title: RNA interference suppression of A100A4 reduces the growth and metastatic phenotype of human renal cancer cells via NF-kB-dependent MMP-2 and bcl-2 pathway.
abstract: OBJECTIVE
  S100A4 is a well established marker and mediator of metastatic disease, but the exact mechanisms responsible for the metastasis promoting effects are less well defined. We tested a hypothesis that the S100A4 gene plays a role in the proliferation and invasiveness of human renal cancer cells (RCC) and may be associated with its metastatic spread.
  
  
  METHODS
  The small interference RNA vector pcDNA3.1-S100A4 siRNA was transfected in to the human renal cancer cell lines ACHN, Ketr-3, OS-RC-2, CaKi-2 and HTB-47, then treated with ABT-737 or BB94. Cell apoptosis and cell viability was detected by flow cytometry and MTT assay. Matrigel was used for cell motility and invasion assay. MMP-2, bcl-2 and S100A4 was detected by RT-PCR and western blot assay. NF-kB subunit p65 activity was detected by confocal microscopy assay. We then determine the effect S100A4 sliencing on tumor growth, lung metastasis development in vivo. Immunohistochemistry was used to detected the expression of S100A4, bcl-2, MMP-2, p65 and CD31.
  
  
  RESULTS
  S100A4 silencing in ACHN cells by RNA interference significantly inhibited NF-kB and NF-kB-mediated MMP-2 and bcl-2 activation and cellular migration, proliferation, and promoted apoptosis. Furthermore, re-expression of S100A4 in S100A4-siRNA-transfected ACHN cells by transient S100A4 cDNA transfection restored the NF-kB and NF-kB-mediated MMP-2 and bcl-2 activation and their high migratory and cellular proliferative ability. An inhibitor ABT-737 (the Bcl-2 antagonist targets Bcl-2) against Bcl-2 suppressed cellular proliferation and promoted apoptosis induced by S100A4 re-expression in S100A4-siRNA-transfected ACHN cells. A inhibitor BB94 against MMPs to neutralize MMP-2 protein suppressed cellular invasion and migration induced by S100A4 re-expression in S100A4-siRNA-transfected ACHN cells. In the prevention model, S100A4 silencing inhibited primary tumor growth by (tumor weight) (76 ± 8%) and (tumor volum) (78 ± 4%) respectively and promoted apoptosis and the formation of lung metastases was inhibited by 89% (p < 0.01). Microvascular density was reduced by 70% (p < 0.01). In addition, S100A4 sliencing inhibited the expression of S100A4 in vivo, followed by the NF-kB, MMP-2 and bcl-2 suppression.
  
  
  CONCLUSIONS
  We conclude that S100A4 plays a crucial role in proliferation and migratory/invasive processes in human RCC by a mechanism involving activation of NF-kB-bcl-2 and NF-kB-MMP-2 pathway.
authors.affiliation: Department of Urology Surgery, the Affiliated Hospital of Medical College, Qingdao University, Qingdao, China. qyfywang@163.com  
authors.forename: X-C
authors.initials: XC
authors.lastname: Yang
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.forename: L
authors.initials: L
authors.lastname: Luo
authors.forename: D-H
authors.initials: DH
authors.lastname: Dong
authors.forename: Q-C
authors.initials: QC
authors.lastname: Yu
authors.forename: X-S
authors.initials: XS
authors.lastname: Wang
authors.forename: K
authors.initials: K
authors.lastname: Zhao
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D005260:Female; D006801:Humans; D007680:Kidney Neoplasms / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D008175:Lung Neoplasms / Q000556:secondary / Q000628:therapy; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism*; D010641:Phenotype; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C077591:S100A4 protein, human; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2