Establishment Of Humen Prostatic Carcinoma Cell Lines With Stable Expression Of Exogenous Gene Via Lentiviral Transduction

BACKGROUNDCancers are great threats to human health, of which prostate cancer(PCa) is one of the most common malignant tumor of the adult males. In the United States, the incidence of PCa ranks the first of cancers in men.In our country, its incidence is increasing every year. In the study on prostate cancer treatment, there are some challenges:the difficulty of diagnosis and the lack of effective and reliable treatment on advanced prostate cancers, especially androgen-independent prostate cancers. The strategy of gene therapy on cancers is transferring some exogenous genes into cancer cells to activate the intracellular apoptotic pathway and thus inducing apoptosis of tumor cells, finally to eliminate or shrink the tumors. Gene therapy has a number of advantages:stronger targeting effect, efficiency for advanced tumors, and systemic treatment. However, there are also some difficulties:Continuing inducing apoptosis, suitable transferring methods for exogenous gene and the lack of detection methodsMitogen-activated protein kinase (MAPK) is an important eukaryotic cell signaling pathway, and p38as one of the family, involving in a variety of biological processes such as stress response, proliferation, and apoptosis in cells. In the development of prostate cancer, the current study found that a variety of therapeutic genes induced apoptosis of prostate cancer through the p38MAPK pathway. So we designed transferring the exogenous p38MAPK genes into the cell and making the cells stably overexpressing p38MAPK protein, observing the effects on cell growth and proliferation. Our study also provides a tool for future therapeutic research in the p38pathway.How to transfer exogenous gene into host cells and make stable expression mostly depends the choices of exogenous gene carrying vector. Now transgene carriers in gene therapy can be roughly divided into two categories:viral vectors and non-viral vectors. Commonly used methods of non-viral vectors, includes the electronic transfer method, calcium phosphate precipitation method and liposome method, which can achieve the effect of transient expression. But transient expression of exogenous gene gradually decreased with cell growth and reproduction, and can not be transmitted to daughter cells. Viral vectors include adenovirus vector, adeno-associated viral vectors and lentiviral vectors. Lentiviral vector which derived from the retrovirus, has the following characteristics:First, lentiviral vectors has no limitation of the cell types:either dividing cell or not; Second, efficiently integration into the host cell genome achieves long-term stable expression and the ability to passage to the daughter cells; Third,the vector does not cause the host immune response; Fourth, the removal of the key points of the original viral replication improves bio-security.For the study of prostate cancer in animal experiments, how to monitor the process of development of the tumor cells or tumor tissue become problems. Common methods include fluorescent protein and bioluminescence. Fluorescent protein as a reporter gene, with a wide range of applications in biology, became the mainstream technology of the cellular and molecular imaging. The far-red fluorescent protein (RFP) becomes the new hot spot among bioluminescence proteins because of longer the excitation and emission wavelengths and better intracellular imaging background. Luciferase is enzymes which catalyze specific substrate to produce bioluminescence in the presence of02and ATP. Collectively, the luminous intensity relates to the number of survival tumor cells and growth state. The most representative luciferase is the firefly luciferase (Flue), which could accurately locate the tumor foci, as a reporter gene providing a tumor early, rapid, accurate, and dynamic means of monitoring.In summary, this paper consists of two parts, first part is constructing a lentiviral vector containing the p38target genes and establishing a prostate cell lines with stable, sustained expression of p38protein by using lentiviral vector technology. The second part is the establishing of prostate cancer stable cell lines with expression of dual report genes:red fluorescent protein-firefly luciferase (RFP-FLUC) also by lentiviral vector technology.Part1:Construction of a recombinant lentiviral vector of p38MAPK and establishment of a human prostatic carcinoma stable cell line expressing foreign p38MAPK ObjectivesTo construct a p38MAPK recombinant lentiviral vector and package lentiviral virus in HEK293T cells. The DU145cells were transduced and a stable cell line EGFP/p38-DU145expressing foreign p38MAPK was established. We observed the effect on proliferation of DU145cells overexpressing of p38and provided tools for further research in the function of p38MAPK and p38MAPK pathway.MethodsConstruction and identification of lentiviral vectorThe plasmid pTYF-EFla-EGFP/p38was constructed by restriction enzyme diges tion, fragment recovering and connecting. The ligation mixture was transformed into E. coli DH5a, and then a number of monoclonals were picked and cultured in specific-resistance medium. For identification recombinant vector was double digested, taking pEGFP/p38plasmid as a control.Production and titration of lentiviral paticlesThe plasmids psPAX2, pMD2.G and lentiviral vector (pTYF-EF1α-EGFP vector as a control) were transfected into293T cells by Lipofectamin2000. Cell supernatants were collected after48h, centrifugalized at3000r/min,4℃for15min, and then saved at-80℃. The end point dilution method was used to measure the virus titer (TU/ml). The virus titer is calculated in this way:virus titer (TU/ml)=number of positive cells×dilution factor.Lentiviral transduction of the target cell lineTo transduce the cells, the total volume of300μl medium was added, which contained100μl of the packaged viral supernatant (MOI=10) and polybrene (8μg/ml). After24h500μl medium without virus solution were replaced. The fluorescence emission of cells was observed72h after transduction.Estabolishent of stably transduced cell linesLimited dilution was used to establish monoclonal cell lines.100μl of cell suspension containing single cells were added to each well in96-well plates. Cells were conventional cultured for20days to select good positive clones with good growth state, and then picked for expanding culture.Detection of totel p38by Western blotting analysisA total of three cells:EGFP/p38-DU145cells, EGFP DU145cells, normal DU145cell were detected. RIPA was used to lysate cell and lysis supernatant was stored at-20℃, then detected by Western blotting with conventional methods.Observation of proliferationEGFP/p38-DU145cells, EGFP-in DU145cells and normal DU145cells were respectively digested. After cell counting, each well of24-well plates was planted with1×104cells. Each type of cell line was inoculated with four plates, and each plate was inoculated with24holes. Three holes in every plate were single-blind randomly selected and counted for every24h. The average numbers of cells per plate were calculated. After eight-day counting, the difference of cell numbers among the three cell lines was taken into statistical analysis and the growth curves were drawn.Statistical analysisThe repeated measures analysis using SPSS13.0statistical software was taken to compare the difference of growth speed and significant difference was confirmed if P<0.05.ResultsIdentification of lentiviral vectorTwo bands near1.7kb and5.5kb in1%agarose gel electrophoresis can be seen after SalI and Nhel double digestion for the lentiviral vector pTYF-EF1α-EGFP/p38. The control vector pEGFP/p38also received the expected results, confirming that the vector was successfully constructed. Production and titration of lentiviral paticles24h after transfection, almost all of293T cells were observed with high brightness of green fluorescence. Dilution end points were the105holes and106holes. Counting the two holes under a fluorescence microscope44and5-positive cells, respectively, were observed. The virus titer was computed in this way:(44×105+5×106)/2=4.7×106TU/ml.Lentiviral transduction of the target cell line72h after Lentiviral transduction, DU145cells was observed under the fluorescence microscope. Under bright-field the epithelial cells grew well. Under fluorescence field, some DU145cells integrating EGFP/p38fusion gene can emit green fluorescence, which distributed throughout the whole cell.Establishments of stably transduced cell linesA cell line selected by limited dilution grew as epithelial cells and did not differ significantly in morphology with normal untreated DU145cells. It grew in good condition with all cells emitting green fluorescence, which is more evenly distributed throughout the cell. After expanding culture and preservation, it was named as EGFP/p38-DU145.Western blotting analysisExpression of the internal reference of the three cells was basically at the same level. The three types of cells had similar bands near39kD, indicating that the endogenous p38protein expression levels did not differ significantly among the three groups of cells. At the same time EGFP/p38DU145cell line also had bands near60kD for exogenous EGFP/p38fusion protein with high expression levels.Observation of proliferationComparing to EGFP-DU145which received the exogenous EGFP gene in the same way and normal DU145cells, the growth of EGFP/p38-DU145cell line expressing exogenous p38were significantly depressed(P<0.05), indicating over-expression of p38MAPK inhibiting the proliferation of DU145cells. ConclusionsIn this experiment, we have successfully constructed a p38MAPK recombinant lentiviral vector and transduced DU145cells. Monoclonal was picked by limited dilution method, and then stable cell line with expression of exogenous p38was selected under fluorescence microscope. The expression of p38was identified by Western blotting, founding that expression of exogenous genes in DU145cells did not change morphology of cells significantly, but inhibiting its growth because of the over-expression of p38protein.Part2Establishment of a humen prostatic carcinoma cell line with stableexpression of dual reporter genes via lentiviral transductionObjectivesTo establish a human prostatic carcinoma cell line which could stably express dual reporter genes,firefly luciferase(Fluc) and far-red fluorescent protein(mKate), and observe the growth characteristics and luciferase luminescence properties in vitro and in vivo.MethodsProduction and titration of lentiviral paticlesLentiviral particles were produced by transient transfection of HEK293T cells with packaging plasmid psPAX2,envelope plasmid pMD2.G and lentiviral vector plenti-cmv-Fluc-2A-mKate-cgk-puro using Lipofectamin2000. Cell supernatants were collected after48h, centrifugalized at3000r/min,4℃for15min, and then saved at-80℃. The end point dilution method was used to measure the virus titer (TU/ml). The virus titer is calculated in this way:virus titer (TU/ml)=number of positive cells×dilution factor.Determination of the optimal puromycin concentration for DU145cellsDU145cell lines were routinely cultured and then added medium containing the concentration of puromycin at1ug/ml,2ug/ml,3ug/ml,4ug/ml,5ug/ml,6ug/ml. The medium containing the above concentration gradient was refreshed every2-3days and morphological changes were observed. Lentiviral transduction of DU145cellsTo transduce the DU145cells, total volume of300μl medium was added, which contained100μl of the packaged viral supernatant (MOI=6) and polybrene (8μg/ml). After24hours,500μ1medium without virus solution were replaced.72h after transduction the fluorescence emission of cells was observedEstablishments of stably transduced monoclonalComplete medium containing optimal concentration of puromycin was added to the transduced DU145cells and replaced every2-3days. The morphology and fluorescence of cells were observed daily until most of the non-resistant cell died. Then Limited dilution was used to establish monoclonal cell lines.100μl of cell suspension containing single cells were added to each well in96-well plates. The cellswere conventional cultured for20days to select good positive clones with good growth state, and then picked one and naming it Fluc-mKate-DU145,for expanding culture in medium containing puromycin.Assessment of cell proliferation and firefly luciferase activity in vitroFluc-mKate-DU145and normal DU145cells were respectively digested. After counting, each well of24-well plate was planted with1×104cells. Each type of cell line was inoculated with four plates, and each plate was inoculated with24holes. Three holes in every plate were single-blind randomly selected and counted for every24h. The average numbers of cells per plate were calculated. After eight-day counting, the difference of cell numbers among the three cell lines was taken into statistical analysis and the growth curves were drawn. Fluc-mKate-DU145cells were planted at1×104-8×104cells per hole in96-well plate. The light-emitting characteristics were measured in Lumina Imaging System. The relative photon number was compared with the number of cells.Establishment of subcutaneously transplanted tumors in nude miceA total of eight six-week-old male nude mice were housed in SPF animal room. They were randomly divided into experimental and control groups well marked. Each group included four mice. Fluc-mKate-DU145and normal DU145cells were digested and suspended in PBS, then injected intraperitoneal into the hip of the mice. Mice were sacrificed after2months for anatomical instruction and pathological examination.Assessment of tumorigenicity and firefly luciferase activity in vivoSince the xenografted tumors became palpable, the diameter, in two directions, was measured every other day using calipers and firefly luciferase activity in vivo was measured in Lumina Imaging System.Statistical analysisThe repeated measures analysis using SPSS13.0statistical software was taken to compare the difference of growth speed and significant difference was confirmed if P<0.05.The correlation between tumor and firefly luciferase activity in vitro or in vivo was compared.ResultsProduction and titration of lentiviral paticles24h after transfection, almost all of293T cells were observed with high brightness of red fluorescence. Dilution end point were the105hole and106hole. Counting the two holes under a fluorescence microscope28and3-positive cells, respectively, were observed. The virus titer was computed in this way:(28×105+3×106)/2=2.9×106TU/ml.Lentiviral transduction of the target cell line72h after lentiviral transduction, DU145cells was observed under the fluorescence microscope. Under bright-field, the epithelial cells grew well. Under fluorescence field, some DU145cells integrating Fluc-mKate gene can emit red fluorescence, which distributed throughout the whole cell.Establishments of stably transduced cell linesA cell line selected by limited dilution grew as epithelial cells and did not differ significantly in morphology with normal untreated DU145cells. It grew in good condition with all cells emitting red fluorescence, which is more evenly distributed throughout the cell. After expanding culture and preservation, it was named as Fluc-mKate-DU145.Assessment of cell proliferation and firefly luciferase activity in vitroComparing to normal DU145cells, the growth of Fluc-mKate-DU145were similar without significantly differences (P>0.1). The relationship between tumor and firefly luciferase activity in vitro was a linear correlation (p<0.05).Establishment of subcutaneously transplanted tumors in nude miceThe xenografted tumors became palpable after3weeks. When mice were sacrificed, there were no significant differences between the experimental group and control group at tumors tumor size and pathological structure.Assessment of cell proliferation and firefly luciferase activity in vivoSubcutaneous tumor volume increased gradually. There were no significant differences between the experimental group and control group at speed of tumor growth. The relationship between the luminous intensity of the tumor and the tumor volume was a nonlinear correlation (r=0.998,p<0.01).ConclusionsIn this study, we successfully constructed human prostate cancer DU145cell line with stable expression of dual reporter genes, Flue and mKate. In vitro observations showed their luciferase luminescence activity is linear correlated to the number of cells. The growth of Fluc-mKate-DU145and normal DU145cells was similar without significantly differences. About20days after subcutaneous injection of tumor cells in nude mice, the subcutaneous tumor became visible, without statistically significant differences in growth rate and pathological structure between experimental group and the control group.The fluorescent luminous intensity increased with the growth of the tumor, providing a good model for prostate cancer in animal experiments.