The Influence And Possible Mechanism Of GDF15 On The Migration 、Proliferation Of Ovarian Cancer Cells

Ovarian malignant carcinoma is one of the most common female gynecological malignancy. Without significantly early symptom and specific early screenning and diagnosis method, it’s difficult to discovery the early lesions. The advanced cases is also lack of effective treatment, therefore the mortality rate of ovarian malignant carcinoma take the first place in gynecological malignancy. Among them, the epithelial ovarian cancer(EOC)accounted for 85%-90%.Growth differentiation factor15(GDF15) is a member of the transforming growth factor-β(TGF-β) superfamily that increased in many malignant carcinomas,include ovarian cancer. The expression of GDF15 in tumor tissues and effusion is negative correlated with survival of ovarian cancer and its overexpression in ovarian cancer cells can prompt the growth and proliferation as well as increase the invasion. Urokinase type plasminogen activator(u PA) is an important member of plasminogen activator system as well as its specificity receptor--urokinase type plasminogen activator receptor(u PAR). Binding of u PA to u PAR triggers the transformation of plasminogen to plasmin and the subsequent activation of metalloproteinases. These events confer tumor cells with the capability to degrade the components of the surrounding extracellular matrix, thus contributing to tumor cell invasion and metastasis. u PA/u PAR can also stimulate cell proliferation and the expression of tumor-promoting genes through FAK/SRC/ERK and other pathways,thus assisting tumor development. It’s reported that GDF15 may contribute to the malignant progression of gastric cancer cells by inducing tumor cell invasion through the up-regulation of the u PA activation system via extracellular signal-regulated kinase-1/2-dependent pathway. Domestic reports about the mechanism of GDF15 on the proliferation, and the relationship between GDF15 and u PA in ovarion cancer cell are rare.Objective: By transient transfection the plasmid of GDF15, we raised the expression of GDF15 in ovarian cancer cell lines SKOV3 and observed the influence of GDF15 on the migration 、 proliferation of ovarian cancer cell and the expression of u PA in ovarian cancer cell to study the possible regulation mechanisms of the migration 、 proliferation of ovarian cancer cells.Methods:1 To extract the plasmid of pc DNA3.1(+)-EGFP-GDF15 、pc DNA3.1(+)-EGFP use Gold Hi Endo Free Plasmid Maxi Kit, then use Agarose gel electrophoresis to its quality.2. The SKOV3 cells were divided into blank control group(SKOV3), experimental group(SKOV3-GDF15) and negative control group(SKOV3-N), respectively give untransfect and transfect with pc DNA3.1(+)-EGFP-GDF15 or pc DNA3.1(+)-EGFP by Lipofectamine?2000, then ob-serve the transfection rate through fluorescence microscope 24 h after tra-nsfection.3 The relative level of GDF15 and u PA m RNA were determined by real-time quantitative PCR using SYBRGreen method 24-48 h after transfection.4 After transfected with pc DNA3.1(+)-EGFP-GDF15 or pc DNA3.1(+)-EGFP 24 h, the cells and untransfected cells was adjusted cell numb-er after trypsinization and anew planked.The MTS experimental was performed after cultured 2h. Setting the time initial adding MTS as 0h, 10μl/well MTS was respectively added to 96 well plate after cultured 0、24、48、72h. Then absorption coefficients were detected at the wave-length of 490 nm by enzyme mark instrument after cultured 1-4h.5 After transfected with pc DNA3.1(+)-EGFP-GDF15 or pc DNA3.1(+)-EGFP 24 h, the wound Healing assay were performed to test the effect of GDF15 on SKOV3 cells migration.1 The identification of pc DNA3.1(+)-EGFP-GDF15、pc DNA3.1(+)- EGFPThe concentration of the plasmid was around 600ng/u L. The electrophoresis banding displays only one strip, and its speed of migrarion was faster. These suggest that the plasmid is mostly composed of plasmid with superhelical structure and it can be used for subsequent expremental, Fig.1.2 Transfection EfficiencyGreen fluorescence was observed by fluorescence microscopy on the cells transfected with pc DNA3.1(+)-EGFP-GDF15 、 pc DNA3.1(+)-EGFP rather than untranfected ones 24 h after transfection,suggesting successfully transfection, Fig.2.3 The expression of GDF15 and u PA m RNA in cells untransfected and transfected with pc DNA3.1(+)-EGFP-GDF15、 pc DNA3.1(+)-EGFP.The relative expression of GDF15 and u PA m RNA in each group was detected RT-PCR analysis. The expression of GDF15 and u PA in SKOV3-GDF15 were significantly higher than that in SKOV3 and SKOV3-N(P<0.05), Fig.3.4 The effect of GDF15 on the proliferation of SKOV3 cells.The proliferation of SKOV3 cells which were transfected with pc DNA3.1(+)-EGFP-GDF15 and empty vector was determined by MTT assay respectively after cultivated 0 、 24 、 48 、 72 h. The proliferation rate of SKOV3-GDF15 had obviously been elevated at different time(P<0.05), and it’s the faster at 24h(it is after transfection 48h), Fig.4, Table 1.5 The effect of GDF15 on the migration of SKOV3 cellsWound Healing assay were performed on the three groups. The results suggested that compared with cells that untransfected and transfected with pc DNA3.1(+)-EGFP, cells that transfected with pc DNA3.1(+)-EGFP –GDF15 migration was significantly increased(P﹤0.05), Fig.5.Conclusion:1 Raising GDF15 could significantly prompt the migration and prol iferation of SKOV3 cells.Results:2 The level of u PA m RNA could be significantly raised with the increase of GDF15 m RNA expression. It suggests that there maybe a regulation mechanism of GDF15/u PA3 The u PA/ u PAR system maybe related with the function of GDF15 on the migration and proliferation of ovarian cancer cells.