Specific inhibition of gene expression of lung resistance-related protein by short interfering RNA.
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.
Department of Pediatrics, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. mean163@163.com
Ning
N
Li
Xin-hua
XH
Qian
Zhi-yuan
ZY
Wang
D018432:Drug Resistance, Multiple / Q000235:genetics*; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection; D020394:Vault Ribonucleoprotein Particles / Q000096:biosynthesis* / Q000235:genetics; D000099187:Major Vault Protein
D049452:Green Fluorescent Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein; C485184:enhanced green fluorescent protein