| Background&objective:Bladder cancer is a common urologic cancer, which directly threaten to the life of patients. At present, the main treatment of bladder cancer is still surgery, and the auxiliary methods are chemotherapy、actinotherapy、 immunotherapy and a variety of methods with combination treatment mode. However, to invasive and metastatic bladder cancer, the prostecdtive efficacy of treatment methods above is less than satisfactory and most patients eventually die of infiltrating and metastasis of the tumor.Survivin gene belongs to the new member of IAPs family, whose molecular weight is the smallest and structure is relatively simple and unique. It is very different from another members in IAPs family in the structure. Literature reports, Survivin has positive function to the occurance and development of cell carcinoma of bladder and is possibly related to metastasis and prognosis of the tumor. Survivin can be an important parameter to filtration、early diagnosis and prognostic ability of bladder cancer. Because Survivin in most cancer tissues with survivin gene has significant express of selectivity, strong function of inhibitor of apoptosis and inhibitor to survivin expression enough to induce the spontaneous apoptosis of cancer cell. This is the characteristic that another apoptosis inhibiting genes not have.RNAi technique is an agene disruption technique that develops rapidly in recent years, which thus leads to gene silencing of level of purpose (PTGS) through making the dsRNA with the regular structure characteristic and having a length of19-25nt, which is siRNA, import into host cells and on the level of specificity degradation to the mRNA of host cells that have homology with it. The technique of RNAi can make target genes stable and silent, not impact the expression of normal genes, and have the characteristics of simplification、specificity and high efficiency. Consequently, the RNAi technique is used in gene treatment for cancer with broad prospects.Anti-tumor gene therapy by the way of magnetic targeting was a new type tumor therapy system, in which the magnetic nanoparticles with satisfactory biocompatibility and low-toxicity side effect can carry genetic therapeutic molecules and initiatively reach locally advanced tumors, then kill the tumor cells under external magnetic fields. In the previous experiments, we found that iron oxide magnetic nanoparticles (CMN) has some satisfactory properties, such as superparamagnetism, biocompatibility, special nanometer effect and stability, etc. It not only can conjugate with the green fluorescent protein plasmid DNA, but also shows good magnetic targeting effect under external magnetic fields which was controled.The gene treatment of cancer contains two areas:one is the choice of target gene; the other is method and strategy of gene treatment. Using the specificity for complementary sequence to mRNA of Survivin genes, siRNA function in tumor cell with apoptotic resistance and block the Survivin gene’s expression, which offers a new treatment method to control tumor cell apoptosis. In the present study, siRNA recombinant plasmid which specific targeted survivin (siRNA-survivin) was used as a loaded gene therapeutic molecule. The effects of magnetic siRNA-CCMN nanoparticles transfection in combination with external magnetic fieldson silencing survivin gene expression of human bladder cancer cells and inducing human bladder cancer cells apoptosis were evaluated, In which CCMN produced by us was used as a gene carrier, respectively in vivo.Materials and Methods:1. The main materials1.1People bladder cancer cell line of BIU-87cells, which is adherent cell. 1.2The main instrument and equipment:Bechtop, Constant temperature CO2incubator, Desktop low temperature high speed centrifuge, H-600TEM, Zeta particle size analysis instrument, Vibrating Sample Magnetometer Signal Processorultra, low temperature refrigerator, ELX-800eliasa, PCRAmplifier, GS-800gel imaging analyzer, DU-800ultraviolet spectrophotometer.1.3The main reagent:RPMI-1640training powder, Sephacryl S-300HR, Polylysine (PLL), carboxymethyl chitosan, OPTI-MEM serum-free medium, Methyl thiazolyl tetrazolium (MTT), E.I.N.A.TM kit to endotoxin plasmid for extracting RT-PCR kit (TaKaRa RNA PCR kit(AMV)Ver3.0), Trizol Kit, DNA in situ end labeling (TUNE) detection kit, Western Blotting and ECL CLIA kit.2. Methods2.1Designing and synthetizing the interference sequences of Survivin-siRNA According to Reynolds’s designing principles of siRNA, make sure the targeted sequences of interference:ACGA GCCAGACTTGGCCCAGT. Use the online designing software offered by Invitrogen Company to design the DNA expression models of the homologous shRNA based on the selected interference target sequence. Through the homology analysis of BLAST, all the work of the synthesis of shRNA DNA template, connection with pSilencerl.0-U6and determination, and metaplasia to escherichia coli DH5a bacterial strain are completed by Wuhan Jing Sai. These recombined plasmids are named after pSilencer1.0-siRNA-survivin.2.2Amplification and extraction of recombinant plasmidInoculating Escherichia coli DH5a strain already transformed into recombinant plasmid in LB Solid culture plat containing0.05g/L kanamycin, puting in37℃5%CO2constant temperature incubator to train overnight. Selecting3almost lmm single colonies to dissolve into5ml LB liquid medium containing0.05g/L kanamycin, make liquid medium oscillate on the table concentrator at a speed larger than225rpm, put in37℃,5%CO2constant temperature incubator to train overnight. On the next day, we can get inocula which is already activation of amplification, use E.I.N.A.TM kit to endotoxin plasmid for extracting plasmid, extracting plasmid based on operation instructions. The concentration and purity of the extracted plasmid DNA can be test through DU-800ultraviolet spectrophotometer.2.3Preparation of CCMNCCMN was produced by the methods of chemical coprecipitation method. Taking carboxymethyl chitosan (1.5g), FeCl3·6H2O (0.234g) and FeCl2-4H2O (0.086g) in3ml of water dissolved. When Magnetic stirring, droping with a certain amount of ammonia. The temperature quickly rose to60℃, reaction time was30min, adjusting pH value to the neutral. Large particles will was removaled after aggregate15minafter low speed centrifugation. Then using the Sephacryl S-300HR gel column to separate and collect the first peak of the gel column. After full dialysis, CCMN was made through frozen vacuum drying.2.4Construction and characters detection of siRNA-CCMN conjugatesAt pH=7, PLL, CCMN and pSilencerl.0-siRNA-survivin were mixed and stirred according to the quality control rate (1:1:2) in the culture solution of RPMI-1640in free of serum. The siRNA-CCMN conjugates compound formed after the mixture reacted in the dark at4℃for1hour. Using H-600TEM, Zeta particle size analysis instrument and Vibrating Sample Magnetometer Signal Processor to detect the diameter, effective diameter, polydispersity and saturation magnetization of siRNA-DMN conjugates, respectively. The ferri ion (Fe) content of conjugates was detected by Spectra-30atomic absorption photometer.2.5Cells culture and transfectionBIU-87cells were seeded into6-well microplate at a density of1×105/well and cultured in RPMI-1640with10%fetal bovine serum in a humidified chamber at37℃and5%CO2. When the cells grow to logarithm growth phase, which were washed by pure RPMI-1640. The siRNA-survivin was transferred into BIU-87cells by CCMN when the siRNA-CCMN containing2μg of siRNA-survivin recombinant plasmid was added into each well. After6hours, cells were cultured in RPMI-1640with10%fetal bovine serum again for48hours. The subsequent procedure was in progress. The study has three groups:The control group, siRNA-CCMN group, siRNA-CCMN+PEMFs group. The following grouping and process were done in the above same manner. The intensity of PEMFs was10mT. The treating methods were described according to the documents provided.2.6Inhibitory effect of siRNA-CCMN on BIU-87cell proliferation by MTT method testIn3groups as the above same, every group having5ventral orifices. Conduct cell transfection, having steps as above. Separately after transfection in24、48、72h, use MTT method to test inhibition rate of cells. Use ELX-800microplate reader at490nm wavelength to test the absorbance value of separated hole.(A), compute inhibition rate of cell proliferation (IR):IR=(A blank control group-A experiment group)/A blank control group×100%.2.7Semiquantitative RT-PCR testing expression level of Survivin mRNAInoculating cells on96hole culture plate.48h after transfection, digest and collect5×104well grown cells in every group, and extract overall RNA according to Trizol reagent instruction. Use DU-800ultraviolet spectrophotometer to test concentration and purity of overall RNA. According to directions of RT-PCR kit TaKaRa RNA PCR kit(AMV)Ver3.0, reverse transcrip-tion (RT) reaction to synthesize cDNA. Then conduct PCR reaction. After the PCR reaction has finished, conduct agarose gel electrophoresis test to reaction product at4℃. Use GS-800gel imaging analyzer to conduct specimen scanning, and use Doiphin1D software to conduct semi-quantitative analysis. The expression intensity of survivin gene mRNA is expressed through optical density of survivin products (A) and optical density of internal controlδβ-actin products ratio. Inhibition rate of survivin gene mRNA expression=(1-survivin expression intensity of experimental group/survivin expression intensity of blank control group)×100%.2.8TUNEL method testing BIU-87cell apoptosis48h after transfection, collect1×106cells, prepare cell climb pieces and use PBS to wash them three times. Then according to directions of TUNEL kit, conduct to operate that. Judgment of result:Observed under a microscope, if nucleus is brown granular coloring, that is apoptosis. Under a microscope, select random five field of vision (×400), every of which is courted100cells, and court the percentage that overall apoptosis makes up. It is apoptosis index (AI)。2.9Western Blotting method testing the expression intensity of Survivin protein72h after transfection, digest and collect almost2×106cells separately every group in1.5ml centrifuge tube, put into Ice precooling forl×PBS lml, wash twice and centrifuge1min. Abandon to supernatant, putting into cell protein cracking fluid of5times in the volume, oscillation blending and ice-bath with30min.14000rpm,4℃centrifugalization of15min. Absorb supernatant into a precooling EP tube, add to2×SDS loading buffer, blending based on1:4, heat at100℃for5min, cool that, and centrifuge at high speed for1min. Make total protein100μg after degeneration to be gel electrophoresis through10%SDS-PAGE. Then conduct to transfer tank for electrophoresis to transfer protein to the nitrocellulose membrane. Thus, put the nitrocellulose membrane in a plate having block buffers5%of skim milk for2h. Use TBST1:400(0.2μg/μl) to dilute primary antibodies,put that into a hermetic bag, place nitrocellulose membrane into the bag and shake gently overnight at4℃. Then use TBST1:1000to dilutesecond antibodies, put that into a hermetic bag. Select Standard A and standard B with optimum in ECL kit to mix with equal volume for1min, and make nitrocellulose membrane protein face down to contact mixed liquorfully for5min. Use GS-800gel imaging analyzer to take photo for the firm, save the image and use Quantity One software to analysis banding pixels, showing the relative amount of protein expression=optical density value of target protein/internal control optical density value.2.10Statistics processingUse SPSS10.0software to make experimental data to be statistics processing. Data of every group is expressed based on x±S, and one-way anova and X2are used to analysis among groups. If p<0.05, differences among groups has statistical significance.Results:1. Concentration and purity of recombinant plasmid DNAMeasure the concentration and purity of plasmid DNA get by amplification and extraction through ultraviolet spectrophotometer. The results are1.208±0.0045μ,g/μl and1.89±0.034。The result shows:the OD260/OD280ratio of each recombinant plasmid DNA is all between1.8and1.9, each recombinant plasmid DNA is showed to be pure. In addition, computing DNA of each recombinant plasmid in0.8～1.3μg/μl is also meet the requirement of cell transfection experiment.2. Character of siRNA-DMNTEM image of siRNA-CCMN showed that its shape was round. The nanoparticles joining with each other and had an average particle size of about10～12nm. The effective diameter and polydispersity of siRNA-CCMN were94.0nm and0.34, respectively. The saturation magnetization was0.18emu/g, the remanent magnetization and coercive field determination were0.11emu/g and2.42, respectively. The atomic absorption photometer detected ferriion (Fe) concentration was340μmol/L.3. The transfection efficiency of siRNA-CCMNAfter siRNA-CCMN has transfected BIU-87cells for4-6h, under fluorescence microscope, we can see more cells having green fluorescence. Transfecting efficiency is about60%in SiRNA-DMN+PEMFs group, and the control group cell without transfecting does not have green fluorescence.4. The inhibiting effect of siRNA-CCMN on BIU-87cell proliferationThe inhibited proliferation rate of every group BIU-87cells detected with MTT method after transfected24h,48h,72h were:normal control group:1.23%,1.66%,1.61%; siRNA-CCMN group:20.54%,27.66%,24.65%; siRNA-CCMN+magnetic field group:33.62%,44.98%,36.57%,respectively. The results showed that: BIU-87cell proliferation level of siRNA-CCMN+PEMFs group was significantly lower than that of siRNA-CCMN group and normal control group (P<0.05). There were significant differences in BIU-87cell proliferation levels between siRNA-CCMN group and normal control group (P<0.05).5. The influence of siRNA-CCMN on expression level of Survivin mRNA in BIU-87cell5.1The results of total RNA extractionThe concentration and purity of total RNA extracted in every group of cell through nucleic acid protein detector are:normal control group:0.599,1.912, siRNA-CCMN group:0.663,1.902,0.625,1.895:siRNA-CCMN+PEMFs group. Each of the OD260/OD280ratio was between1.8-2, showed that the total RNA with higher purity of the extracted.5.2Survivin mRNA expression level after cell transient transfection for48h(1) After transient transfection of every group of cell for48h, gel electrophoresis results for using semiquantitative RT-PCR to test cell Survivin mRNA expression level is shown in figure.As shown in figure, we cannot see additional nonspecific banding appearing. The first, second and third lane are separately siRNA-CCMN, control group、siRNA-CCMN+PEMFs group, withp-actin amplification banding it refers to. Every group’s internal control β-actin amplification banding’s luminance in the area of587bp is equal, with expression level of no significant difference; while for the Survivin amplification banding at311bp, the banding luminance of siRNA-CCMN+PEMFs group is significantly weaker than another two groups. After transient transfection of every group of cell for48h, using semiquantitative TR-PCR to test cell Survivin mRNA expression relative level are:Normal control group:0.773, siRNA-CCMN:0.545, inhibition rate was32.62%, the siRNA-CCMN+PEMFs group:0.379, the inhibition rate is50.97%. The results show that, the expression level of Survivin mRNA in siRNA-CCMN+PEMFs group was significantly reduced than normal control group, and siRNA-CCMN group, the difference was statistically significant (P<0.05), the inhibition rate was50.97%. There was also a significant difference between siRNA-CCMN group and normal control group, the expression level (P<0.05), the inhibition rate was32.62%.6. The influence of siRNA on BIU-87cell apoptotic rateEvery group’s apoptosis index value (AI) is:normal control group:2.84, siRNA-CCMN:13.46, siRNA-CCMN+PEMFs:28.40. The results show that the apoptotic rate of BIU-87cells in the siRNA-CCMN+PEMFs group was significantly higher than that in normal control group and siRNA-CCMN group, the difference was statistically significant (P<0.05); siRNA-CCMN group was significantly higher than that in the normal control group (P<0.05).7. The influence of siRNA-CCMN on BIU-87cell’s Survivin protein expression7.1Histogram of Western Blotting method testing Survivin protein expression is shown, the banding gray of siRNA-CCMN+PEMFs group distinctly becomes shallow, which is weaker than control group and siRNA-CCMN group. It prompts that Survivin’s protein expression is restrained.7.2Western Blotting method testing Survivin protein’s expression strength. The result are:Normal control group:0.874, siRNA-CCMN:0.475, siRNA-CCMN+PEMFs:0.253. The results show that, the intensity of Survivin protein expression in BIU-87cells in siRNA-CCMN+PEMFs group is significantly lower than that of normal control group and siRNA-CCMN group, the difference was statistically significant (P<0.05). The siRNA-CCMN group were significantly lower than that of normal control group (P<0.05)..Conclusion:1. This study used siRNA recombinant plasmid which specificly targeting survivin (siRNA-survivin) as a loaded gene therapeutic molecule in the present study. CCMN produced by us was used as a gene carrier, respectively in vivo. The effection of magnetic siRNA-CCMN which was transfected in BIU-87cells on silencing survivin gene expression of human bladder cancer cells and inducing human bladder cancer cells apoptosis in combination with external magnetic fields were evaluated. 2. This study used the method of chemical coprecipitation to instruct CCMN. The method of chemical coprecipitation was simple and the preparation conditions were easily controlled. The CCMN had satisfactory compatibility, degradation and non-immunoantigenicity properties and possessed good hydrophilic property, superparamagnetism, especial nanometer effect and better single dispersion. The active groups of high polymer of CCMN could conjugate with bioactive molecules, such as plasmid by chemical modification. The compounds could enter intra-cellular by the cell taking in and succeed in transferring the GFP gene into human bladder cancer cells BIU-87. The CCMN after chemical modification could conjugate stably with pSilencerl.0-siRNA-survivin by oxidation-reduction reaction. The siRNA-CCMN conjugates could effective inhibite proliferation of BIU-87cells and induce apoptosis. The effects of combination with external magnetic fields on killing BIU-87cells were enhaced. Compared with existing carriers, CCMN have safety, specificity and feasibility which provided the experimental data for the feasibility of CCMN as gene drug nanometer carrier and established the foundation for the magnetic targeted treatment of tumor.3. The experimental results show, siRNA-CCMN targeting Survivin after it successfully transfected with human bladder cancer cell line BIU-87cells can significantly down-regulated mRNA transcription and protein expression level of Survivin gene mRNA, induced BIU-87cell apoptosis, inhibited cell proliferation. With a constant external magnetic field, the effects of siRNA-CCMN were strengthened. It provides a possible new way and experimental basis for magnetic targeting gene target to bladder cancer treatment. |
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