title: The involvement of miR-100 in bladder urothelial carcinogenesis changing the expression levels of mRNA and proteins of genes related to cell proliferation, survival, apoptosis and chromosomal stability.
abstract: BACKGROUND
  MicroRNAs (miRNA) are small non-coding RNAs that play an important role in the control of gene expression by inhibiting protein translation or promoting messenger RNA degradation. Today, miRNAs have been shown to be involved in various physiological and pathological cellular processes, including cancer, where they can act as oncogenes or tumor suppressor genes. Recently, lowered expression of miR-100, resulting in upregulation of FGFR3, has been correlated with low-grade, non-invasive bladder urothelial cancer, as an alternative oncogenesis pathway to the typical FGFR3 gene mutation. Our aim is to analyze the role of miR-100 in bladder cancer cell lines in controlling the expression of some of its possible target genes, including FGFR3 and its relationship with proliferation, apoptosis and DNA ploidy.
  
  
  METHODS
  The bladder cancer cell lines RT4 and T24 were transfected with pre-miR 100, anti-miR 100 and their respective controls using a lipid-based formulation. After transfection mRNA and protein levels of its supposed target genes THAP2, BAZ2A, mTOR, SMARCA5 and FGFR3 were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. Cell proliferation, apoptosis and DNA ploidy were analyzed by flow cytometry. For statistical analysis, a t-test was applied, p < 0.05 was considered significant.
  
  
  RESULTS
  After miR-100 transfection, there was a significant reduction in the mRNA of mTOR (p = 0.006), SMARCA5 (p = 0.007) and BAZ2A (p = 0.029) in RT4, mTOR (p = 0.023) and SMARCA5 (p = 0.015) in T24. There was a reduction in the expression of all proteins, variable from 22.5% to 57.1% in both cell lines. In T24 miR-100 promoted an increase in cell proliferation and anti-miR 100 promoted apoptosis characterizing miR-100 as an oncomiR in this cell line representative of a high-grade urothelial carcinoma.
  
  
  CONCLUSIONS
  miR-100 transfection reduces expression of BAZ2A, mTOR and SMARCA5 mRNA and protein in BC cell lines. miR-100 would be classified as an oncomiR in T24 cells representative of high grade urothelial carcinoma promoting increase in cell proliferation and reduction in apoptosis. The knowledge of miRNA role in tumors will allow their use as tumor markers and targets for new therapies.
authors.affiliation: Laboratory of Medical Research, Department of Urology - LIM55, University of Sao Paulo Medical School, Sao Paulo, Brazil ; Department of Pathology, University of Sao Paulo Veterinary Medicine and Zootechnics School, Sao Paulo, Brazil.
authors.forename: Denis R
authors.initials: DR
authors.lastname: Morais
authors.affiliation: Laboratory of Medical Research, Department of Urology - LIM55, University of Sao Paulo Medical School, Sao Paulo, Brazil.
authors.forename: Sabrina T
authors.initials: ST
authors.lastname: Reis
authors.affiliation: Laboratory of Medical Research, Department of Urology - LIM55, University of Sao Paulo Medical School, Sao Paulo, Brazil.
authors.forename: Nayara
authors.initials: N
authors.lastname: Viana
authors.affiliation: Laboratory of Medical Research, Department of Urology - LIM55, University of Sao Paulo Medical School, Sao Paulo, Brazil.
authors.forename: Camila Berfort
authors.initials: CB
authors.lastname: Piantino
authors.affiliation: Department of Pathology, University of Sao Paulo Veterinary Medicine and Zootechnics School, Sao Paulo, Brazil.
authors.forename: Cristina
authors.initials: C
authors.lastname: Massoco
authors.affiliation: Laboratory of Medical Research, Department of Urology - LIM55, University of Sao Paulo Medical School, Sao Paulo, Brazil.
authors.forename: Caio
authors.initials: C
authors.lastname: Moura
authors.affiliation: Laboratory of Medical Research, Department of Urology - LIM55, University of Sao Paulo Medical School, Sao Paulo, Brazil.
authors.forename: Nelson
authors.initials: N
authors.lastname: Dip
authors.affiliation: Laboratory of Medical Research, Department of Urology - LIM55, University of Sao Paulo Medical School, Sao Paulo, Brazil.
authors.forename: Iran A
authors.initials: IA
authors.lastname: Silva
authors.affiliation: Laboratory of Medical Research, Department of Urology - LIM55, University of Sao Paulo Medical School, Sao Paulo, Brazil.
authors.forename: Miguel
authors.initials: M
authors.lastname: Srougi
authors.affiliation: Laboratory of Medical Research, Department of Urology - LIM55, University of Sao Paulo Medical School, Sao Paulo, Brazil.
authors.forename: Katia Rm
authors.initials: KR
authors.lastname: Leite
keywords: Bladder cancer; Gene expression; MicroRNA; PCR; Protein expression; Western blotting; miR-100