Experimental Studies On Breast Carcinoma's Gene Therapy Targeted By Chitosan Nanoparticles Mediated XIAP SiRNA

Breast carcinoma is one of the malignant tumors which have a high prevalence and mortality rate in countries throughout the world, among which its mortality in China ranks the first place. The therapies for breast cancer have been under constant study. The current therapy for breast cancer has a high reoccurrence and metastasis rate postoperation. Therefore, our tentative study on the pathologic mechanism of breast cancer and its therapies are necessary.XIAP gene is a new member of inhibitors of apoptosis protein (IAP) family. It is observed to express mainly in the developing embryonic tissues and the common human malignant tumor tissues. It has been found in previous studies that XIAP possesses a double regulatory activity on cell apoptosis and cell division cycle by exerting a direct inhibitory effect on the activities of caspase, especially caspase-3, caspase-7 or caspase-9, to block the corporate pathway of the down-stream cell apoptosis induced by various stimulants. Besides, XIAP has a specific expression in the cells in G2/M stage of cell division circle, and is involved in the regulation of cell gene's transcripition by combining with the microtouble of cell mitosis spindle. And XIAP can directly inhibit the activity of caspase-9 to reduce tumor cells' sensitivity to chemotherapeutic agents. XIAP gene's selective expression in tissues and its special activity on the inhibition of cell apoptosis suggest that it is likely to be identified as a special index in diagnosis and a potential therapeutic target in breast carcinoma.It has been demonstrated that occurrence of breast cancer combines with many oncogenes' activation and anti-oncogenes' inactivation. Therefore, we select the oncogenes and anti-oncogenes which play important roles in the occurrence of breast cancer, and emply antisense oligonucleotide technology, gene knock-down, gene substitute and gene transduction, the oncegenes' expression can be inhibited and knocked down and a gene therapy for breast cancer can be achieved.A number of gene carriers /vectors have been studied for gene delivery to the breast cancer, including viral and non-viral gene carriers. Viral vectors, particularly adenovirus and lentivirus, have demonstrated high level of transgene expression. Viral vectors have the ability to infect a wide variety of cell types in vivo. However, viral vectors are associated with severe problems such as host immune response against the vectors leading to the rapid rejection of transduced cells, intrinsic toxicity of the viral proteins, and limited packaging size. Non-viral gene carriers have been increasingly proposed as safer alternatives. Nanosphere encapsulating DNA gene delivery methods was established by prof Leong in 1998, because of ease of synthesis, flexibility in the size of the transgene to be delivered, and most importantly, the low toxicity and minimum host immune response. In addition, they can better address the pharmaceutical issues such as storage stability and scale-up synthesis.In this study , we examine the feasibility of breast-targeted gene delivery by chitosan-XIAP siRNA nanoparticles. We provide a new theoretical basis for gene therapy of breast carcinoma and for others' malignant tumors.The advantage of the target-oriented therapy with tumor apoptosis-inhibiting gene as its target is that it can increase tumor cells' apoptosis and inhibit their proliferation, which it has no bad effect on normal tissues. RNA interference (RNAi) can specifically and efficiently degrade mRNA, resulting in post-transcriptional gene silencing. Besides, its blocking action on gene expression has been successfully observed in rats and human cells cultured in vitro, and the knockdown of cell genes has been achieved. The present study was designed mainly to identify whether XIAP gene expression in the breast cancer cells can be knockdown so as to induce cell apoptosis and inhibit proliferation.Aims:To study XIAP gene's possible mechanism in breast cancer cell proliferation and cell apoptosis and to provide a tentative theoretical basis for gene therapy for breast cancer. At the same time, to investigate the feasibility, safety, transfection efficiency, and delivery method of a novel nanosphere complexes-based gene therapy, compared with plasmid DNA.Methods:1. SABC immunohistochemical staining was emplyed to detect XIAP gene in 119 breast cancer tissues, 58 adjacent noncancerous tissues of deparaffinized sections, 8 breast cancer tissues and MCF-7 , bcl-2 and caspase-9 in 30 cases of breast cancer. XIAP mRNA and protein expression were examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively.2. Human breast cancer cells line (MCF-7) was transfected with the eukaryotic expression vector of dsRNAi of XIAP gene through a lipofectin transfection method to construct cell model. And then SABC immunohistochemical staining, RT-PCR, Western blot method, Flow cytometry(FCM) analysis, TUNEL technology, and the cell growth curve were applied to detect XIAP protein expression, its mRNA expression, cell proliferation and cell growth in MCF-7 before and after transfection.3. BALB/c nude mice were injected subcutaneously with scramble control, blank plasmid and XIAP siRNA cell suspension respectively to construct nude mouse breast carcinoma animal model. The formation and growth of the tumors were observed by measuring and weighing. Besides, XIAP protein expression was detected by SABC immunohistochemical staining.4. To prepare the chitosan nanoparticle with a complex coacervation process;Tomeasure its diameter, the diametric distribution gene range and its form by scanning electron microscope and laser atomic examing apparatus. The XIAP siRNA plasmid is absorbed to the chitosan nanoparticle by electrostatic forces;The potential of absorbing XIAP siRNA on nanoparticles was analyzed by agarose gel electrophoresis and spectrophotometer. To check its protection ability to DNA by DNase I digestive experiment. To transfect it to MCF-7 cell and evaluate the possibility and efficiency of chitosan nanoparticle as a vector in gene transference compared to cationic liposome by fluorescence microscope. The expression of XIAP gene transfected by XIAP siRNA in the MCF-7 cell line was examined through RT-PCR and Western blot assay. MTT method was used to evaluate cell toxicity.Results1. An up-regulation expression of XIAP gene was observed in breast cancer tissues and there was no correlation between this expression and the age and gender of the patients, the size of the tumors, metastasis or not , bcl-2 expression, and clinical stages. Instead, this expression was closely associatedwith that of caspase-9.2. XIAP protein expression and its mRNA expression disappeared in MCF-7 cells transfected by shRNA of XIAP gene. The MCF-7 cell model was characterized by a block in its protein expression and transcription, and occurrence of some typical manifestations of cell apoptosis and a remarkably-inhibited cell growth.3. The tumorigenic rate in nude mice injected with MCF-7 transfected by scramble control and blank plasmid was 100%, and the growth index was 19.4%. The vessels on the surface of the tumors were large and clear, and tumor infiltrated into the deep muscular tissues. The tumorigenic rate in nude mice injected with MCF-7 cells suspension transfected by dsRNAi of XIAP gene was 50%, its incubation period of tumor was lengthened, and the tumor-inhibiting rate was 87.2%.4. Chitosan nanoparticles is showed to be spherical particles distributed evenly,the least nanoparticles is 45nm in diameter, d(0.5) is 98nm, by scanning electron microscope and laser atomic examining apparatus. Chitosan can be used as effective protectant for gene transfection in vitro by FCM. There was no XIAP expression in MCF-7 transfected by chitosan/XIAP siRNA by western blot assay. MCF-7 cells' transfection efficiency by the chitosan nanoparticles,was nearly with that of cationic liposome. It was found that nanosphere-complexes had a significantly inhibitory effect on nanosphere-MCF-7 cell lines by using MTT method. It can be seen from the experiments that MCF-7 cells can be transfected efficiently by chitosan/XIAP siRNA. Some better results were gained at charge ratio(+/-)of 2:1 and the optimal interval of transfection was for 8 hours. It was showed serum was not a key influencing factor for the experiment.Conclusions:1. The results of the detect of XlAP in protein and its mRNA in the breast cancer tissues and human breast carcinoma cells line demonstrate that XIAP gene contributes to the occurrence of breast cancer by enhancing the breast cancer cells' proliferation and inhibiting their apoptosis. The findings suggest that detecting XIAP gene expression in the breast cancer tissues may be to some extent valuable to diagnose breast cancer in the clinical application.2. In the breast cancer tissues, XIAP expression is closely correlated with that of caspase-9, but there is no correlation between XIAP expression and that of bcl-2, which suggests that the combination of XIAP gene, caspase-9 and bcl-2or any of them, are involved in the occurrence of breast carcinoma.3. The results of the transfection by XIAP gene in vitro and the tumorigenic experiment in nude mice in vivo confirm that XIAP gene can enhance the proliferation of breast cancer cells and inhibit their apoptosis both in vivo and in vitro, which provides new evidence to prove that XIAP gene is involved in the occurrence and development of breast carcinoma, also prove that dsRNAi both in vivo and vitro can specifically knock down the XIAP expression in breast carcinoma cells, which may serve as a scientific evidence for XIAP gene as the target gene in the gene therapies for breast cancer.4. Homogeneous and small in diameter chitosan nanoparticles has been prepared and loaded with plasmid effectively. It can protect the plasmid from being digested. MCF-7 cells are transfected successfully by the chitosan nanoparticles, and its transfection efficiency is nearly with that of cationic liposome. Nanosphere complexes-Based gene delivery system as non-viral vector may provide a novel gene therapeutic strategy ,and can possibly apply for clinic usage.