ABSTRACT
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 real-time 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 OPN-specific 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 Lovo-OPN-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. After 24, 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 Lovo-NC 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 ± 177.36 µg/L vs 4084.74 ± 349.54 µg/L and 4011.41 ± 424.48 µg/L, respectively), MMP-9 (3782.89 ± 300.64 µg/L vs 5062.90 ± 303.02 µg/L and 4986.38 ± 300.75 µg/L, respectively) and uPA (1152.69 ± 120.79 µg/L vs 1380.90 ± 147.25 µg/L and 1449.80 ± 189.92 µg/L, respectively) (all P < 0.05). CONCLUSION: Knockdown of OPN gene expression suppresses colon cancer cell growth, adherence, invasion, and expression of angiogenic factors.
Subject(s)
Cell Proliferation , Colonic Neoplasms/metabolism , Gene Knockdown Techniques , Neovascularization, Pathologic , Osteopontin/metabolism , Cell Adhesion , Cell Line, Tumor , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Colonic Neoplasms/therapy , Down-Regulation , Gene Expression Regulation, Neoplastic , Genetic Therapy/methods , Humans , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Neoplasm Invasiveness , Osteopontin/genetics , RNA Interference , RNA, Messenger/metabolism , Transfection , Urokinase-Type Plasminogen Activator/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
OBJECTIVE: To investigate the effects of human colon carcinoma-associated fibroblasts (CAFs) on proliferation, adhesion, migration and invasion of colon carcinoma Lovo cells. METHODS: The co-culture models among colon CAFs, NFs and Lovo cell were established by conditioned medium (CM) of human colon CAFs and colon normal fibroblasts (NFs). Lovo cells in the blank control group was treated with serum-free culture medium. The effects of human colon CAFs on proliferation, adhesion, migration and invasion of colon carcinoma Lovo cells were detected by cell proliferation assay, adhesion assay, migration assay and Transwell invasion assay. RESULTS: After co-culture with colon CAFs, the absorbance (A) value of Lovo cells was (0.667±0.059) in 48 h and (0.709±0.030) in 72 h. The A value of Lovo cells adhesion to fibronectin was (0.588±0.067). The cell mobility rates were (35.2±8.7)% in 12 h and (64.6±7.1)% in 24 h. The number of invasive cell was (56.2±4.8). All the above parameters were increased compared with those in the blank control group and NFs group (all P<0.01). CONCLUSION: Human colon CAFs can promote the proliferation, adhesion, migration and invasion of colon carcinoma Lovo cells.
