摘要
AIM: To investigate the effects of osteopontin (OPN) gene expression knockdown on colon cancer Lovo cells in vitro.
AIM:To investigate the effects of osteopontin(OPN)gene expression knockdown on colon cancer Lovo cells in vitro.METHODS:Four candidate small interfering RNA(siRNA)constructs targeting the OPN gene and a scrambled control sequence(NC-siRNA)were synthesized and inserted into a pGPU6/GFP/Neo expression vector.After confirmation by restriction enzyme digestion and DNA sequencing,the recombinant plasmids were subsequently transfected into a human colon cancer cell line(Lovo)using a liposome transfection method.Stably transfected cells were maintained with G418 selection and referred to as Lovo-OPN-1,-2,-3,-4,and Lovo-NC cells.Knockdown efficiency of each of the four siRNA constructs was determined by realtime reverse transcription polymerase chain reaction assays and western blotting,and the construct with the most effective silencing was used for subsequent experiments.Cell proliferation,adhesion,and Matrigel invasion assays were performed to analyze the effects of OPN knockdown in stably transfected Lovo cells.The levels of four angiogenic factors,namely vascular endothelial growth factor(VEGF),matrix metalloproteinase(MMP)-2,MMP-9 and urokinase plasminogen activator were detected by enzyme-linked immunosorbent assays(ELISA).RESULTS:Recombinant vectors containing OPNspecific and scrambled siRNA sequences were successfully constructed and stably transfected into Lovo cells.Compared with the control Lovo and Lovo-NC cells,the levels of OPN mRNA and protein expression in LovoOPN-1,-2,-3,and-4 were significantly reduced(all P<0.05),with the most efficient reduction observed in Lovo-OPN-4 cells(P<0.05).Relative to untransfected Lovo cells,OPN mRNA expression levels in Lovo-NC and Lovo-OPN-4 cells were 1.008±0.067 and 0.160±0.023,respectively.The relative OPN protein expression levels in Lovo,Lovo-NC,and Lovo-OPN-4 cells were 3.024±0.211,2.974±0.630,and 0.121±0.008,respectively.Moreover,transfection with the scrambled sequence had no effect on the expression of OPN.After24,48,72,and 96 h of cultivation,absorption values at 450 nm to assess proliferation of Lovo-OPN-4 cells were 0.210±0.017,0.247±0.024,0.314±0.037,and 0.359±0.043,respectively,which were significantly lower than those of Lovo(0.244±0.031,0.313±0.024,0.513±0.048 and 0.783±0.051)and LovoNC cells(0.241±0.029,0.309±0.022,0.563±0.023,and 0.735±0.067)(all P<0.05).The absorption values at 595 nm,which were measured in a cell adhesion assay,showed that adhesion of Lovo-OPN-4 cells(0.215±0.036)was significantly decreased compared to Lovo(0.490±0.037)and Lovo-NC cells(0.462±0.043)(P<0.05).The number of invasive Lovo-OPN-4 cells(16.1±1.9)was also significantly decreased compared to Lovo(49.9±5.4)and Lovo-NC cells(48.8±4.5)(P<0.05).ELISA assays showed significant reductions in Lovo-OPN-4 cells compared to Lovo and Lovo-NC cells with regard to the expression of VEGF(1687.85±167.84 ng/L vs 2348.54±143.80 ng/L and 2284.39±138.62 ng/L,respectively),MMP-2(2966.07±177.36μg/L vs 4084.74±349.54μg/L and 4011.41±424.48μg/L,respectively),MMP-9(3782.89±300.64μg/L vs5062.90±303.02μg/L and 4986.38±300.75μg/L,respectively)and uPA(1152.69±120.79μg/L vs1380.90±147.25μg/L and 1449.80±189.92μg/L,respectively)(all P<0.05).CONCLUSION:Knockdown of OPN gene expression suppresses colon cancer cell growth,adherence,invasion,and expression of angiogenic factors.
基金
Supported by Grants from the National Natural Science Foundation of China,No.81260364,No.81270472 and No.81070310
the"Chunhui"Program of Ministry of Education in China,No.Z2012007
the Natural Science Foundation of Inner Mongolian Autonomous Region,No.2012MS1123 and No.2013MS1132
