title: Osteopontin knockdown suppresses the growth and angiogenesis of colon cancer cells.
abstract: OBJECTIVE
  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).
  
  
  CONCLUSIONS
  Knockdown of OPN gene expression suppresses colon cancer cell growth, adherence, invasion, and expression of angiogenic factors.
authors.affiliation: Xin-Lin Wu, Wan-Xiang Wang, Xing-Kai Meng, Ming-Xing Hou, Pei-De Dong, Jun-Jing Zhang, Zhao-Yang Wang, Department of General Surgery, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China.
authors.forename: Xin-Lin
authors.initials: XL
authors.lastname: Wu
authors.affiliation: Xin-Lin Wu, Wan-Xiang Wang, Xing-Kai Meng, Ming-Xing Hou, Pei-De Dong, Jun-Jing Zhang, Zhao-Yang Wang, Department of General Surgery, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China.
authors.forename: Kai-Jin
authors.initials: KJ
authors.lastname: Lin
authors.affiliation: Xin-Lin Wu, Wan-Xiang Wang, Xing-Kai Meng, Ming-Xing Hou, Pei-De Dong, Jun-Jing Zhang, Zhao-Yang Wang, Department of General Surgery, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China.
authors.forename: Ai-Ping
authors.initials: AP
authors.lastname: Bai
authors.affiliation: Xin-Lin Wu, Wan-Xiang Wang, Xing-Kai Meng, Ming-Xing Hou, Pei-De Dong, Jun-Jing Zhang, Zhao-Yang Wang, Department of General Surgery, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China.
authors.forename: Wan-Xiang
authors.initials: WX
authors.lastname: Wang
authors.affiliation: Xin-Lin Wu, Wan-Xiang Wang, Xing-Kai Meng, Ming-Xing Hou, Pei-De Dong, Jun-Jing Zhang, Zhao-Yang Wang, Department of General Surgery, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China.
authors.forename: Xing-Kai
authors.initials: XK
authors.lastname: Meng
authors.affiliation: Xin-Lin Wu, Wan-Xiang Wang, Xing-Kai Meng, Ming-Xing Hou, Pei-De Dong, Jun-Jing Zhang, Zhao-Yang Wang, Department of General Surgery, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China.
authors.forename: Xiu-Lan
authors.initials: XL
authors.lastname: Su
authors.affiliation: Xin-Lin Wu, Wan-Xiang Wang, Xing-Kai Meng, Ming-Xing Hou, Pei-De Dong, Jun-Jing Zhang, Zhao-Yang Wang, Department of General Surgery, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China.
authors.forename: Ming-Xing
authors.initials: MX
authors.lastname: Hou
authors.affiliation: Xin-Lin Wu, Wan-Xiang Wang, Xing-Kai Meng, Ming-Xing Hou, Pei-De Dong, Jun-Jing Zhang, Zhao-Yang Wang, Department of General Surgery, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China.
authors.forename: Pei-De
authors.initials: PD
authors.lastname: Dong
authors.affiliation: Xin-Lin Wu, Wan-Xiang Wang, Xing-Kai Meng, Ming-Xing Hou, Pei-De Dong, Jun-Jing Zhang, Zhao-Yang Wang, Department of General Surgery, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China.
authors.forename: Jun-Jing
authors.initials: JJ
authors.lastname: Zhang
authors.affiliation: Xin-Lin Wu, Wan-Xiang Wang, Xing-Kai Meng, Ming-Xing Hou, Pei-De Dong, Jun-Jing Zhang, Zhao-Yang Wang, Department of General Surgery, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China.
authors.forename: Zhao-Yang
authors.initials: ZY
authors.lastname: Wang
authors.affiliation: Xin-Lin Wu, Wan-Xiang Wang, Xing-Kai Meng, Ming-Xing Hou, Pei-De Dong, Jun-Jing Zhang, Zhao-Yang Wang, Department of General Surgery, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Shi
mesh_terms: D002448:Cell Adhesion; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D009361:Neoplasm Invasiveness; D009389:Neovascularization, Pathologic; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; C506895:SPP1 protein, human; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D053495:Osteopontin; D014568:Urokinase-Type Plasminogen Activator; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9
keywords: Angiogenesis; Colon carcinoma; Lovo cells; Osteopontin; RNA interference