title: [Effect of TIMP-2, MT1-MMP and MMP-2 expression on the in vitro invasive capacity of acute monocytic leukemia SHI-1 cells].
abstract: OBJECTIVE
  To study the effect of matrix metalloproteinase 2 (MMP-2), membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2) expressions on the in vitro invasive capacity of acute monocytic leukemia SHI-1 cells.
  
  
  METHODS
  SHI-1, NB4, K562, M937 and THP-1 human leukemia cell lines were cultured in vitro. The mRNA and protein expressions of TIMP-2, MMP-2 and MT1-MMP in different cells were detected by quantitative RT-PCR and western blot. A retroviral vector carrying human TIMP-2 cDNA was constructed and transfected into SHI-1 cells. Three subclone cells (S1, S2 and S3) were screened by G418 and selected by limiting dilution. RNA interference (RNAi) was used to knock down the expression of MMP-2, MT1-MMP and TIMP-2. Cell invasion capacity was performed through a reconstituted human basement membrane assays. Zymography was used to analyze the expression of MMP-2 in the supernatant of co-culture.
  
  
  RESULTS
  The expressions of MMP-2, MT1-MMP and TIMP-2 in SHI-1 cells were higher than that in other leukemic cells at both mRNA and protein levels (P < 0.05). The amount of proMMP-2 and activated MMP-2 in the conditioned media from SHI-1 cells co-cultured with bone marrow stromal cells (BMSCs) was more than that from other cells (P < 0.05). The in vitro invasive capacity of SHI-1 cells were higher than that of other cells (P < 0.05). The mRNA levels of TIMP-2 were increased by about 3 fold, 2 fold and 1.5 fold in S1, S2 and S3 cells, respectively (P < 0.05), while the protein levels were by about 2.6 fold, 1.5 fold and 1.3 fold than that of SHI-1 cells, respectively (P < 0.01). The invasion rates of subclone cells demonstrated a 1.5 - 2.5 fold' elevation (P < 0.05) and activated MMP-2 from their supernatants increased by 1.5 - 2.0 fold (P < 0.01). The knock-down efficiency of siRNA was 85% to 98%. The down-regulation of TIMP-2, MMP-2 and MT1-MMP decreased the invasion rates of SHI-1 cells by 60% - 70%, 50% - 60% and 40% - 50%, respectively (P < 0.05). No activated MMP-2 in the supernatants from any knock-down cells could be found.
  
  
  CONCLUSIONS
  SHI-1 cells constitutively overexpress MMP-2, MT1-MMP and TIMP-2 at both mRNA and protein levels. After co-cultured with BMSCs the SHI-1 cells increased MMP-2 activation and cell invasion. An increase of TIMP-2 expression in SHI-1 cells reflects an activating effect on cells invasion and MMP-2 activation.
authors.affiliation: The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Suzhou 215006, China.
authors.forename: Chun-Ling
authors.initials: CL
authors.lastname: Wang
authors.forename: Zi-Xing
authors.initials: ZX
authors.lastname: Chen
authors.forename: Zhen-Jiang
authors.initials: ZJ
authors.lastname: Li
authors.forename: Jian-Nong
authors.initials: JN
authors.lastname: Cen
mesh_terms: D018920:Coculture Techniques; D006801:Humans; D007948:Leukemia, Monocytic, Acute; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D053504:Matrix Metalloproteinases, Membrane-Associated; D012333:RNA, Messenger
chemical_list: D012333:RNA, Messenger; D053504:Matrix Metalloproteinases, Membrane-Associated; D020778:Matrix Metalloproteinase 2