Construction Of SiRNA-Stat3 And Endostatin Co-expression Plasmid And Preliminary Study For Prostate Cancer Therapy

Prostate cancer is a serious genitourinary system cancer frequently occurred in the 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. In China, the annual incidence and mortality rates of prostate cancer has risen as well. General therapies for prostate cancer include radical surgery, hormone therapy, cryotherapy, high intensity focused ultrasound therapy, chemotherapy and so on. However, prostate cancer is difficult to find in the early stage, and the late stage cancer will ultimately develop into hormone-refractory prostate cancer after the treatment. There is no way to cure the prostate cancer in clinical. So gene therapy is thought highly increasingly.Stat3 is a significant member of signal transducers and activators of transcription (STAT) family, which plays an important role in promoting proliferation, differentiation, antiapoptosis, and cell cycle progression. If stat3 signaling pathway is actived persistently, its overexpression has been suggested to be associated with prostate cancer progression. Many tumors, including prostate cancer, Stat3 and its downstream gene such as Bcl-2, cyclin D1, Bcl-xL, c-Myc, VEGF often have abnormal over-expression or aberrantly active, these genes influence cell cycle progression or inhibit apoptosis. This suggests that the aberrantly active Stat3 and downstream genes have a close relationship with the carcinogenesis. Aberrantly active Stat3 plays an important role, so we suppose the suppression of Stat3 and block Stat3 signaling pathway may inhibition the cancer malignant development by RNA interference. However, in mammalian cells, RNAi can not completely block gene expression, especially for abnormally expression gene. We have developed a multi-factor combined therapy in order to increase the efficacy of prostate cancer.Carcinogenesis and metastasis depend on angiogenesis, and inhibition of angiogenesis drugs has made remarkable effect in recent years. Endostatin, a kind of angiogenesis inhibitor, has attracted much more attention because of its good effect. Endostatin is an endogenous angiogenesis inhibitor. Furthermore, the effect of Endostatin in tumor inhibition is better than angiostatin. Endostatin can act on endothelial cells specificially, especially on microvascular endothelial cells, inhibit migration and induce apoptosis, thus inhibit angiogenesis, and as a result, inhibit tumor growth. At present, the application of Endostatin in treatment of tumors and the synergistic treatment of cancer with other vascular inhibitory factor, radiotherapy, chemotherapy, antisense RNA have achieved very satisfied effect. In this research, the gene and Stat3-specific siRNA were selected to construct a together-expression vector system and obtained a good synergistic effect.With the deepening of the study, the curative effect of cancer has made great improvement. But the conventional treatment of cancer drugs often leads to cell death in normal tissue while mediating apoptosis or necrosis in tumor cells. Serious side effects limit the application of some drugs. Moreover, limit the improvement of curative effect of cancer. In gene therapy, the search for efficient and low-toxic carriers which can deliver drugs to local tumor specially and develop curative effect is an important direction in cancer research. Attenuated Salmonella with the characteristics of low toxicity, invasion, targeting, etc, and as a gene therapy vector, can not only make the carrying purpose gene to express in local tumor specially, but also has inhibitory effect because of its own competition with cancer for nutrition.In order to obtain a synergistic effect of Endostatin and si-Stat3 gene, the experiment construct Endostatin gene with U6 promoter-mediated gene si-Stat3 in pcDNA3.1 vector, construct siRNA-Stat3 and Endostatin together-expression plasmid successfully. Copy prostate xenograft model in mice, take the pSi-Scramble, pSi-Stat3 and Endostatin plasmid as the control group, transfer plasmid to attenuated Salmonella by electric transforming way, inject to mice intravenously, to observe its specific comprehensive targeting treatment effect of entity tumor.Objective:Construction of siRNA-Stat3 and Endostatin co-expression plasmid (referred as pEndo-Si-Stat3). To observe the effective treatment for prostate cancer in mice by inject the pEndo-Si-Stat3 co-expression plasmid carried by attenuated Salmonella typhimurium and to find its potential mechanism. Methods:The co-expression plasmid pEndo-Si-Stat3 was constructed using recombinant DNA technology. Mice were injected i.v. with attenuated S. typhimurium carrying different plasmids. To observe the effective treatment for prostate cancer in mice subcutaneous tumor, RT-PCR and Western blot analysis were used to detect the mRNA and proteins expression levels of Stat3, Endostatin and related gene Bcl-2 and VEGF. The expression of Endostatin in tumor tissue was detected by ELISA kit. The influence of heart, liver, spleen, lung, kidney and tumor changes was also detected by attenuated Salmonella using HE staining. The apoptosis of tumor cells was detected by TUNEL assay. The expression of Stat3,Endostatin,Caspase3,PCNA and CD34 were detected by Immunohistochemistries.Results:The co-expression plasmid pEndo-Si-Stat3 was constructed successfully by restriction enzyme digestion and sequencing. It was show that the co-expression plasmid pEndo-Si-Stat3 carryied by attenuated Salmonella could be effectively gathered in the tumor in colony distribution experiments. Average tumor weights and volumes in the mice treated with pEndo-Si-Stat3 carried by Salmonella were significantly lower than the control groups. RT-PCR and Western blot experiments showed that co-expression plasmid could reduce Stat3 gene and protein expression, while it could raise Endostatin gene and protein expression in tumor tissues. ELISA experiments showed that the expression of Endostatin was significantly higher than the control groups in tumor tissue (P <0.01). TUNEL detection showed that the tumor tissue cells developed apoptosis treated with pEndo-Si-Stat3 carried by Salmonella than the control groups significantly. RT-PCR and the Western blot experiments showed that the expression of Stat3 gene reduce and its downstream gene Bcl-2 and VEGF expression were also inhibited. We find that there was almost no change in histological of heart, liver, spleen, lung, kidney and tumor by attenuated Salmonella using HE staining. Immunohistochemistries experiments show that the co-expression plasmid pEndo-Si-Stat3 can raise the expression of Caspase3 and reduce the expression of PCNA and CD34.Conclusions:In this project, the co-expression plasmid pEndo-Si-Stat3 was constructed successfully. It is show that co-expression plasmid pEndo-Si-Stat3 carried attenuated Salmonella have the effect of inhibiting tumor cell proliferation and promoting apoptosis in vivo. The mechanism is may be that the combination of siRNA-Stat3 and Endostatin can reduce Stat3 gene and protein expression, thus reduce Bcl-2 and PCNA expression, and raise Caspase3 expression, ultimately, promoted apoptosis of tumor cells. While co-expression plasmid can raise Endostatin gene and protein expression, thus reduced CD34 and VEGF expression, so that it inhibits endothelial cell proliferation and angiogenesis, ultimately inhibits tumor growth.