| Background and ObjectionNasopharyngeal carcinoma is one of the most common malignant tumors, with80%of incidents occurring in China. Given its particular anatomical location, Nasopharyngeal cancer is primarily treated through radiation. In recent years—ith the development of diagnostic techniques, the improvement of radiotherapy technology, and the application of effective treatment—he treatment of nasopharyngeal cancer has improved significantly; however, the five-year survival rate is still around70-80%. If gene therapy is used to improve the radiosensitivity of nasopharyngeal carcinoma cells while killing cancer cells at the same time, a better recovery period can be expected.In recent years, with the progress and development of molecular biology, tumor immunology, and other fields related to gene expression, cancer gene therapy has become possible. Gene therapy is now one of the most important means of biological treatment methods aside from surgery, radiotherapy, chemotherapy, and immunotherapy. Among the large number of gene therapies, suicide gene therapy is the most impressive; however the clinical efficacy is inadequate. In recent years, scholars have attempted to use radiation to achieve the transfer of the spatial regulation of in vivo genes, to improve the efficiency of gene targeting expression, and to increase the sensitivity of tumors to radiation. Through gene therapy, encouraging results can be expected.RNA interference （RNA interference, RNAi） is a highly conservative evolutionary process by double-stranded RNA （double-stranded RNA,dsRNA）-induced homologous mRNA degradation of highly specific phenomenon. There have been dramatic breakthroughs in the research of RNAi in recent years:it has placed top ten in Science magazine’s list of global sciencfic progress in2001, and has ranked first for global scientific progress in2002. Specific genes can be removed by using RNAi, so the technology has been widely used to explore gene function for gene therapy of infectious diseases and malignant tumors. RNAi is an efficient sequence-specific gene knockout technology in the rapidly developing field of infe’ctious diseases and cancer gene therapy.The proto-oncogene, Pokemon （zbtb7genes） is located on human chromosome19p13.3region, with a full-length of21600bp. The mRNA length of the Pokemon gene is4456bp and consists of2exons which contain an open reading frame encoded with a Pokemon based on protein polypeptide with584amino acids, and a molecular weight of61.5kD. Studies have shown that Pokemon may have a carcinogenic effect due to inhibition of the transcription of cancer suppressor gene ARF. The proto-oncogene Pokemon has been found highly expressed in some human tumors such as lymphoma, breast cancer, lung cancer, colon cancer, prostate cancer, and bladder cancer. Pokemon gene is highly expressed in nasopharyngeal carcinoma cells. When genomic DNA damage occurs, p53, which is the Pokemon gene effector becomes involved in the repair of DNA. Should the repair fail, p53will activate programmed cell death, inducing apoptosis. Studies show that p53can inhibit tumor cell growth and increase the rate of apoptosis in tumor cells. Pokemon is essential to the formation of cancer. It is critical for Pokemon to be present in order for other oncogenes to cause cancer. No other cancer genes have a similar functionality.Therefore, this project uses Pokemon gene silencing of RNAi （RNA interference, RNA interference） technology to restructure the siRNA fragment of the silenced Pokemon gene and wild-type p53gene into a defective virus; after constructing a recombinant virus, expose the recombinant virus nasopharyngeal carcinoma cells to radiation therapy. Depending on the gene expression of Pokemon, p53, and P14ARF before and after the detection treatment of mRNA and protein levels, we can analyze whether there is improved sensitivity of the nasopharyngeal carcinoma cells to radiotherapy.Methods and materials1. Cultivation of NPC cell linesNPC cell lines CNE-2was maintained in RMPI-1640medium which was supplemented with10%heated inactivated fetal calf serum, incubator conditions:5%CO2,37°c temperature the cell line were digested by condition.2.Synthesis of siRNA fragment argeting the Pokemon gene Based on the Pokemon gene published on NCBI GenBank （NM015898） full length sequence, follow the principle of siRNA design, using online software filter out3series referred to the Rui Bo bio-synthesis technology, select on the GenBank sequences with BLAST than on, prove that the selected sequence has no homology with other genes. Experiments as well as to GACATCATAGGTCGCATGC as a negative control group of interference sequences, the sequence is not linked to any human gene sequence homology.3. fragments of siRNA transfectionTaking the logarithmic growth phase cells seeded in6-well plates and96-well plates until the cells reached85%-90%of the fusion, the preparation steps in accordance with the LIPOFECTAMINE RNAIMAX kits and reagents ratio, by adding the transfection reagent and siRNA fragments four hours after adding fresh medium.4. Pokemon gene and protein expression detectionReverse transcriptase-polymerase chain reaction （RT-PCR） can detect the RNA expression of Pokemon gene. CNE-2cells were collected24hours after transfection, total RNA extracted with TRIzol method. Take the amplified product of5μl2%agarose gel electrophoresis. Western blots to detect the expression of Pokemon protein. CNE-2cells were collected24hours after transfection, cell lysate extraction of total cellular protein, the Bradford method detect the total protein concentration, taking50μg total protein electrophoresis. KODAK IMAGE STATION2000R imager can take pictures, and use SensiAnsys image analysis software electrophoretic bands of gray value, detection of the Pokemon gene and protein expression intensity. 5. Detection of the P14ARF, p53geneReverse transcriptase polymerase chain reaction （RT-PCR） is used to detect the expression of P14ARF, p53gene RNA expression. Collection24h CNE-2cells after transfected with total RNA using TRIzol extraction. Amplified products are preparated, after adding5μ.12%by agarose gel electrophoresis. Immunoblotting （Western blot） to detect expression of Pokemon. Collection CNE-2of24h cells after Transfection, cell lysis liquid extraction cell protein, detection of total protein concentration by Bradford, and then50μg protein electrophoresis are took. And then photographed on KODAK IMAGE STATION2000R imager, using SensiAnsys analysis of electrophoresis image analysis software with the grayscale values, P14ARF, the p53gene and protein expression strength test.6. Detection of cell proliferationCell proliferation was detected by MTT. Will go dye processing group and the blank control group is logarithmic growing of cell to3,000a/hole vaccination96hole Board, respectively at6, and12, and24, and48and the72h respectively abandoned to medium, each hole joined5g/L of MTT solution continues to training4H, abandoned to MTT, all hole joined150μ1of DMSO, at room temperature Xia shocks10minutes, in enzyme standard instrument A490nm Department determination all hole of sucking photometric value （-a value）, draws cell of growth curve, and calculation cell of growth suppression rate7.Colony formation assay:For colony formation assay,200cells seeded into6-well plate.being cultured for110-14days, surviving colonies （>50cells/colony） experiments were counted with1%crystal violet staining. Triplicate independent were performed8. Transwell migration assay:For cell migration assay, cells were starved with serum free medium for24hrs before the assay. Cells （5X104） were suspended in0.5ml serum-free medium and loaded on the upper compartment of transwell chamber.The lower compartment was filled with complete medium as chemoattractant. After12hrs, migration cells were fixed, stained, and counted under a microscope.Triplicate independent experiments were done9. Detection of cell apoptosisFlow cytometric detection of cell apoptosis. Collection logarithmic growing CNE-2cell1x106, in accordance with reagents box steps1000rpm centrifugal10minutes, joined pre-cold PBS washing3times, will cell heavy hanging at200μl of Binding Buffer, joined10μ1Annexin V-FITC, mixed uniform, avoidance light at room temperature reaction15minutes, before takiing the sample to the machine about5minute,10μl iodine of pyridine and300μl of Binding Buffer are joined.Later the samples are detected within1hour.To each group of apoptosis in cells as a percentage of total number of cells apoptosis rate calculation10.Construction and identification of recombinant adenovirus①Construction of recombinant plasmid pAdTrack-CMV-PokemonSynthesis of pre-Pokemon transcription template just chain and the antisense strand of DNA Oligo sequence, after anneal to form DNA double-stranded parallel with the pAdTrack carrier Kpn I and double enzyme Xho I cuts, connections to build a carrier pAdTrack-CMV-Pokemon②Construction of recombinant adenovirus plasmid pAd-si-PokemonThe correct identification sequence containing green fluorescent protein（GFP） mark pAdTrack-CMV-Pokemon Kpn I cleavage of plasmid after linearization, skeleton and adenovirus plasmid transformation Electroporation total pAdEasy-1. pAd-si-Pokemon can be identified by the correct clone name. Preparation of adenovirus vector as negative control group③Gland packing and titer of virus detectionRecombinant plasmid are preparated by using LIPOFECTAMINE RNAIMAX5g （Invitrogene） methods of transfection293A cell virus in packaging. After amplification and purification by experimental determination of virus titer using virus Plaque formation④Identification of recombinant adenovirus expressionAfter the purification of viruses to MOI （multiplicity of infection） to10293A cell tissue infection, infection with the48h, cells in a Trizol extraction of RNA, reverse transcriptase PCR amplification using primers after. On the use of immunofluorescence and Western Blotting assay in clear expression of Pokemon11. Function in vitro experiment①the detection of cell proliferationMTT method dets proliferation of CNE-2cell. Will is logarithmic growing of go dye group and the control group cell vaccination96hole Board （3,000a/hole）, select6h, and12h, and24h, and48h and the72h time points abandoned to medium, each hole joined MTT solution （5g/L） continues to training4h, abandoned to MTT solution, each hole joined DMSO solution150μ1, shocked at room temperature10minutes, enzyme standard instrument A490nm Department determination each hole of sucking photometric photometric value （-a value）, and draws CNE-2cell growth graphs②Colony formation assay:For colony formation assay,200cells seeded into6-well plate.being cultured for110-14days, surviving colonies （>50cells/colony） experiments were counted with1%crystal violet staining. Triplicate independent were performed③Transwell migration assay:For cell migration assay, cells were starved with serum free medium for24hrs before the assay. Cells （5X104） were suspended in0.5ml-serum-free medium and loaded on the upper compartment of transwell chamber.The lower compartment was filled with complete medium as chemoattractant. After12hrs, migration cells were fixed, stained, and counted under a microscope.Triplicate independent experiments were done④The detection of cell apoptosis:Flow cytometric detection of cell apoptosis. Collection logarithmic growing CNE-2cell1×106, in accordance with reagents box steps1000rpm centrifugal10minutes, joined pre-cold PBS washing3times, will cell heavy hanging at200μ1of Binding Buffer, joined10μ1Annexin V-FITC, mixed uniform, avoidance light at room temperature reaction15minutes, before taking the sample to the machine about5minutes,10μ1iodine of pyridine, and300μ1of Binding Buffer joined.Later the sample are detected within1h. To each group of apoptosis in cells as a percentage of total number of cells apoptosis rate calculation12. Detection of the CNE-2cell’s radiosensitivity using clone assayUsing Siemens line Accelerator respectively using with0, and2, and4, and6, and8, and10Gy of dose of6MvX-Ray irradiation all hole CNE-2cell, and will CNE-2pancreatic enzyme digestive made single cell hanging liquid, in accordance with dose size respectively will1×101, and1×102, and2×103, and1×104, and1×105, and1×106a cell vaccination in60mm training dish in the, each irradiation dose are set3a parallel samples. With Siemens Accelerator irradiation （radiation types and energies:6MV x-ray, beam20cmx20cm,SSD=100cm, frame angle of180degrees, equivalent film under1.5cm）. Then dish in the incubator at37°c,5%CO2training2weeks. After2weeks the Group CNE-2using ethanol fixation95%, count after the Giemsa staining （>50cells count as a surviving colony）, calculate survival score （surviving Frac-tion, SF） and clone formed rate. Click the target mathematical models （single-hit multitarget model） calculates the value of Do （average lethal dose） and Dq （prospective domain dose）. GraphPad Prism4.0cell survival curve fitting software13.Statistical analysis:All analyses were carried out using the SPSS13.0software package.A P value less than0.05was considered statistically significant.All detas are expressed as mean±SD and analyzed by one-way ANOVA;x2test was used, Results1.Three siRNA series were transfected to CNE-2, the density of electrophoresis of PCR products were:inhibition rate of Si-h-ZBTB7A001group was47.51%, inhibition rate of Si-h-ZBTB7A002group was74.88%, inhibition rate of Si-h-ZBTB7A003group was44.52%,expression inhibition rate differences are significant. Comparing three groups, Si-h-ZBTB7A002group had better inhibition effect.2.When siRNA were transfected, expression of P14ARF was higher than the negative control group and the blank group.（P<0.05） expression of P14ARF was higher than the negative control group and the blank group.（P<0.05）, expression of p53was higher than the negative control group and the blank group.（P<0.05）, expression of P53protein than the negative control group and the space group-expression3.When Sirna were transfected. MTT method by measuring of CNE-2cell interference before and after growth curve displayed RNAi cell proliferation speed slowed down, cell growth was suppression （P<0.05）; flat clone experimental displayed, RNAi CNE-2cell formed of cell clone number （47.33±1.15） significantly is less than negative controlled group （83.67±1.15） and blank Group （84.33±1.53）（P<0.05）;after12hours, CNE-2cell penetrating matrix rubber of cell number （36.33±1.53） more negative controlled group （94.67±2.52） and blank Group （94.33±3.51） significantly reduced. Detection of cell apoptosis by flow cytometry, RNAi CNE-2cells apoptosis rate （23.533±0.016） than the negative control group （9.187±0.002） and the space group （9.227±0.003） obviously rise.4.The recombinant adenovirus plasmid pAd-si-Pokemon were amplificated and purificated.The titer of virus is4.6X10VP/ml.By RT-PCR amplification of a gene fragment of the same size and purposes.Ultrafiltration cell supernatant after electrophoretic transfer color display, inoculating recombinant adenovirus pAd-si-Pokemon293cells supernatant on left and right there is a clear, consistent with the Pokemon of the expected effects of protein size. 5.The effection to the function of cell biology. MTT method by measuring of CNE-2cell interference before and after growth curve displayed RNAi cell proliferation speed slow, cell growth was suppression （P<0.05）; flat clone experimental displayed, pAd-si-Pokemon CNE-2cell formed of cell clone number （54.00±2.65）、pAd-p53（40.33±1.53）significantly is less than negative control group （83.33±2.08）（P<0.05）; deck12hours, pAd-si-Pokemon （45.00±1.00）. pAd-p53（33.00±2.64） CNE-2cell penetrating matrix rubber of cell number more negative control group （82.67±1.53） reduced. Detection of cell apoptosis by flow cytometry, apoptosis in CNE-2cells pAd-si-Pokemon （22.6±0.005）、 pAd-p53（35.6±0.008） than the negative control group （9.2±0.003） is significantly increased.6. Do values before and after the CNE-2cells were treated with adenovirus with pAd-of Pokemon, with pAd-53infection before and after are as follows:1.989persons0.017,2.309persons0.477;, Dq values were2.363persons0.047,2.773persons0.031. With pAd-of Pokemon group and the group with pAd-53radiosensitization than SER values were1.25,1.16.Conclusion1.The confirm siRNA of human Pokemon gene was synthesized and insert in CNE-2cell can silence Pokemon gene expression, It can prevent CNE-2cell proliferation, inhibite of cell migration and enhance cell apoptosis.2.Recombinant adenovirus was packed and amplified and high purity, and then high transfection efficiency was made.3.Recombinant adenovirus can prevent CNE-2cell proliferation, inhibite of cell migration and enhance cell apoptosis.4.Recombinant adenovirus can increase CNE-2cells radiation sensitivity,it’s effect is more apparent Comparing with simple p53import. |
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