The Study On 67KDa Laminin Receptor Antisense Oligodeoxynucleotides Inhibiting The Invasion And Metastasis Of The Ovarian Carcinoma Cells

Objective To observe the growth features, morphology under microscope of four kinds of ovarian carcinoma cells cultured in vitro. To detect the expression of 67kDa laminin receptor (67kDa LN-R) of the cells respectively by immunochemistry and RT-PCR methods. To determine the positive cell lines expressing 67kDa LN-R will create a basis for further study of its effect on invasion and metastasis of ovarian carcinoma.Methods The frozen HRA,SKOV3,3AO,OVCAR3 ovarian carcinoma cells were resuscitated and cultured in RPMI1640 mediums supplemented with 10% bovine calf serum, 1 × 10 5 IU/L penicillin and lOOmg/L streptomycin to logarithmic phase in CO2 culturing box. After Giemsa staining, shapes of the four different cells were compared. The monolayers of the tumor cells were made, of which the expression of 67kDa LN-R was detected by immunochemistry method. About 106 ovarian carcinoma cells were harvested respectively, of which the expressions of 67kDa LN-ROvarian carcinoma cell; Cell culture; 67kDa laminin receptor; ExpressionMTT assay and FCM we have observed the changes of cell morphology, the survival situation and the induced-apoptosis effect. 3. The total RNAs were extracted from the cells in 24-48 hours after transfection and then semi-quantitative RT-PCR, immunocytochemistry and FCM method were performed to evaluate the 67kDa laminin receptor gene and protein expression levels respectively to identify the efficiency of ASODN. 4. The invasiveness of transfected cells was measured quantitatively by Matrigel invasion assays (Transwell chamber). 5. The immunocytochemistry assay was used to evaluate the expression levels of MMP-2 and KAIl that were related to the invasion and metastasis of tumor cells, to analyze the invasion ability of transfected cells.Results The ovarian carcinoma cells transfected into 67kDa LN-R ASODN for 24h-48h became round, in which toxic granulars and apoptosis bodies appeared. The cells regained the normal situation of growth 48h to 72h after changing the medium. MTT assay demonstrated that 67kDa LN-R ASODN could partially inhibit the growth of HRA cells. The 67kDa LN-R gene and protein expression and invasiveness of HRA cells treated with ASODN of different final concentration were significantly decreased compared with that of that transfected with SODN and the controls (p<0.05)Furthermore, the inhibiting effects were significantly different among the cells treated with ASODN of different concentration (p<0.05). The immunocytochemistry showed that the expression levels of MMP-2 in transfected cells were lower than the controls; otherwise, the expression levels of KAIl became much higher compared with that of the controls.Conclusions The expression of 67kDa LN-R of HRA cells decreased obviously at protein and mRNA levels after transfected into 67kDa LN-R ASODN . 67kDa LN-R ASODN complementary to the start codon region of 67kDa laminin receptor mRNA has a significant inhibitory effect on the invasiveness of human ovarian carcinoma cell line in a dose-dependent manner . It is suggested that 67kDa LN-R ASODN is the regulator of MMP-2 and KAIl expression.