title: MMP-9 directed shRNAs as relevant inhibitors of matrix metalloproteinase 9 activity and signaling.
abstract: BACKGROUND
  The main function of matrix metalloproteinases is the degradation of extracellular matrix components, which is related to changes in the proliferation of cells, their differentiation, motility, and death. MMPs play an important role in physiological processes such as embryogenesis, angiogenesis and tissue remodeling. The increase of MMPs activity is also observed in pathological conditions including tumorigenesis where MMP-2 (gelatinase A) and MMP-9 (gelatinase B) show the ability to degrade the basement membrane of vessels and they are involved in metastasis. The aim of our study was to verify the changes of MMP-9 enzymatic activity and the mobility of cells after inhibition of MMP-9 gene expression.
  
  
  METHODS
  The oligonucleotide shRNA insert had been designed to silence MMP-9 gene expression and was cloned into the pSUPER.neo expression vector. The construct was introduced into the HeLa (CCL-2) cervical cancer cells by lipotransfection. Simultaneously in control cells MMP-9 were inhibited by doxycycline. Changes in activity of MMP-9 were analyzed by gelatin zymography and wound-healing assay.
  
  
  CONCLUSIONS
  Gelatin zymography allowed us to confirm that activity of MMP-9 in cells transfected by shRNA-MMP-9 and treated by doxycycline were similar and significantly lower in comparison with control cells. Phenotypic tests of migration in vitro confirm statistically significant (P<0.05) changes in cell migration - control cells healed 3 to 5 times faster in comparison with transfected or doxycycline treated cells. Our studies show the significant role of MMP-9 in mobility and invasiveness of tumor cells, thus indicating a potential target point of interest for gene therapy.
authors.affiliation: Department of Biotechnology and Genetic Engineering, Medical University of Silesia, Katowice, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Nowak
authors.forename: Anna
authors.initials: A
authors.lastname: Galilejczyk
authors.forename: Daniel
authors.initials: D
authors.lastname: Sypniewski
authors.forename: Ilona
authors.initials: I
authors.lastname: Bednarek
mesh_terms: D002465:Cell Movement / Q000502:physiology; D004318:Doxycycline / Q000494:pharmacology; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000235:genetics* / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors / Q000494:pharmacology*; D009361:Neoplasm Invasiveness / Q000503:physiopathology; D009369:Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology; D009389:Neovascularization, Pathologic; D034741:RNA, Small Interfering / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D014945:Wound Healing / Q000502:physiology
chemical_list: D061965:Matrix Metalloproteinase Inhibitors; D034741:RNA, Small Interfering; D020780:Matrix Metalloproteinase 9; D004318:Doxycycline