|
|
|
| Effect of siRNA Combined-silencing MMP-9 and FAK on Invasion and Migration of Mouse Melanoma Highly Metastatic Cells B16F10 in vitro |
| TANG He-jing1, TANG Zhao-yong1, LIU Long-xing1, ZHANG Xiao-mei2, WANG Yi-ting2, FANG Liao-qiong1,2 |
1. College of Biotechnology, Southwest University, Chongqing 400016, China;
2. College of Biomedical Engineering, Chongqing Medical University State Key Laboratory of Ultrasound Engineering in Medicine Co-founded by Chongqing and the Ministry of Science, Chongqing 400016, China |
|
|
|
Abstract Objective: To observe the effect of combined-silencing MMP-9 and FAK on invasion and migration of mouse melanoma highly metastatic B16F10 cells in vitro. Methods: Construct recombinant plasmid vectors pGV102-MMP9-siRNA and pGV102-FAK-siRNA respectively. Culture mouse melanoma B16F10 cells, then the plasmid vectors were transfected into cells using Lipofectamine TM2000 method. This experiment was divided into 5 groups of blank control group, negative control group, Anti-MMP-9 experimental group, Anti-FAK experimental group, and Anti-MMP-9 &FAK experimental group. G418 was used to screen those GFP+ cells. In addition, the transfection efficiency were assayed with flow cytometry, and the cellular morphology of transfected cells were observed by confocal microscopy. mRNA level of MMP-9 and FAK of all groups B16F10 cells was detected by RT-PCR. Finally invasion and migration of transfected B16F10 cells in vitro were tested by transwell experiment. Results Transfection efficiencies of the three experimental groups were to 92.41±1.64%, 95.72±0.21%, 91.52±0.11% respectively by flow cytometry via G418 screening. Moreover, screened cells morphology was good. Compared with the blank control group, MMP-9 and FAK mRNA level of three experimental groups' cells was significantly lowered (P<0.01); their invasion and migration also weakened dramaticly (P<0.01), and Anti-MMP-9 &FAK experimental group was very distinctly lower than Anti-MMP-9 experimental group and Anti-FAK experimental group. Conelusion Combined-silencing MMP-9 and FAK could obviously inhibit invasion and migration of mouse melanoma cell B16F10 in vitro.
|
|
Received: 25 June 2014
Published: 25 September 2014
|
|
|
|
|
|
[1] 朱耀德. 恶性黑色素瘤. 徐州医学院学报, 1982, 1: 81-87. Zhu Y D. Malignant Melanoma. Acta Academiae Medicinae Xuzhou,1982,1:81-87.
[2] Wittekindt C, Jovanovic N, Guntinas-Lichius O, et al. Expression of matrix metalloproteinase-9 and blood vessel density in laryngeal squamous cell carcinomas. Acta Otolaryngol, 2011, 131(1): 101-106.
[3] Hawinkels L J, Zuidwijk K, Verspaget H W, et al. VEGF release by MMP-9 mediated heparan sμlphate cleavage induces colorectal cancer angiogenesis. Eur J Cancer, 2008, 44: 1904-1913.
[4] Yang S, Zhao Z, Wu R, et al. Expression and biological relationship of vascular endothelial growth factor-a and matrix metalloproteinase-9 in gastric carcinoma. J Int Med Res, 2011, 39(6): 2076- 2085.
[5] Ilic D, Furuta Y, Kanazawa S, et al. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK deficient mice. Nature, 1995, 377(6549): 539-544.
[6] Nikkola J, Vihinen P, Vuoristo M S, et al. High serum levels of matrix metalloproteinase-9 and matrix metalloproteinase-1 are associated with rapid progression in patients with metastatic melanoma. Clin Cancer Res, 2005, 1: 5158-5166.
[7] Redondo P, Lloret P, Idoate M, et al. Expression and serum levels of MMP-2 and MMP-9 during human melanoma progression. Clin Exp Dermatol, 2005, 30: 541-545.
[8] Shufeng Li, Wei Dong, Yiwei Zong, et al. Polyethylenimine-complexed plasmid particles targeting focal adhesion linase function as melanoma tumor therapeutics. Mol Ther, 2007, 15(3): 515-523.
[9] 张楠楠, 魏丽丽, 强荣兵, 等. 黏着斑激酶与肿瘤侵袭转移. 医学分子生物学杂志, 2009, 6(3): 269-273. Zhang N N, Wei L L, Qiang R B, et al. The role of focal adhesion kinase in cancer invasion and metastasis. Journal of Medical Molecular Biology. 2009,6(3):269-273.
[10] Hauck C R, Sieg D J, Hsia D A, et al. Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor stimulated signaling promoting the migration of invasive human carcinoma cells. Cancer Res, 2001, 61(19): 7079-7090.
[11] Zhao-Yong Tang, et al. RNAi-mediated MMP-9 silencing inhibits mouse melanoma cell invasion and migration in vitro and in vivo. Cell Biol Int, 2013, 1-6.
[12] Terao S, Shirakawa T, Goda K, et al. Recombinant interleukin-2 enhanced the antitumor effect of ADV/RSV-HSV-tk/ACV therapy in a murine bladder cancer model. Anticancer Res, 2005, 25(4): 2757-2760.
[13] Huang Q, Pu P, Xia Z, et al. Exogenous wt-p53 enhances the antitumor effect of HSV-TK/GCV on C6 glioma cells. J Neurooncol, 2007, 82(3):239-248.
[14] Lai K C, Huang A C, Hsu S C, et al. Benzy isothiocyanate(BITC) inhibits migration and invasion of human colon cancer HT29 cells by inhibiting matrix metalloproteinase-2/-9 and urokinase plasminogen(uPA) through PKC and MAPK signaling pathway. J Agric Food Chem, 2010, 58(5): 2935-2942.
[15] Chen J S, Huang X H, Wang Q, et al. FAK is involved in invasion and metastasis of hepatocellular carcinoma. Clin Exp Metastasis, 2010, 27(2): 71-82.
[16] 何振辉,何太平,翁闪凡,等.芦荟大黄素对高转移乳腺癌细胞MDA-MB-231体外转移潜能的影响.中国药理学通报.2013,29(8):1114-1118. He ZH H, He T P, Weng SH F, et al. Effect of Aloe emodin on metastatic abilities of human high metastatic breast cancer MDA-MB-231 cells in vitro. Chinese Pharmacological Bulletin. 2013,29(8):1114-1118.
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
