南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (01): 1-5.

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短干涉RNA特异性抑制白血病细胞中肺耐药相关蛋白基因表达(英文)

李宁;钱新华;王志远;   

  1. 南方医科大学南方医院儿科； 南方医科大学教务处 广东 广州 510515； 广东 广州 510515；
  • 出版日期:2006-01-20 发布日期:2006-01-20
  • 基金资助:
    基金项目：Financially supported by Science and Technology Research Program of Guangzhou Municipality (2002J1-C0031)

Specific inhibition of gene expression of lung resistance-related protein by short interfering RNA

LI Ning, QIAN Xin-hua, Wang Zhi-yuan Department of Pediatrics, Nanfang Hospital, Department of Teaching Affairs, Southern Medical University, Guangzhou 510515, China   

  1. 南方医科大学南方医院儿科； 南方医科大学教务处 广东 广州 510515； 广东 广州 510515；
  • Online:2006-01-20 Published:2006-01-20

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摘要： 背景和目的双链RNA特异性地降解相应序列的mRNA,导致转录后水平的基因沉默.肺耐药相关蛋白的过度表达与白血病细胞多药耐药密切相关.本研究旨在探讨自行设计的LRP特异性siRNA能否发挥特异性降解LRPmR-NA、抑制蛋白质表达的作用.方法构建LRP真核表达载体pcDNA3.0/LRP.以pcDNA3.0/LRP、pEGFP-C1、二者的特异性siRNA分别组和,转染K562细胞.以RT-PCR方法和流式细胞术检测LRPmRNA和蛋白质水平的变化,以荧光显微镜检测GFP的表达的变化.结果K562细胞转染pcDNA3.0/LRP后,LRP mRNA水平明显升高,蛋白质表达由阴性转为阳性,阳性率30%;LRP特异性siRNA与pcDNA3.0/LRP共同转染K562细胞,LRP mRNA和蛋白质表达均明显降低,由阳性转为阴性;LRP特异性siRNA与pEGFP-C1共同转染K562细胞,GFP的表达与pEGFP-C1单独转染无差异;GFP特异性siRNA与pcDNA3.0共同转染K562细胞,LRP mRNA水平和蛋白质表达与pcDNA3.0/LRP单独转染无差异.结论LRP特异性siRNA在K562细胞中能够特异性地降解LRP mRNA、抑制其蛋白质表达.该研究将为利用siRNA逆转LRP引起的白血病细胞MDR奠定理论基础.

Abstract: Objective To investigate inhibitory effect of short interfering RNA (siRNA) on the expression of lung resistance-related protein (LRP) in leukemia cells. Methods The eukaryotic vectors of LRP, pcDNA3.0/LRP, were constructed. The trans-fection protocol of K562 cells grown in standard conditions consisted of different combinations of pcDNA3.0/LRP, pEGFP-C1 expressing mammalian enhanced green fluorescent protein (GFP), and their gene-specific siRNAs. RT-PCR and flow cytometry were employed to evaluate the mRNA and protein expression of LRP and fluoroscopy was performed for assay of GFP expression in the transfected cells. Results Compared with untreated K562 cells, pcDNA3.0/LRP-transfected cells showed increased LRP mRNA and protein expression and the positive cell percentage reached 30%. In the cells co-transfected with LRP gene-specific siRNA and pcDNA3.0/LRP, both LRP mRNA and protein expression decreased significantly to a level defined as negative results; the GFP expression showed no significant difference between the cells transfcted with pEGFP-C1 and those co-transfected with LRP gene-specific siRNA and pEGFP-C1. LRP mRNA and protein expressions were also similar between the cells transfcted with pcDNA3.0/LRP and those co-transfected with GFP gene-specific siRNA and pcDNA3.0/LRP. Conclusions The LRP gene-specific siRNA we designed is capable of degrading LRP mRNA and inhibiting the protein expression effectively and specifically, which shed light on the potential application of siRNA for gene-specific therapy to reverse LRP-induced multidrug resistance of leukemia cells. 更多

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