Abstract
Recent evidence shows that altered microRNA-9 (miR-9) expression is implicated in the progression of gastric cancer. However, the exact roles and underlying mechanisms of miR-9 in the proliferation, invasion and metastasis of gastric cancer still remain unknown. In this study, miR-9 was found to be down-regulated and inversely correlated with the expression of cyclin D1 and v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets1) in gastric cancer tissues and cell lines. Bioinformatics analysis revealed the putative miR-9 binding sites in the 3′-untranslated regions (3′-UTR) of cyclin D1 and Ets1 mRNA. Ectopic expression or knockdown of miR-9 resulted in responsively altered expression of cyclin D1, Ets1 and their downstream targets phosphorylated retinoblastoma and matrix metalloproteinase 9 in cultured gastric cancer cell lines SGC-7901 and AGS. In the luciferase reporter system, miR-9 directly targeted the 3′-UTR of cyclin D1 and Ets1, and these effects were abolished by mutating the miR-9 binding sites. Over-expression of miR-9 suppressed the proliferation, invasion, and metastasis of SGC-7901 and AGS cells in vitro and in vivo. Restoration of miR-9-mediated down-regulation of cyclin D1 and Ets1 by transient transfection, rescued the cancer cells from decrease in proliferation, migration and invasion. Furthermore, anti-miR-9 inhibitor promoted the proliferation, migration and invasion of gastric cancer cells, while knocking down of cyclin D1 or Ets1 partially phenocopied the effects of miR-9 over-expression. These data indicate that miR-9 suppresses the expression of cyclin D1 and Ets1 via the binding sites in their 3′-UTR, thus inhibiting the proliferation, invasion and metastasis of gastric cancer.
Highlights
Gastric cancer is the fourth most common cancer in the world [1]
These results indicated that miR-9 was down-regulated and inversely correlated with expression of cyclin D1 and Ets1 in gastric cancer tissues and cell lines
Knockdown of miR-9 with anti-miR-9 inhibitor increased the luciferase activities in GES-1, SGC-7901 and AGS cells (Fig. 3C and Fig. S4C), while mutation of miR-9 recognition site abolished these effects (Fig. 3C and Fig. S4C). These results indicated that miR-9 directly and interacted with the target sites in the 39-untranslated regions (39-UTR) of cyclin D1 and Ets1
Summary
In spite of the improvement in surgical and multimodal therapy, the prognosis of advanced gastric cancer still remains poor due to the recurrence, invasion and metastasis, with a 5-year survival rate below 30% [1]. MicroRNAs (miRNAs), a recently identified category of small and highly conserved noncoding RNAs, can participate in the post-transcriptional regulation of gene expression through partial complementary binding with the 39 untranslated regions (39-UTRs) of target mRNA, resulting in translational repression or mRNA degradation [3]. Emerging evidence shows that miRNAs are involved in the biological processes related to apoptosis, proliferation, differentiation, invasion and metastasis, while deregulation of which is crucial to cancer initiation and progression [3]. It is currently urgent to investigate the roles of miRNAs and their target genes in tumor progression by experimental models
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