Original Article
Copyright ©2014 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Aug 14, 2014; 20(30): 10440-10448
Published online Aug 14, 2014. doi: 10.3748/wjg.v20.i30.10440
Osteopontin knockdown suppresses the growth and angiogenesis of colon cancer cells
Xin-Lin Wu, Kai-Jin Lin, Ai-Ping Bai, Wan-Xiang Wang, Xing-Kai Meng, Xiu-Lan Su, Ming-Xing Hou, Pei-De Dong, Jun-Jing Zhang, Zhao-Yang Wang, Lin Shi
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
Kai-Jin Lin, Department of Internal Medicine, Fuqing City Hospital, Fuqing 350300, Fujian Province, China
Ai-Ping Bai, Department of Gastroenterology, the First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China
Xiu-Lan Su, Clinical Medicine Research Center, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China
Lin Shi, Department of Pathology, the Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010059, Inner Mongolian Autonomous Region, China
Author contributions: Wu XL and Lin KJ contributed equally to this work; Wu XL, Bai AP, Meng XK and Shi L designed the research; Lin KJ, Wang WX, Meng XK, Su XL, Hou MX, Dong PD, Zhang JJ, Wang ZY and Shi L performed the research and analyzed the data; Wu XL, Bai AP and Shi L wrote the paper.
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; and the Natural Science Foundation of Inner Mongolian Autonomous Region, No. 2012MS1123 and No. 2013MS1132
Correspondence to: Lin Shi, MD, Department of Pathology, the Affiliated Hospital of Inner Mongolia Medical University, 1 Tongdao North Street, Inner Mongolian Autonomous Region, Huhhot 010059, Inner Mongolian Autonomous Region, China. shilinsd2003@aliyun.com
Telephone: +86-471-6637678 Fax: +86-471-6637678
Received: January 5, 2014
Revised: March 28, 2014
Accepted: April 28, 2014
Published online: August 14, 2014

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.

Keywords: Colon carcinoma, Lovo cells, Osteopontin, Angiogenesis, RNA interference

Core tip: This study investigated the anti-tumor effects of osteopontin (OPN) knockdown in human colon cancer (Lovo) cells. Despite recent evidence that shows OPN expression is closely related to the development of colorectal cancer, the mechanism remains elusive. The results of the present study showed that siRNA-mediated downregulation of OPN inhibited the expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, MMP-9 and urokinase plasminogen activator in Lovo cells, which may lead to decreased invasion and angiogenesis of colon cancer.