Published online Dec 14, 2014. doi: 10.3748/wjg.v20.i46.17439
Revised: June 23, 2014
Accepted: July 29, 2014
Published online: December 14, 2014
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.
Core tip: Abnormal expression of microRNAs (miRNAs) is one of the key mechanisms in cancer development, invasion and metastasis. The results of this study identify aberrant expression of 26 miRNAs in two human gastric cancer cell lines, which included upregulation of miR-374b-5p. The invasive and metastatic ability of cancer cells was decreased by transfection of miR-374b-5p antisense oligonucleotides. Furthermore, reversion-inducing cysteine-rich protein with Kazal motif was identified as a target of miR-374b-5p. These data suggest that miR-374b-5p may be a potential biomarker and diagnostic tool for gastric cancer classification and diagnosis.