title: MiR-374b-5p suppresses RECK expression and promotes gastric cancer cell invasion and metastasis.
abstract: OBJECTIVE
  To profile expression of microRNAs (miRNAs) in gastric cancer cells and investigate the effect of miR-374b-5p on gastric cancer cell invasion and metastasis.
  
  
  METHODS
  An miRNA microarray assay was performed to identify miRNAs differentially expressed in gastric cancer cell lines (MGC-803 and SGC-7901) compared with a normal gastric epithelial cell line. Upregulation of miR-374b-5p was newly identified and confirmed via quantitative real-time reverse transcription-PCR (qRT-PCR). MGC-803 cells were transfected with a synthesized anti-miR-374b-5p sequence or a control vector using Lipofectamine reagent, or treated with transfection reagent alone or phosphate-buffered saline as controls. Rate of transfection was verified after 48 h by qRT-PCR. Cells were then subjected to transwell migration, wound scratch and cell counting kit-8 assays. A bioinformatic analysis to identify miR-374b-5p target genes was performed using miRanda, PicTar and TargetScan software. A dual luciferase reporter assay was performed to evaluate the influence of miR-374b-5p on target gene activation, and qRT-PCR and Western blot were used to evaluate the levels of target mRNA and protein following transfection with miR-374b-5p antisense oligonucleotides.
  
  
  RESULTS
  The microarray profiling revealed downregulation of 14 (fold change < 0.667; P < 0.05) and upregulation of 12 (fold change > 1.50; P < 0.05) miRNAs in MGC-803 and SGC-7901 cells compared with GES-1 controls. The upregulation of miR-374b-5p (fold change = 1.75 and 1.64 in MGC-803 and SGC-7901, respectively; P < 0.05) was confirmed by qRT-PCR. Compared with the control groups, the restoration of miR-374b-5p expression with anti-miR-374b-5p significantly suppressed the metastasis, invasion and proliferation of MGC-803 cells. The bioinformatic analysis predicted that the 3' untranslated region (UTR) of reversion-inducing cysteine-rich protein with Kazal motif (RECK) contains three miR-374b-5p target sequences. RECK was verified as a target gene in a dual luciferase reporter assay showing that activation of RECK 3'UTR-pmirGLO was increased by co-transfection with miR-374b-5p. Finally, transfection of miR-374b-5p antisense oligonucleotides increased mRNA and protein levels of RECK in MGC-803 cells (P < 0.05).
  
  
  CONCLUSIONS
  These findings indicate that upregulation of miR-374b-5p contributes to gastric cancer cell metastasis and invasion through inhibition of RECK expression.
authors.affiliation: Juan Xie, Zhi-Hui Tan, Xia Tang, Yan-Ping Liu, Li Zhang, Guo-Qing Li, Department of Gastroenterology, the Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan Province, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Xie
authors.affiliation: Juan Xie, Zhi-Hui Tan, Xia Tang, Yan-Ping Liu, Li Zhang, Guo-Qing Li, Department of Gastroenterology, the Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan Province, China.
authors.forename: Zhi-Hui
authors.initials: ZH
authors.lastname: Tan
authors.affiliation: Juan Xie, Zhi-Hui Tan, Xia Tang, Yan-Ping Liu, Li Zhang, Guo-Qing Li, Department of Gastroenterology, the Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan Province, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Tang
authors.affiliation: Juan Xie, Zhi-Hui Tan, Xia Tang, Yan-Ping Liu, Li Zhang, Guo-Qing Li, Department of Gastroenterology, the Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan Province, China.
authors.forename: Ming-Shu
authors.initials: MS
authors.lastname: Mo
authors.affiliation: Juan Xie, Zhi-Hui Tan, Xia Tang, Yan-Ping Liu, Li Zhang, Guo-Qing Li, Department of Gastroenterology, the Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan Province, China.
authors.forename: Yan-Ping
authors.initials: YP
authors.lastname: Liu
authors.affiliation: Juan Xie, Zhi-Hui Tan, Xia Tang, Yan-Ping Liu, Li Zhang, Guo-Qing Li, Department of Gastroenterology, the Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan Province, China.
authors.forename: Run-Liang
authors.initials: RL
authors.lastname: Gan
authors.affiliation: Juan Xie, Zhi-Hui Tan, Xia Tang, Yan-Ping Liu, Li Zhang, Guo-Qing Li, Department of Gastroenterology, the Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan Province, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.affiliation: Juan Xie, Zhi-Hui Tan, Xia Tang, Yan-Ping Liu, Li Zhang, Guo-Qing Li, Department of Gastroenterology, the Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan Province, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Juan Xie, Zhi-Hui Tan, Xia Tang, Yan-Ping Liu, Li Zhang, Guo-Qing Li, Department of Gastroenterology, the Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan Province, China.
authors.forename: Guo-Qing
authors.initials: GQ
authors.lastname: Li
mesh_terms: D020413:3' Untranslated Regions; D001665:Binding Sites; D045744:Cell Line, Tumor; D002465:Cell Movement; D016000:Cluster Analysis; D019295:Computational Biology; D015536:Down-Regulation; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; D058851:GPI-Linked Proteins; C587995:MIRN374 microRNA 374, human; D035683:MicroRNAs; C115460:RECK protein, human
keywords: Gastric cancer; Invasion and metastasis; RECK; miR-374b-5p; microRNAs microarray