Down-regulation of Egr-1 by siRNA inhibits growth of human prostate carcinoma cell line PC-3.
The inhibitory effect of a specific small EGR-1 interfering RNA (siRNA) on cell proliferation and the expression of EGR-1 in human prostate carcinoma cell lines PC-3 and LNCaP was investigated. To knockdown Egr-1 expression, a siRNA targeting against Egr-1 was synthesized and transfected into PC-3 and LNCaP cells. The down-regulation of Egr-1 expression at both mRNA and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The transcription activity was determined by luciferase expression. Cell proliferation inhibition rates were determined by soft agar and methyl thiazolyl tetrazolium (MTT) assay. The effect of Egr-1 siRNA on cell cycle distribution and cell apoptosis was determined by flow cytometry (FCM). RNA interference efficiently suppressed the Egr-1 expression in PC-3 and LNCaP cells. At 96 h after transfection, the expression inhibition rate was 44.52% at mRNA level detected by RT-PCR and 40.17% at protein level by Western blot analysis. The cell proliferation inhibition rates at 24, 48, 96 and 120 h after Egr-1 siRNA and non-silencing siRNA transfection, were 5, 25.06, 65.61 and 78.36%, respectively for PC-3 cells and 23, 40.3, 75.9 and 67.4%, respectively for LNCaP cells. The apoptosis rate was similar for both PC-3 and LNCaP and the number of cells was increased in G0/G1 phase from 38.2 to 88.6%, and decreased in S and G2/M phase at 96 h after transfection. Down-regulation of Egr-1 results in significant inhibition of tumor growth in vitro. The inhibition of Egr-1 expression can induce apoptosis of PC-3 cells. The use of Egr-1 siRNA deserves further investigation as a novel approach to cancer therapy.
Parra
Eduardo
E
Biomedical Experimental Laboratory, Department of Biology, Faculty of Sciences, Tarapaca University of Arica, Avenida General Velasquez 1775, Arica, Chile. eparra@uta.cl
Ortega
Arnaldo
A
Saenz
Leonardo
L
D017209:Apoptosis; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D051766:Early Growth Response Protein 1 / Q000096:biosynthesis*; D005434:Flow Cytometry / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue