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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。本研究旨在探讨白行设计的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LRP mRNA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LRPmRNA、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。
Background and Purpose Double-stranded RNA specifically degrades mRNA of corresponding sequences, resulting in the silencing of genes at the post-transcriptional level. Overexpression of lung resistance-related proteins is closely related to multi-drug resistance of leukemia cells. This study aimed to investigate whether LRP-specific siRNAs designed by white line can play a specific role in degrading LRPmR-NA and inhibiting protein expression. Methods The LRP eukaryotic expression vector pcDNA3.0 / LRP was constructed. K562 cells were transfected with pcDNA3.0 / LRP and pEGFP-C1, respectively. The mRNA and protein levels of LRP were detected by RT-PCR and flow cytometry. The changes of GFP expression were detected by fluorescence microscope. Results The expression of LRP mRNA in K562 cells was significantly increased after transfection with pcDNA3.0 / LRP, and the protein expression turned from negative to positive. The positive rate of LRP mRNA was 30%. LRP specific siRNA and pcDNA3.0 / LRP co-transfected K562 cells with LRP mRNA The expression of GFP and pEGFP-C1 were not significantly different between the LRP-specific siRNA and pEGFP-C1 transfected K562 cells; GFP-specific siRNA was co-transfected with pcDNA3.0 Staining K562 cells, LRP mRNA level and protein expression and pcDNA3.0 / LRP alone transfected no difference. Conclusion LRP specific siRNA can specifically degrade LRP mRNA in K562 cells and inhibit its protein expression. This study will lay the theoretical foundation for the use of siRNA to reverse LRP-induced MDR in leukemia cells.