| Background The latest American Cancer Society estimates for prostate cancer (Pca) in the United States are for 2009, about 192,280 new cases of PCa will be diagnosed,27,360 men will die of PCa. While in other countries, according to certain reports, in order to get better information about this disease, autopsy studies of Chinese, German, Israeli, Jamaican, Swedish, and Ugandan men who died of other causes were done and it was found that PCa was present in thirty percent of men in their 50s. Modern methods of detection and treatment mean that many PCas are now found earlier and can be treated more effectively. Androgen deprivation therapy is effective for most PCas. However, there is no effective treatment for when PCa becomes androgen independent. Hormone-refractory invasive PCa is the end stage and accounts for the majority of PCa patient deaths. Therefore, a new target for therapy of androgen independent PCa is needed. It is considered that androgen-independent progression is due to many kinds of androgen receptor mutation, overexpression of receptor tyrosine kinase (RTKs) and focal adhesion kinase (FAK), neuroendocrine and Interleukin-6 (IL-6). Previously, it has been reported that the Bombesin (BBS), Zoledronic acid, Inositol hexakisphosphate, and Plumbagin (PL) were investigated as novel agents for therapy of hormone-refractory PCa. But the the results were not entirely satisfactory. Although extensive work has been done in the area, the question then became how to tackle such a daunting challenge effectively. Moreover, a recent in vitro study indicated that the amphoterin-RAGE interaction was involved in the development of PCa. It is noteworthy that among the three PCa cell lines (LNCaP, PC-3, and DU145), the DU145, a hormone-independent PCa cell line, showed higher expression of RAGE and amphoterin mRNA compared to the other cell lines. Therefore, the RAGE gene played an important role in the androgen-independent progression.Objective To construct four shRNA-psiU6 plasmids targeting RAGE and a negative control plasmid as shRNA control. To observe the influence in biological behavior of the prostate cancer cells after the silencing RAGE gene by RNA interference.Methods A subclone cell line with high expression of RAGE was screened by limiting dilution technique, real-time fluorescence quantitative-PCR(RFQ-PCR) and flow cytometre (FCM), and DU145 cells stimulated by AGE-BSA were taken as the control. Four kinds of RAGE-specific short-chain oligonucleotides were synthesized, intergrated into the plasmid vecter of psi-U6 and transfected to the subclone cell line with high expression of RAGE. The transfection efficiency was observed by Fluorescence microscope. The expression of RAGE mRNA and protein, cell cycle, cell morphology, proliferation and migration were detected by RFQ-PCR, Western blot, cell counting kit-8(CCK-8), inverted microscope, FCM, and scratch test.Results1. A subclone cell line with high expression of RAGE, SD-2C1, was screened successfully.2. The results showed that the expression of the RAGE gene were decreased significantly, especially under the influence of RAGE shRNA-1. The reduction rate of RAGE mRNA was about 84%, RAGE protein was about 27%, compared with negative control (P＜0.05).3. In vitro, CCK-8 test showed that cell proliferation was significantly reduced after transfection of the shRNA vectors.4. In vitro, FCM results suggested that arrest of G2/M phase was reduced in the RAGE shRNA-1 group (P＜0.05).5. In vitro, the cell morphology was changed significantly after transfection.6. In vitro, Scratch test suggested that there was no significant change in cell migration ability (P＞0.05).Conclusions In this study, RAGE shRNA expression plasmids, constructed from psi-U6 plasmid, can successfully be transfected into SD-2C1 cells and can effectively inhibit the expression of RAGE protein and mRNA. There is a significant inhibiting effect of RNA interference on the proliferation, cycle and morphology of DU145 cells in vitro. Data indicate that silencing of this gene provide a new molecular target for therapy or prevention of the androgen independent PCa.
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