Aim: To construct recombinant vectors expressing siRNA that target Sp1 gene and to generate a stable Sp1 knockdown PC-3 cell line to analyze the role of Sp1 in the expression of CD59.
Methods: Sp1 siRNA was cloned into pSUPER vector. The prostate cancer cell line PC-3 was transfected with this recombinant plasmid using liposome and the stable cells were selected by G418 medium. mRNA and protein levels of Sp1 and CD59 were detected by RT-PCR and Western blot, and cell function was analyzed by dye release assay.
Results: The pSUPER-siRNA expressing vector was successfully constructed, and a stable cell line PC-3 with Sp1 knockdown was selected and analyzed for the expression of GFP. The siRNA vector effectively inhibited the CD59 gene expression from both mRNA and protein levels. Dye release assay suggested that decreased CD59 expression caused less redistance to complement mediated cytolysis.
Conclusion: The siRNA vector targeting Sp1 gene effectively inhibited CD59 expression and diminished CD59-mediated anti-complement activity. These results may pave the way for studying the role of Sp1 in the expression of CD59.