Osteopontin Promotes The Invasiveness Of Human Ovarian Carcinoma Cell Line SKOV3

ObjectiveTo detect the expression of osteopontin(osteopontin,OPN) and other metastasisrelated genes urokinase-type plasminogen activator(urokinase plasminogen activator, uPA) and heparanase(heparanase,HPA)in ovarian cancer cell strain SKOV3,and to study the OPN on ovarian cancer invasion and metastasis potential impact and to explore its possible mechanism.Materials and MethodsThe ovarian serous cystadenocarcinoma cell strain SKOV3 cells cultured in conventional recovery containing 10%calf serum in RPMI1640 culture medium with 0.25%trypsin digestion passage.Using Lipofectamine 2000 liposome-mediated transfection of recombinant plasmid pcDNA3.1(-)/OPN,while empty vector transfected pcDNA3.1(-) as control,and the use of G418 selection were stable transfected cell strains.Using reverse transcription polymerase chain reaction RT-PCR detection of OPN mRNA expression level,Touching gel imaging analysis strip gray value,and compared to calculation results withβ-actin to analyze OPN expression intensity.To detect OPN protein expression by Western-blot,and exposure to display strap by dropwise luminescence agentia in darkroom.Serum-free culture medium to the cultured cells,in 4℃,12000r/min conditions,centrifugation 5min,sub-cell supernatant for ELISA to determinate OPN,uPA and HPA,using microplate reader at 450nm wavelength measured absorbance(OD value ).And using matrix gel invasion assay detected two groups of cell invasiveness,methanol fixed,eosin staining,relative to the number of invasive cells to express the invasive ability of tumor cells(TC).The experimental results compare the rate of use ofχ~2 test,between the two samples are quite a few t test,P＜0.05 for the difference has statistical significance. Results1.RT-PCR results showed that the positive expression rate of OPN were signifieantly higher in the recombinant plasmid transfection group(1.268±0.046) than that in control group(0.550±0.027)(P＜0.05).The difference had statistical signifycance. Western-blot results showed that the positive expression rate of OPN were signifieantly higher in the recombinant plasmid transfection group(0.642±0.079) than that in control group(0.371±0.033)(P＜0.05).The difference had statistical signify- cance.2.ELISA assay detection of cell culture clear supernatant,the positive expression rate of OPN were signifieantly higher in the recombinant plasmid transfection group(3.605±0.205)ng/ml than that in control group(1.602±0.119)ng/ml(P＜0.05).The difference had statistical significance(P＜0.05);The positive expression rate of uPA were significantly higher the recombinant plasmid transfection group(5.342±0.031 ng/ml) than that in control group(1.956±0.047)ng/ml(P＜0.05).The difference had statistical significance(P＜0.05);The expression of HPA had not statistical significance between the recombinant plasmid transfection group(2.762±0.145ng/ml) and control group(2.692±0.140ng/ml)(P＞0.05).3.Matrix gel invasion assay results showed that the cell populations can to penetrate plasmalemma in the recombinant plasmid transfection group(36.459±4.447) are mroe than in control group(18.128±6.017)(P＞0.05).Conclusions1.OPN can promote the invasion potency of ovarian cancer.2.OPN may be by upregulating the expression of uPA to promote invasion and metastasis of ovarian cancer.3.Can further explore the treatment in order to OPN as target invasion and metastasis of ovarian cancer,and to provide an effective theoretical basis for further experimental studies.