Construction Of A Stable Human Neuroblastomas Cell Line By DDX1 Gene Knockdown And Its Effects On Biological Behavior Of Neuroblastomas

Background:Neuroblastoma is one of the childhood refractory malignant solid tumors, and with poorer long-term outcomes. Therefore, it is of great significance to carry out the research on neuroblastoma of children. Previous studies have shown that MYCN gene mutation is closely related to the occurrence, development and prognosis of neuroblastoma. Recent studies have found that DDX1(Dead box 1) gene is co amplified with MYCN gene in neuroblastoma, suggesting that DDX1 gene expression may be of great significance in the occurrence and development of neuroblastoma. In this study, we explore and elucidate the relationship between DDX1 gene and the biological behavior of the tumor cells and the drug resistance of the tumor cells based on the high expression of DDX1 gene in neuroblastoma. To provide the experimental basis for further studies of neuroblastoma pathogenesis and DDX1 gene, to explore the expression of DDX1 gene may be used as neuroblastoma clinical prognostic analysis of potential value factors or therapeutic targets.Part ? The expression of DDX1 in neuroblastomaObjective:Investigate the expression of DDX1 in tumor tissue and adjacent tumor tissues of neuroblastoma clinical samples, and explore the relationship between DDX1 and neuroblastoma.Methods: 1. Take 5 cases of patients with neuroblastoma tumor tissue and tumor adjacent tissue(peritumoral tissue at least 2 cm from the tumor tissue of non-tumor tissue), fixed in 4% formaldehyde solution. The above samples were embedded in paraffin, sliced, with DDX1 antibodies do immunohistochemistry, then the expression of DDX1 beside tumor tissue and tumor tissue was observed.2. In this study, semi-quantitative immunohistochemical standard.A:degree of staining:non-staining recorded as 0 point, a light color record as 1 point, coloring modest counts as 2 points, the color depth is recorded as 3 points; B:positive cell ratio:positive cell ratio <10% recorded as 0 point, the proportion of positive cells between 10%-30% recorded as 1 point, the proportion of positive cells between 31%-60% recorded as 2 points, the proportion of positive cells> 61% of the record 3 points; the final score is(A+ B) / 2, if the final score was 0-1 then recorded as "-", 1.1-2.0 noted as "+", 2.1-3.0 denoted as "++", 3.1-5.0 recorded as "+++."Results: Next to the 5 cases the tumor tissues immunohistochemistry final score are "-" and the average is 0.5, and the tumor tissues' DDX1 expression were positive and average is 1.8. Therefore, immunohistochemical analysis showed 5 cases of neuroblastoma tissues' expression of DDX1 gene was significantly higher than the corresponding adjacent tumor tissue. This suggests DDX1 may serve as an oncogene, play a catalytic role in the development of neuroblastoma, the support provided clinical evidence for our follow-up study.Conclusions:DDX1 highly expressed in neuroblastoma tumor tissues, which may contribute to the development of neuroblastoma.Part ? Construction of a stable human neuroblastomas cell line by DDX1 gene knockdownObjective:Establish DDX1 gene knockdown SK-N-BE(2) cell line, to prepare for the follow-up study of the function of DDX1 gene on neuroblastoma.Methods:1. Design sh targets, building p Lenti6.3-EGFP-sh DDX1 interference lentiviral vector, and packaging lentiviral by 293 T cell.2. Using packaged virus to infect SK-N-BE(2) cells, successfully infected cells stably express GFP green fluorescence.3. Using PCR and Western blot to detect the efficiency of the knockdown targets, and select the most efficient target for subsequent experiments.Results:PCR and Western blot experiments have demonstrated the highest efficiency knockdown target is the second one, reaching more than 60%. So, we have successfully established a DDX1 knockdown SK-N-BE(2) cell line to continue the study of the function of DDX1 in neuroblastoma.Conclusions:Infection by lentiviral, we got stabled DDX1 knockdown SK-N-BE(2) cell lines successfully.Part ? The effect of DDX1 on SK-N-BE(2) proliferation, apoptosis and cell cycleObjective:Study the difference between SK-N-BE(2) / Blank, SK-N-BE(2) / sh V and SK-N-BE(2) / sh DDX1 in cell cycle, apoptosis and proliferation, to determine the effect of DDX1 in neuroblastoma cell cycle, apoptosis and proliferation.Methods:1. Seeded SK-N-BE(2) / Blank, SK-N-BE(2) / sh V and SK-N-BE(2) / sh DDX1 cells in 96-well plates,which growth in good condition and in the logarithmic growth phase, each well was inoculated to 2 000 cells, and set up 5 sub holes. Using CCK-8 kit to detect the cell numbers in 12 h, 24 h, 36 h, 48 h, and take the average, with time(hour) as abscissa, OD 450 nm reading as vertical axis, draw the growth curves of these 3 cells.To investigate the effects of DDX1 for the proliferation of neuroblastoma cells.2. Seeded SK-N-BE(2) / Blank, SK-N-BE(2) / sh V and SK-N-BE(2) / sh DDX1 cells in 6-well plates,which growth in good condition and in the logarithmic growth phase, each well was inoculated to 3×105 cells. After 24 h, using flow cytometry(PI staining) to detect the apoptosis of SK-N-BE(2)/Blank?SK-N-BE(2)/sh V and SK-N-BE(2)/sh DDX1 cell lines. To observe the effects of DDX1 for the apoptosis of neuroblastoma cells.3. Seeded SK-N-BE(2) / Blank, SK-N-BE(2) / sh V and SK-N-BE(2) / sh DDX1 cells in 6-well plates,which growth in good condition and in the logarithmic growth phase, each well was inoculated to 3×105 cells. After 24 h, using flow cytometry(PI staining) to detect the proportion in G1?S?M?G2 stage of SK-N-BE(2)/Blank?SK-N-BE(2)/sh V and SK-N-BE(2)/sh DDX1 cell lines. To observe the effects of DDX1 for the cell cycle of neuroblastoma cells.Results:1. SK-N-BE(2) / sh DDX1 cell proliferation was significantly weaker than SK-N-BE(2) / Blank and SK-N-BE(2) / sh V cells, that is to say, DDX1 knockdown reduce the cell proliferation of neuroblastoma.2. SK-N-BE(2) / shDDX1 apoptosis was significantly higher than the number of SK-N-BE(2) / Blank and SK-N-BE(2) / sh V cells, which means that DDX1 knockdown increase tumor cell apoptosis of neuroblastoma.3. Compared with SK-N-BE(2)/Blank and SK-N-BE(2)/sh V cells, the cell cycle of SK-N-BE(2) / sh DDX1 cells arrest, and proliferation is affected.Conclusions:After DDX1 expression is restrained, neuroblastoma cell cycle affected, cell apoptosis increased, and cell proliferation reduced.Part ? The effect of DDX1 on SK-N-BE(2) cell migration, invasion and resistance capabilityObjective:To discuss the effect of DDX1 on SK-N-BE(2) cell migration, invasion and resistance capability.Methods:1. Serum-free DMEM medium 5: 1 dilution of Matrigel, take the dilution 40?L spread evenly in Transwell chambers(BD Company) of the upper chamber, 37 for ?30 min spare. The pretreated cells was digested cell suspension 5×108 / L, and the upper chamber of cell suspension per well was added 400 ?L, 600 ?L DMEM medium was added at room, and another lower chamber with 10% fetal calf serum 50 ?L, 36 h after, with a cotton swab and wipe the interior cell Matrigel, 4% paraformaldehyde 10 min, crystal violet staining, PBS wash away the excess dye, random Ly selected field of view camera count, the above experiment was repeated three times random Ly.2. The pretreated cells was digested cell suspension 5 × 108 / L, and the upperchamber of cell suspension per well was added 400 ?L, 600 ?L DMEM medium was added at room, and another lower chamber with 10% fetal calf serum 50 ?L, 36 h after the lower chamber migrating cells with 4% paraformaldehyde 10 min, crystal violet staining, PBS wash away the excess dye, random Ly selected field of view camera count, the above experiment was repeated three times random Ly.3. Seeded SK-N-BE(2) / Blank, SK-N-BE(2) / sh V and SK-N-BE(2) / sh DDX1 cells in 96-well plates,which growth in good condition and in the logarithmic growth phase, each well was inoculated to 6 000 cells, and set up 5 sub holes. Drugs were add into cell culture plate, make a final concentration of doxorubicin as 1.0 ?g / m L, final concentration of cisplatin as 2.5 ?g / m L. Using CCK-8 kit to detect the number of cells 24 h after drug treatment.Results:1. Cell invasion of SK-N-BE(2) / sh DDX1 was significantly weaker than SK-N-BE(2) / Blank and SK-N-BE(2) / sh V cells, that is to say, DDX1 knockdown reduce the cell invasion of neuroblastoma.2. Cell migration of SK-N-BE(2) / sh DDX1 was significantly weaker thanSK-N-BE(2) / Blank and SK-N-BE(2) / sh V cells, that is to say, DDX1 knockdownreduce the cell migration of neuroblastoma.3. 24 h after doxorubicin and cisplatin treatment, the number of living cells of SK-N-BE(2) / sh DDX1 cell line was significantly lower than the SK-N-BE(2) / Blank and SK-N-BE(2) / sh V cell line, that is to say, knockdown DDX1 could increase the doxorubicin and cisplatin drug sensitivity of SK-N-BE(2) cells, and decrease the resistant ability of neuroblastoma cells.Conclusions:Knockdown DDX1 could decrease the cell invasion, migration and resistance capability of neuroblastoma cells.Full text induction:1. Subject research basis: According to the research of children with neuroblastoma annual incidence rate of only 0.3 to 0.5/10 million, but the project is still at the early stage of the research has accumulated to 5 cases of newly diagnosed pediatric cases, verification of neuroblastoma does exist high expression of DDX1, so this research obtains the theoretical and experimental basis as fully as possible.2. Preparation of experimental conditions: the successful establishment of DDX1 gene knocked down of SK-N-BE(2) cell line, in order to carry out the research of DDX1 gene on the function of neuroblastoma, and provide effective experimental conditions.3. The research results are summarized: By comparing the results of cell lines suggest that knocked down of DDX1 can make the neuroblastoma cell proliferation, proliferation ability and invasion or migration abilities diminished, and could increase the sensitivity of the tumor cells to adriamycin and cisplatin chemotherapy. The expression of DDX1 gene was closely related to the occurrence and development of neuroblastoma, as well as the clinical prognosis. In order to provide the experimental basis for the further research of DDX1 gene, the potential value of DDX1 gene expression may be used as a prognostic factor for the diagnosis and treatment of neuroblastoma.