Double transfection improves small interfering RNA-induced thrombin receptor (PAR-1) gene silencing in DU 145 prostate cancer cells.
The efficiency of small interfering RNA (siRNA)-induced gene knockdown is hampered by low transfection efficiency. We established a novel and simple double transfection method using specific siRNA duplexes targeted against human thrombin receptor PAR-1 in DU 145 prostate cancer cells. The initial siRNA transfection of cell suspensions followed by re-transfection of adherent cells on the following day resulted in undetectable PAR-1 mRNA and absent receptor protein. PAR-1 mRNA expression was silenced for up to five days. Functional studies showed that PAR-1 gene silencing in DU 145 cells abolished the modulating effects of thrombin on cell adhesion to the extracellular matrix proteins, fibronectin and laminin, thus demonstrating the essential role of PAR-1 in mediating thrombin effects on DU 145 cell adhesion.
Liu
Jian
J
Faculty of Medicine, Institute of Clinical Chemistry and Pathobiochemistry, University of Rostock, Ernst-Heydemann-Strasse 6, D-18057 Rostock, Germany.
Schuff-Werner
Peter
P
Steiner
Michael
M
D001483:Base Sequence; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D017931:DNA Primers; D005434:Flow Cytometry; D020868:Gene Silencing / Q000502:physiology*; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology; D034741:RNA, Small Interfering / Q000502:physiology*; D044463:Receptor, PAR-1 / Q000235:genetics*; D014162:Transfection
D017931:DNA Primers; D034741:RNA, Small Interfering; D044463:Receptor, PAR-1