title: [Silencing of tumor metastasis suppressor gene 1 promotes invasion of prostate cancer cell in vitro and its molecular mechanisms].
abstract: OBJECTIVE
  To explore the effect of small interference RNA (siRNA) targeting homosapiens longevity assurance homologue 2(LASS2, or TMSG1) on the invasion of PC-3M-2B4 (a variant subline of human prostate carcinoma cell line PC-3M with low metastatic potential) and its molecular mechanisms.
  
  
  METHODS
  PC-3M-2B4 cells were transfected with siRNA by using lipofectamine 2000. The expression of LASS2 mRNA and protein was detected after transfection by real-time fluorogentic quantitative PCR (RFQ-PCR) and Western blot to screen the effective siRNA fragment. The V-ATPase activity of PC-3M-2B4 cells was detected by V-ATPase activity assay kit. The concentration of extracellular hydrogen ion was measured by pH-sensitive fluorescence probe bis-carboxyethyl-carboxyfluorescein (BCECF). The matrix metalloproteinase-2 (MMP-2) protein in the supernatant and cells was analyzed by Western blot. The activity of MMP-2 was examined by Gelatin zymography. Furthermore, the migration and invasion of cells were evaluated by in vitro wound migration assay and invasion assay.
  
  
  RESULTS
  RFQ-PCR and Western blot revealed dramatic reduction (84.5% and 60% ) in the levels of LASS2 mRNA and protein after transfection of siRNA-2 in PC-3M-2B4 cells. The V-ATPases activity and extracellular hydrogen ion concentration were significantly increased in PC-3M-2B4 cells transfected with the siRNA-2 compared with other control groups (P<0.05); There were no differences in the expression and secretion of MMP-2 protein between LASS2-siRNA treated cells and other control groups. However, the activity of MMP-2 was up-regulated in LASS2-siRNA treated cells compared with other control groups( P<0.05); and the capacity for migration and invasion in LASS2-siRNA treated cells was significantly higher than in other control groups (P<0.05).
  
  
  CONCLUSIONS
  Silencing of LASS2 can promote invasion of prostate cancer cells in vitro through the increase of the V-ATPases activity, extracellular hydrogen ion concentration and in turn the activation of secreted MMP-2, indicating that LASS2 is a novel tumor metastasis suppressor gene.
authors.affiliation: Department of Pathology, Peking University School of Basic Medical Sciences, Beijing 100191, China.
authors.forename: Xiao-yan
authors.initials: XY
authors.lastname: Xu
authors.forename: Jiang-feng
authors.initials: JF
authors.lastname: You
authors.forename: Fei
authors.initials: F
authors.lastname: Pei
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics*; D009361:Neoplasm Invasiveness; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D051049:Sphingosine N-Acyltransferase / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; C438311:CERS2 protein, human; D051049:Sphingosine N-Acyltransferase; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D025262:Vacuolar Proton-Translocating ATPases