title: Restoration of miR-517a expression induces cell apoptosis in bladder cancer cell lines.
abstract: The aim of this study was to find novel tumor suppressor microRNAs through screening genes epigenetically silenced by methylation in bladder cancer (BC) cell lines using microRNA microarrays. Since miR-517a and miR-520g, both located on chromosome 19q13.42, were found to highly up-regulated genes after treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dc), we hypothesized that they are tumor-suppressor microRNAs and performed a gain-of-function study using these mature microRNAs. The miR-517a restoration showed significant inhibition of cell proliferation in the transfectants compared to miR-control-transfected cells (p<0.0001 both in BOY and T24 cells). Furthermore, ectopic overexpression of miR-517a markedly induced apoptosis in the miR-517a-transfected BC cell lines. In addition, we carried out oligo microarray analysis using miR-517a transfectants and miR-control transfectants (BOY and T24), from which 35 down-regulated genes and 19 up-regulated genes were identified. These included amphiregulin (AREG) and BCL2-associated transcription factor 1, transcript variant 1 (BCLAF1), previously reported to be concerned with apoptosis, in both cell lines by miR-517a restoration. These data suggest that miR-517a functions as a tumor suppressor through inhibition of cell proliferation and induction of apoptosis under the regulation of AREG and/or BCLAF1 in BC cells. Anti-apoptotic effects may be maintained by down-regulation of miR-517a due to DNA hypermethylation in human BC cells, suggesting that restoration of miR-517a may be a novel therapeutic strategy for human BC.
authors.affiliation: Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Yoshitomi
authors.forename: Kazumori
authors.initials: K
authors.lastname: Kawakami
authors.forename: Hideki
authors.initials: H
authors.lastname: Enokida
authors.forename: Takeshi
authors.initials: T
authors.lastname: Chiyomaru
authors.forename: Ichiro
authors.initials: I
authors.lastname: Kagara
authors.forename: Shuichi
authors.initials: S
authors.lastname: Tatarano
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Yoshino
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Arimura
authors.forename: Kenryu
authors.initials: K
authors.lastname: Nishiyama
authors.forename: Naohiko
authors.initials: N
authors.lastname: Seki
authors.forename: Masayuki
authors.initials: M
authors.lastname: Nakagawa
mesh_terms: D066258:Amphiregulin; D017209:Apoptosis / Q000235:genetics*; D045744:Cell Line, Tumor; D066255:EGF Family of Proteins; D015870:Gene Expression; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D046228:Microarray Analysis; D012097:Repressor Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics; D001749:Urinary Bladder Neoplasms / Q000235:genetics*
chemical_list: C587848:AREG protein, human; D066258:Amphiregulin; C119386:BCLAF1 protein, human; D066255:EGF Family of Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; C546349:MIRN517 microRNA, human; D035683:MicroRNAs; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins