Double transfection improves small interfering RNA‐induced thrombin receptor (PAR‐1) gene silencing in DU 145 prostate cancer cells

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Abstract
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.