Inhibition of Matrix Metalloproteinase 9 Expression in Rat Dermal Fibroblasts Using Small Interfering RNA

• DOI: 10.7547/1020299
• Publication Date: 2014-04-01T13:44:28Z
• Authors: Xiao-Ying Xie; Meng Ren; Chuan Yang; Shaoyun Hao; Ping Zhu; Li Yan
• Keywords: 0301 basic medicine; Wound Healing; Gene Expression; Fibroblasts; Immunohistochemistry; Extracellular Matrix; Rats; Rats, Sprague-Dawley; 03 medical and health sciences; Matrix Metalloproteinase 9; Diabetes Mellitus; Animals; RNA, Small Interfering; Cells, Cultured; Skin

Background:Matrix metalloproteinases (MMPs) degrade extracellular matrix components. Increased MMP-9 content in diabetic skin contributes to skin vulnerability and refractory foot ulcers. To identify ways to decrease MMP-9 levels in skin, inhibition of MMP-9 expression in dermal fibroblasts using small interfering RNA was investigated in vitro.Methods:A full-thickness wound was created on the midback of streptozotocin-induced diabetic rats; skin biopsies were performed 3 days later. Skin MMP-9 expression was observed by immunohistochemical analysis. Dermal fibroblasts from 1-day-old normal Sprague Dawley rats cultured with high glucose and homocysteine concentrations were transfected with small interfering RNA complexes. Cells were collected 30, 48, and 72 hours after transfection, and reverse transcription–polymerase chain reaction, Western blot analysis, and gelatin zymography for MMP-9 were performed.Results:Expression of MMP-9 was increased in diabetic rat skin, especially around wounds. After 30-, 48-, and 72-hour transfection with each MMP-9–specific small interfering RNA, reverse transcription–polymerase chain reaction showed markedly decreased MMP-9 messenger RNA expression, protein abundance, and activity. Of four MMP-9 small interfering RNAs, one sequence had a stable high inhibition rate (>70% at 30 and 48 hours after transfection).Conclusions:Expression of MMP-9 was increased in diabetic rat skin, especially around wounds, and was markedly inhibited after MMP-9 small interfering RNA transfection in vitro (P< .05). These findings may provide new treatments for diabetic skin wounds. (J Am Podiatr Med Assoc 102(4): 299–308, 2012)