Effect Of Gene MAPK7 To Osteosarcoma Cell Proliferation And Migration

Objective: Osteosarcoma is the most common primary malignant bone tumor,which is one of malignant tumors having a high incidence in children and adolescents. It is commonly characterized by rapid growth and easy transfer, thus resulting in its high mortality. Therefore,to further explore the proliferation and invasion molecular biology mechanisms of osteosarcoma, is an urgent need to the new generation of cancer therapy targeting some molecular.Abnormal amplification of chromosome 17p11.2 region is the genetics characterize of many clinical osteosarcoma cases. Further research in this area suggests several potential tumor genesis genes including PMP22, TOP3 A and MAPK7 so on. It reported that PMP22 and TOP3A can regulate the proliferation of osteosarcoma. While some clinical explores indicates the gene MAPK7 to be a prognostic markers in osteosarcoma. But how the gene MAPK7 promotes the proliferation and metastasis in osteosarcoma is still unclear.In this paper, we will use osteosarcoma cell line SOSP-M as an in vitro model. We upregulate and knock down MAPK7 expression in cells by means of overexpressing and silencing MAPK7 gene. We use MTT cell proliferation assay, Wound scratch assay and matrigel invasion assay to detect the proliferation, migration and invasion of tumor cells. In addition, we use flow cytometry technology to detect cell apoptosis and cell cycle after MAPK7 overexpression and interference.Methods:1. Culture osteosarcoma cell line SOSP-M cells. Overexpress and knock down MAPK7 gene of cells as an in vitro model, detecting the role of MAPK7 gene in tumor proliferation,migration and invasion in cell level.2. Overexpression and knocking down of gene MAPK7: Custom PCDNA-MAPK7plasmids(1ug/ml) and siRNA-MAPK7(1ug/ml). Transfect PCDNA-MAPK7 plasmids into SOSP-M cells to overexpress gene MAPK7, transfect si RNA-MAPK7 into SOSP-M cells to knock down gene MAPK7. Use transfection reagent Lipofectamine2000 TMto transfect.3. Detection of MAPK7 protein expression levels : the cultured SOSP-M cells were randomly divided into five groups: the blank group: no transfection treatment, the overexpression control group: transfect cells with blank plasmid, overexpression group:transfect cells with PCDNA- MAPK7 plasmids, Scramble SiRNA group:transfect cells with Scramble Si RNA, knock down group: transfect cells with si RNA-MAPK7. Collect cells after transfected for 24 h. Total protein is extracted. Use western blot analysis to detect expression of the target protein MAPK7 expression levels with GAPDH as internal control.4. Detection of MAPK7 gene expression levels: the cultured SOSP-M cells were randomly divided into five groups: the blank group: no transfection treatment, the control group: transfect cells with blank plasmid, overexpression group: transfect cells with PCDNAMAPK7 plasmids, Scramble SiRNA group:transfect cells with Scramble SiRNA, knock down group: transfect cells with siRNA-MAPK7. Collect cells after transfected for 24 h. Total RNA is extracted and reverse transcribed into cDNA, and then do Real-time PCR analysis to detect the expression of the target gene MAPK7 mRNA expression levels with GAPDH as internal control.5. Observation of cell proliferation: the five groups’ cells are passed into 96-well plates.Use MTT assay. Measure absorbance values of each well at 570 nm wavelength, record the results, with time as the abscissa and absorbance value as the ordinate for the cell growth curve, calculating the rate of growth inhibition, to explore cell proliferation 24 h after transfection in each group.6. Observation of cell migration: the five groups’ cells are passed into 6-well plates. Use wound scratch assay, randomly selecting eight horizons in each group, when transfect for 24 h.Calculate cell migration rate to compare the cell migration in each group.7. Observation of cell invasion ability: the five groups’ cells are passed into transwell chambers that coated with basement membrane. Culture cells for 48 h culture, then the cells are washed off the membrane, stained for 30 min. Count the number of cells in the field of each 200 X, comparing the invasion between groups.8. We use flow cytometry technology detect cell apoptosis and cell cycle to analysis apoptosis of cells and cell cycle progression. We use RealtimePCR and Western blot analysis test epithelial cell mass, that is to say the expression level of molecular markers of EMT process.9. All collected data were firstly tested for the normal distribution using one-sample K-S test. Enumeration data were analyzed by chi-square test or rank-sum test. Measurement data were tested by student t-test(for two groups) or analysis of variance(ANOVA, for more thanthree groups). Further between-group-comparison was then performed by post-hoc Tukey test.A statistical significance was defined when p<0.05.Results:1. The analysis of MAPK7 expression levels.Compared with the other group, the level of gene MAPK7 is significantly increased in the group that the cells are transfected with PCDNA-MAPK7 plasmids. While the cells transfected with siRNA-MAPK7 has an significantly decrease. The results shows the PCDNA-MAPK7 plasmids and siRNA-MAPK7 can increase or knockoff the expression of gene MAPK7 in osteosarcoma cell line SOSP-M cells.2.The gene MAPK7 can promote the proliferation of tumor cell.MTT cell proliferation assay shows that compared with blank group,cells transfected with control have no changes in cell proliferation, but there is an increasing cell proliferation which is about 1.79 times that of the overexpression control group when transfected with PCDNA-MAPK7 plasmids. The cell proliferation is suppressed in cells transfected with siRNA-MAPK7 which is about 51.8% that of the Scramble SiRNA group. These results show that the gene MAPK7 has an important effect on proliferation of tumor cell.3.The gene MAPK7 can promote the migration of tumor cell.In wound scratch assay, we find more migrating cells in scratch of the group that the cells are transfected with PCDNA-MAPK7 plasmids. The cells transfected with si RNAMAPK7 almostly has no migration in scratch. These results shows that the gene MAPK7 has an effect on migration of tumor cell.4. The gene MAPK7 can promote the invasion of tumor cell.The results of matrigel invasion assay exhibit, compared with control group the group thransfected with PCDNA-MAPK7 plasmids have obviously more invasion cells within each200 X field of view 12.6 cells showing a stronger invasion ability. The group thransfected with siRNA-MAPK7 shows suppression in cell invasion within each 200 X field of view 2.1 cells.5. The gene MAPK7 can suppress the apoptosis of osteosarcoma SOSP-M.To examine the impact of MAPK7 expression levels on osteosarcoma SOSP-M apoptosis,we use flow cytometry technology to detect cell apoptosis in cells after MAPK7 overexpression and interference. MAPK7 overexpression significantly inhibited cell apoptosis, compared with the control overexpression group SOSP-M apoptotic cellsdecreased by about 30%; si RNA- MAPK7 transfected cells significantly contributed to the apoptosis, compared with the Scramble SiRNA group SOSP-M apoptotic cells increased by about 2.1 times. The difference was statistically significant(* p <0.05).6. The gene MAPK7 can promote the cell cycle of osteosarcoma SOSP-M.In order to study the mechanism of the gene MAPK7 regulation to cell proliferation, we further investigated whether MAPK7 is involved in the regulation of cell cycle. To examine the impact of the gene MAPK7 on SOSP-M cell cycle, we use flow cytometry technology to detect cell apoptosis in cells cycle after MAPK7 overexpression and interference.In SOSP-M cells, compared with the control group over-expressed MAPK7, the proportion of cells in G1 phase decreased from 59.4% to 37.5%, the proportion of cells in S phase increased from 21% to 34.5%.And after transfected with siRNA-MAPK7 the cell cycle progression was significantly inhibited. In other words, when MAPK7 was interferenced, more cells accumulated in the G1 phase, and can not successfully enter S phase. Therefore, we think MAPK7 impacts SOSP-M cell from G1 phase into S phase, thus impact the cell cycle progression(* p <0.05).Conclusion:1. The gene MAPK7 can promote the proliferation in osteosarcoma.2. The gene MAPK7 can promote the migration in osteosarcoma.3. The gene MAPK7 can promote the invasion in osteosarcoma.4. The gene MAPK7 can suppress the apoptosis of osteosarcoma SOSP-M.5. The gene MAPK7 can promote the cell cycle of osteosarcoma SOSP-M.