Plasmid-Based Stat3 SiRNA Delivered By Hydroxyapatite Nanoparticles Suppresses Prostate Tumor Growth

Prostate cancer is one of the most globally common cancers in elderly men. Nowadays, people's lifespan is much longer than before with the improvement of living conditions and life quality. However, the aging trend of society leads to the prostate cancer growing rapidly. In western world, the incidence of prostate cancer has been ranked first among all the cancers behind lung cancer. In China, the annual incidence and mortality rates of prostate cancer have risen as well. While the patients with prostate cancer can be detected in earlier stages, thanks to the application of diagnosis such as prostate-specific antigen (PSA) screening over the recent years and therefore they may be cured or maintained successfully by standard medical treatments including radical prostatectomy, androgen deprivation, chemotherapy, local radiation therapy, and cryoablation, but there is still no effective treatment modality currentlv for those who have advanced disease when being diagnosed or who have failed the primary curative attempts, and the prognosis is dismal to those patients. Novel and improved treatment for this common disease is desperately needed. As prostate cancer represents an accumulation of genetic mutations in cells that have gone awry, one novel approach against prostate cancer is gene therapy.Recent studies have indicated that constitutive activation of the signal transducer and activator of transcription 3 (Stat3) pathway can contribute to tumorigenesis in prostate cancer. There is evidence to implicate that stat3 is overexpressed in a variety of tumours. Furthermore, we used a vector-based siRNA-Stat3 expression system to suppress Stat3 expression, as well as the expression of its downstream mediators, cyclin D1. VEGF that contribute to promote cell cycle progression, and prevent facilitate angiogenesis. Our results indicate that blocking Stat3 expression using a specific RNAi approach significantly reduces prostate cancer cell proliferation and induces cell apoptosis. Thus. Stat3 may represent a new molecular target for therapeutic intervention of prostate cancer.RNAi is a post-transcriptional gene-silencing (PTGS) mechanism found in plants, some invertebrate and mammalian cells. RNAi was triggered by a novel mechanism through which small double-stranded RNA molecules bind to specific mRNA sequences and block the respective gene's protein expression. SiRNA is a key molecule in the RNAi pathway. Inhibiting the translation of the cancer gene is an effective strategy in cancer therapy. Thus, blocking stat3 signaling in tumor cells by RNAi can suppress the tumongenesis in prostate cancer.The biggest challenge in the use of siRNA-based cancer therapy is the difficulty of siRNA delivery. At present, an efficient, targeted gene delivery system is the key issue of cancer therapy. To help siRNA reach the cancer cells, where they can become effective and induce gene silencing, different delivery systems have been developed. They can be classified into two main groups:viral and non-viral delivery systems. Actually, there are high transduction efficacy due to the inherent ability of viruses to transport genetic material into cells, which is one of their advantageous properties. But the potential of mutagenicity or oncogenesis. several host immune responses, and the high cost of production limit their application. For these reasons, different kinds of non-viral siRNA delivery systems have been tested to avoid viral vectors(for example, the injection of chemically stabilised or modified RNA. encapsulating siRNA in microparticles. nanoparticles or liposomes. binding siRNA to cationic or other particulate carriers). In recent years with the rapid development of nanotechnology. nanotechnology-based research has become a hot topic. The number of reports is increasing in Nano-particles as a gene transporter on gene therapy. But there is no report that hydroxyapatite nanoparticles carried specific siRNA expression vector, and with RNA silence of tumor cells.Our previous study showed that Stat3 specific siRNA can markedly suppress Stat3 expression and inhibit the growth of prostate cancer cells. In this study. HAP nanoparticles were used to mediate DNA-vector-based Stat3-specific RNAi to treat prostate cancer, with the goal of investigating the antitumour efficacy of HAP nanoparticle-mediated RNA interference, and to explore its possible mechanism. The study is the first report that hydroxyapatite nanoparticles as the siRNA-Stat3 vector for cancer treatment, which a newly effective carrier for the application of RNAi technology.MethodHydroxyapatite nanoparticles morphology was observed by Electron microscopy The binding ability of modified nanoparticles and DNA, and the transfection efficiency were detected by fluorescence microscopy. HAP nanoparticles were used to mediate DNA-vector-based Stat3-specific RNAi to treat prostate cancer cells in vivo and in vitro. The effect on the growth of mouse prostate cancer cells of siRNA-Stat3 delivered by hydroxyapatite was determined in this study. RM1 tumour blocks were transplanted into C57BL/6 mice. CaCl2 modified hydroxyapatite-carrying siRNA-Stat3 plasmids were injected into tumours, and tumour growth and histology were determined. The expression levels of Stat3, pTyr-Stat3, Bcl-2, Bax, Caspase3, VEGF and cyclin D1 were measured by western blot and qPCR analysis. Amounts of apoptosis in cancer cells were analysed with immunohistochemistry and the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay. The cells were also analyzed for cell cycle phase distribution and apoptosis rate by flow cytometry.Results1. Hydroxyapatite nanoparticles showed a needle-like shape under the EM and CaCl2 modified hydroxyapatite-carrying siRNA-Stat3 plasmids DNA.2. Hydroxyapatite nanoparticles could carry plasmids and deliver them into the tumor cells.3. The results showed that siRNA-Stat3 plasmids delivered by hydroxyapatite nanoparticles were capable of suppressing prostate tumor cell growth and inducing the apoptosis effects in vivo. The nanoparticles combined with the plasmid exhibited a synergistic anti-tumor effect.4. The results showed that hydroxyapatite-delivered siRNA-Stat3 significantly suppressed tumour growth in vitro. Stat3 expression was dramatically down-regulated in the tumours. The immunohistochemistry and TUNEL results showed that Stat3 siRNA-induced apoptosis.ConclusionHydroxyapatite nanoparticles showed inhibition on tumor cells alone and could carry the Stat3 specific siRNA plasmids into the tumor cells, which exhibited an effectively synergistic anti-tumor effect. In this study, we present an innovative approach of prostate therapy by using the modified hydroxyapatite nanoparticles as a carrier of siRNA plasmids in vitro and in vivo.