| [Background]In China, colorectal cancer has become the most common gastrointestinal cancer, with the incidence rate increased year by year, whose annual incidence rate is 59/10 million, and five-year survival rate is about 32%. To find effective ways to prevent and control colorectal cancer is a priority, in which gene therapy is an important research direction of cancer treatment. Affected by many factors, gene therapy is far from perfect. On the one hand, tumorigenesis mechanism is very complex. It is difficult to determine the specificity targeted gene. Apoptosis disorder is generally regarded as an important mechanism of carcinogenesis. Inducement of tumor cell apoptosis can be an effectual way to treat cancer. Bcl-2-associated athanogene 1 (Bag-1) is a positive regulator of Bcl-2 which is an anti-apoptotic gene. Bag-1 is highly expressed in colorectal cancer, which plays a critical role in promoting metastasis, poor prognosis, especially in anti-apoptotic function, and is perhaps a valuable gene target for colorectal cancer therapy. On the other hand, gene therapy is limited by gene delivery systems. Currently virus-based gene carriers have serious security risks which makes it difficult to wildely applied. The non-viruses gene carrier, magnetic nanoparticle gene carrier, with low toxicity and low immunity, is a novel gene carrier with huge potential. Preparing a new and effective magnetic nanoparticle gene vector loading RNAi recombinant plasmid which can specific block Bag-1 gene for experimental study in vitro and vivo is a good way to analyse the efficacy of the treatment of colorectal cancer and further clarify the mechanism of the genesis and development of colorectal cancer. Except that, it is significant to establish a novel and effective methods of gene therapy for colorectal cancer.[Objective]Prepare a new composite magnetic gold nanoparticle and apply for silencing Bag-1 gene via magnetic gold nanoparticle-delivered siRNA plasmid for colorectal cancer therapy in vivo and in vitro[Methods]1. Applied chemical co-precipitation method and molecular surface modification techniques for preparing functional composite magnetic gold nanoparticles (PEG/ Fe3O4@Au), then test their characteristics and analyse their performance as gene vectors.2. Using bacterial culture to amplify then extracting Bag-1 gene RNAi plasmid (pGPH1/GFP/Neo-Bag-l-homo-825), then incubating the RNAi plasmid with magnetic gold nanoparticles for preparing magnetic gold nanoparticle mediated RNAi plasmid transfection system.3. Different colorectal cancer cell lines (LoVo, HCT116) and normal colorectal tissue cell line (FHC) were applied to test the toxicity of magnetic gold nanoparticles by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Lipofectamine 2000 was selected as a control.4. Magnetic gold nanoparticle-mediated RNAi plasmids were transfected into LoVo cells. Transfected cells were observed under a fluorescence microscope, and cells’ transfection efficiency and apoptosis rate were analyzed by flow cytometry. The expressions of Bag-1 gene in mRNA and protein levels were detected by real-time PCR and Western blot assays.5. Four-to-five-week-old male Balb c/nude mice (14～16 g) which were kept in SPF environment were injected subcutaneously with 100 μL of LoVo cell suspension (lx 106) into the right flank.After the tumor mean diameter reached about 5 mm, the LoVo tumor-bearing mice were randomly divided into five groups and received the following treatment by intratumoral injection:normal saline (n= 4), nanoparticles (n= 4), plasmid (n=4), nano-plasmid complexes (n= 4), and nano-plasmid complexes (n= 4) which were kept in a cage with about 5000 gauss magnetic filed under it. The mice were treated with five doses at intervals of 3 days. The tumor volume was calculated as 0.52×length×width2 and was recorded every 3 days. Four weeks later, they were sacrificed by dislocation of the cervical vertebra and tumor tissue of each group were divided into two parts and respectively treated with 4% paraformaldehyde for tissue fixed and directly preserved at -80P<0 conditions after liquid nitrogen fixed.6. Observing different groups of tumor tissue morphology by HE staining. Detect expressions of Bag-1 gene and tumor-associated C-myc,β-catenin gene of tumor tissues in different groups by immunohistochemistry (IHC).7. Real-time PCR and Western blot were applied to detect the expressions of Bag-1 gene and tumor-associated C-myc, p-catenin gene expression at the mRNA and protein levels of tumor tissues in different groups.[Results]1. Magnetic gold nanoparticles (PEG/Fe3O4@Au) were round or nearly round particles with about 100nm uniform size distribution under TEM. Laser light scattering particle size analyzer showed that the nanoparticle size is about (95.38± 7.16) nm, zeta potential (35.03±3.12) mV, which can load and release the plasmid DNA.2. The extracted plasmids were proved to be the constructed Bag-1 gene RNAi recombinant plasmids (pGPHI/GFP/Neo-Bag-l-homo-825). After incubating with magnetic gold nanoparticles, the magnetic gold nanoparticle/plasmid complexes were relatively steady and can be used for cell transfection.3. The magnetic gold nanoparticles were substantially non-toxic to different human colorectal carcinoma cell lines (LoVo, HCT116) and normal colorectal tissues (FHC) which was similar to Lipofectamine2000 transfection reagent. RNAi plasmid loaded magnetic gold nanoparticles were significantly inhibiting the activity of human colorecta cancer cells but no significantly affected to normal human colon tissue cells.4. The RNAi plasmid loaded magnetic gold nanoparticles were successfully transfected into LoVo cells and the exogenous gene was smoothly expressed. Cell transfection efficiency was about 22% by flow cytometry analysis. Cell apoptosis of RNAi plasmid loaded magnetic gold nanoparticle group was significantly increased (P <0.05), compared to to bare magnetic gold nanoparticle transfection group, naked RNAi plasmid group and control group. And cell apoptosis rates among the other three groups were not statistically significant. Real-time PCR and Western blot test showed that the expressions of Bag-1 gene in RNAi plasmid loaded magnetic gold nanoparticle group were significantly decreased in mRNA (P<0.05) and protein levels (P<0.05).5. When the sizes of tumor growing about 5mm, treatment were began to give to each group. Four weeks later, xenograft tumor sizes in the nano-plasmid complexes group and nano-plasmid complexes group were significantly smaller than the other three groups (P< 0.05),while the difference between the two groups was not statistically significant.6. HE staining did not find a significant difference among different groups, but in IHC assays, the expressions of Bag-1 gene, and tumor-associated C-myc, P-catenin genes in nano-plasmid complexes group and nano-plasmid complexes with magnetic field group were significantly reduced, while there was no significant difference between them.7. RT-PCR and Western blot assays showed that the expressions of Bag-1 gene at the mRNA level (P<0.05) and protein levels (P<0.05) in nano-plasmid complexes group and nano-plasmid complexes with magnetic field group were significantly lower than the other three groups, but the difference between the two groups was not statistically significant. In further study, C-myc and P-catenin, mainly molecules of Wnt/β-catenin pathway, were decreased notably when Bag-1 were silenced in nanoparticle plasmid complex transfected Balb c/nude tumor xenograft.[Conclusion]Magnetic gold nanoparticles, as gene vectors, were relatively easy to synthesize and have lower cost, lower cytotoxicity. These all made magnetic gold nanoparticles potential gene vectors. In the present study, the RNAi plasmid loaded magnetic gold nanoparticles were successfully transfected into LoVo cells and the exogenous gene was smoothly expressed. Vivo and vitro experiments confirmed Bag-1 gene is an anti-apoptotic gene and closely linked to the genesis and development of colorectal cancer. Experiments also suggest this mechanism may be related to Wnt/β-catenin signaling pathway disorder. The present study successfully prepared a novel magnetic gold nanoparticle gene vector and magnetic gold nanoparticle delivered siRNA plasmid silencing Bag-1 is an effective gene therapy method for colorectal cancer. |
PDF Full Text Request