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. 2014 Dec 14;20(46):17439-47.
doi: 10.3748/wjg.v20.i46.17439.

MiR-374b-5p suppresses RECK expression and promotes gastric cancer cell invasion and metastasis

Juan Xie  1 , Zhi-Hui Tan  1 , Xia Tang  1 , Ming-Shu Mo  1 , Yan-Ping Liu  1 , Run-Liang Gan  1 , Yi Li  1 , Li Zhang  1 , Guo-Qing Li  1
Affiliations

MiR-374b-5p suppresses RECK expression and promotes gastric cancer cell invasion and metastasis

Juan Xie et al. World J Gastroenterol. .

Abstract

Aim: 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).

Conclusion: These findings indicate that upregulation of miR-374b-5p contributes to gastric cancer cell metastasis and invasion through inhibition of RECK expression.

Keywords: Gastric cancer; Invasion and metastasis; RECK; miR-374b-5p; microRNAs microarray.

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Figures

Figure 1
Figure 1
Differential expression of microRNAs in gastric cancer cells. A: microRNA (miRNA) expression in gastric cancer cell lines SGC-7901 and MGC-803 was compared with the normal GES-1 cell line using cluster analysis. All cell samples were detected in triplicate, such that the nine lanes include GES-1 (G1-G3), SGC-7901 (S1-S3) and MGC-803 (M1-M3). The color scale denotes the extent of relative upregulation (red) and downregulation (green); B: Histogram of miR-374b-5p expression detected by cluster analysis; C: Confirmation of miR-374b-5p expression via quantitative real-time reverse transcription-polymerase chain reaction (n = 3); aP < 0.05 vs GSE-1.
Figure 2
Figure 2
Effects of miR-374b-5p on MGC-803 cell metastasis and invasion. MGC-803 cells were transfected with a miR-374b-5p antisense oligonucleotide or negative control-oligo vector, or treated with phosphate-buffered saline (untreated), or transfection reagent only. A: Cells were subjected to a transwell invasion assay and detected at 48 h after seeding (left: Coomassie Brilliant Blue staining, × 200; right, quantification); B: Wound margin distance was measured after 48 h in a scratch assay (Coomassie Brilliant Blue staining, × 40); C: Absorbances in a cell counting kit-8 assay 96 h after transfection; (n = 3), aP < 0.05 vs Anti-miR-374b-5p.
Figure 3
Figure 3
Regulation of RECK expression in MGC-803 cells by miR-374b-5p. A: The miR-374b-5p binding site within the 3′ UTR of human (hsa) RECK cDNA was predicted with PicTar, TargetScan and miRanda software; B: Cells were co-transfected with RECK 3’UTR-pmirGLO and a plasmid containing either miR-374b-5p or miR-NC plasmid, aP < 0.05 vs miR-NC (n = 3); C: Western blot of proteins; D: qRT-PCR from cells transfected for 48 h with anti-miR-374b-5p or a negative control vector, or treated with phosphate-buffered saline (untreated), or transfection reagent only; (n = 3), cP < 0.05 vs Anti-miR-374b-5p.
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