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

Juan Xie, Zhi-Hui Tan, Xia Tang, Ming-Shu Mo, Yan-Ping Liu, Run-Liang Gan, Yi Li, Li Zhang, Guo-Qing Li
World Journal of Gastroenterology: WJG 2014 December 14, 20 (46): 17439-47

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.

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