Specific inhibition of gene expression of lung resistance-related protein by short interfering RNA
Ning Li1, Xin-hua Qian, Zhi-yuan Wang
1Department of Pediatrics, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. mean163@163.com
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 transfection 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 transfected 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 transfected 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.
Insights
Short interfering RNA (siRNA) effectively inhibits lung resistance-related protein (LRP) expression in leukemia cells. This targeted approach shows potential for overcoming LRP-induced multidrug resistance in leukemia therapy.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- Lung resistance-related protein (LRP) is implicated in multidrug resistance in leukemia.
- Targeting LRP expression is a potential strategy to overcome chemoresistance.
Purpose of the Study:
- To investigate the inhibitory effect of short interfering RNA (siRNA) on LRP expression in leukemia cells.
- To assess the specificity and efficacy of siRNA in downregulating LRP.
Main Methods:
- Construction of eukaryotic vectors for LRP expression (pcDNA3.0/LRP).
- Transfection of K562 cells with LRP vectors and LRP-specific siRNA.
- Evaluation of LRP mRNA and protein levels using RT-PCR and flow cytometry.
Main Results:
- Transfection with pcDNA3.0/LRP increased LRP expression.
- Co-transfection with LRP-specific siRNA significantly decreased LRP mRNA and protein levels.
- siRNA demonstrated specificity, with no significant impact on unrelated gene (GFP) expression.
Conclusions:
- LRP-specific siRNA effectively degrades LRP mRNA and inhibits protein expression.
- Designed siRNA is specific and potent, offering a potential therapeutic strategy.
- siRNA-mediated LRP inhibition may reverse multidrug resistance in leukemia.
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