| Backgroud Prostate adenocarcinoma is the most commonly diagnosed noncutaneous cancer and second leading cause of cancer related deaths in men. Death due to prostate cancer is attributed to metastatic disease in which primary tumors evade the prostate capsule and eventually metastasize. This presents a large problem considering that current diagnostic procedures do not readily determine whether an individual with indolent disease will develop aggressive metastatic disease. To reduce mortality due to prostate cancer, it is imperative to develop an in-depth understanding of molecular events that occur during prostate cancer progression and metastasis to identify key features and molecular alterations suitable for use as diagnostic markers or therapeutic targets.CD59 is widely expressed in blood cells and many other tissue cells, protecting host cell membranes from homologous MAC. CD59 is sometimes over expressed on tumor cells, and its expression has been linked to promoting tumor growth and the protection of tumor cells from mAb therapy. Other investigators have shown that CD59 is over expressed in invasive prostate cancer cells. However, the mechanism for this expression is not known. The regulation of CD59 is complex and occurs through both transcriptional and post-transcriptional mechanisms. CD59 lacks the typical TATA box and contains a GC-rich sequence. It was previously shown the importance of the additional 35 bp sequence between the-35 and-70 promoter constructs for elevated expression of the CD59 gene. We analyzed the 35 bp positive regulatory sequence in the CD59 promoter for potential binding to transcription factors. Using Genomatix Software, we identified transcriptional activator Sp1 that may bind to the positive regulatory sequence.Spl plays a key role in maintaining expression of genes that lack TATA box. Spl interacts with GC-rich Sp1 binding sites in multiple promoters to regulate gene expression, and there are an increasing number of studies showing that Spl interacts with other nuclear proteins, including promoter-bound transcription factors, to attenuate tissue-specific expression of selected genes. Spl is over expressed in gastric and pancreatic cancers, and has a prominent role in regulating genes involved in angiogenesis such as vascular endothelial growth factor (VEGF). Interestingly, Sp1 has also been shown to regulate genes involved in prostate cancer metastasis.Objective To construct recombinant vectors expressing siRNA that target Spl gene and a stable-inhibit cell line PC-3 in order to analyze the role of Spl in the expression of CD59.Methods The 63 bp encoded targeting Sp1 gene shRNA sequence was cloned and transferred into pSUPER vector by DNA recombinant technique. The prostate cancer cell PC-3 was transfected with this recombinant plasmid by liposome and the stable strains was selected by G418 medium. Sp1 and CD59 protein was detected by RT-PCR and Western blot, CD59 was detected by flow cytometry and its function was detected by MTT and dye release assay.Results The pSUPER-siRNA expressing vector was successfully constructed, and a stable cell line PC-3 was selected and detected the expression of GFP. The siRNA vector effectively inhibited Spl and CD59 gene expression from mRNA and protein in level. MTT assay and Dye release assay suggested that CD59's protection to complement mediated cytolysis decreased.Conclusion The siRNA vector targeting Spl gene could consistently inhibit CD59 expression. Furthermore, it decreased CD59's protection against complement. These results may pave the way for studying the role of Spl in the expression of CD59. |
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