模型(K562/NaB)的基础上[5],设计合成了LRP特异性siRNALRP,并转染上述细胞模型,观察siRNALRP抑制LRP基因和蛋白表达、消除LRP改变细胞内药物蓄积和分布作用的效果,以期为逆转LRP介导的肿瘤细胞MDR、提高儿童难治性和复发性白血病化疗效果探索新的方法,并探讨以siRNA介导的RNAi用于肿瘤细胞MDR治疗的可行性.
1材料和方法
1.1材料
AML细胞系K562细胞购自中国科学院上海细胞生物研究所;单克隆抗体LRP56为Monosan公司产品;固定和破膜试剂盒、藻红蛋白(phycoerythrin,PE)荧光标记羊抗鼠IgG抗体为Caltolog Laboratories产品;NaB为Sigma公司产品.
LRP特异性短干涉RNA(siRNALRP)自行设计,由Dharmacon公司合成. 浓度20 μmmol/L,序列:5′ GCU CUU UUC AGU GCC AGA C dTdT (正义链),dTdT CGA GAA AAG UCA CGG UCU G 5′ (反义链).
1.2方法
1.2.1K562细胞培养和高表达
LRP的K562多药耐药细胞模型(K562/N)[10]K562细胞在含100 mL/L小牛血清的RPMI 1640培养基于37℃, 50 ml/L CO2条件下培养. K562细胞在含2 mmol/L NaB的培养液中处理3 d,制作K562细胞高表达LRP的多药耐药细胞模型,命名为K562/NaB. 阴性对照组不加NaB.
1.2.2siRNA转染
实验K562/NaBsiRNA组:取K562/NaB细胞接种于24孔板内,每孔500 μL,浓度为3×108/ L. 分别以无血清RPMI 1640培养液 50 μL稀释siRNALRP, Lipofectamine各1 μL,混匀静置后加入细胞悬液. 再加入NaB使终浓度为2 mmol/L,进行细胞培养. 每24 h以含2 mmol/L NaB的RPMI 1640培养基500 μL换液,连续3 d. 转染后24, 48, 72 h分别取细胞进行相关检测.
K562/NaBH2O组:取K562/NaB细胞,以无RNase水代替siRNALRP转染细胞,操作方法、实验条件同siRNA转染组. 取转染72 h细胞进行检测.
对照组: K562/NaB细胞作为阳性对照;原始的K562细胞作为阴性对照.
1.2.3RTPCR方法检测