Influence of antisense osteopontin gene eucaryotic expression vector on cell proliferation and invasion of human renal cancer cells

Background and purpose:Overexpression of osteopontin(OPN) is correlated with cell proliferation,invasion,and metastasis in a variety of malignant tumors.However,the role of OPN in human renal cancer has not been fully understood.To verify the feasibility of OPN in renal cancer gene therapy,we constructed antisense OPN gene eucaryotic expression vectors and transfected it into human renal cancer ACHN cells.The impacts of the vector on the proliferation and invasion of ACHN cells were studied.Methods:The total RNA were extracted from ACHN cells.The target fragments of OPN were amplifi ed by RT-PCR method and the fragments were inserted into eucaryotic expression vector pcDNA3.1(-).Antisense OPN gene reconstructed plasmids were constructed and transfected into ACHN cells with LipofactamineTM 2000.The mRNA and protein expressions of OPN were analyzed by RT-PCR and Western blot;MTT method was used to analyze cell growth;the cell cycle and ratios of apoptotic cells were assessed by flow cytometry;the abilities of proliferation and invasion of cells were detected by soft agar clone formation experiments and Transwell chamber invasive assays.Results:The OPN expression(0.29±0.04) in cells transfected with recombinant vector was signif icantly inhibited,compared with the cells transfected with empty vector(0.86±0.05) and the control groups(0.88±0.04)(P0.05).The growth velocity of ACHN/OPN cells was obviously slowed down.Flow cytometry showed that ACHN/OPN cells were blocked in the S phase and apoptotic ratio increased signif icantly(P0.05).The results through transwell assays and soft agar clone formation experiment suggested that the abilities of invasion and proliferation of ACHN/OPN cells decreased signifi cantly(P0.05),compared with the control groups.Conclusion:OPN plays an important role in the proliferation and invasion of human renal cancer ACHN cells.The malignant phenotype of ACHN cells can be partly inhibited by antisense OPN gene.