MicroRNA-148a is downregulated in gastric cancer, targets MMP7, and indicates tumor invasiveness and poor prognosis.
Bottom Line: Downregulation of miR-148a was significantly correlated with an advanced clinical stage, lymph node metastasis, and poor clinical outcome.Custom oligonucleotide array analysis revealed that MMP7 expression was markedly downregulated in miR-148a-overexpressing GC cells; MMP7 was found to be a direct and functional target of miR-148a, participating in cell invasion.These results suggest that miR-148a contributes to the maintenance of homeostasis in normal stomach tissue and plays an important role in GC invasion by regulating MMP7 expression.
Affiliation: Department of Molecular Pathology, Hiroshima University Institute of Biomedical and Health Sciences, Hiroshima, Japan.Show MeSH
Related in: MedlinePlus
Mentions: To confirm the regulation of MMP7 by miR-148a, MKN-45 and MKN-74 cells were transfected to induce miR-148a overexpression, then examined for MMP7 expression level by Western blotting. In both cell lines, MMP7 expression was significantly downregulated; levels were similar to that of MMP7-specific siRNA treated cells (Fig. 3a). The MMP7 mRNA levels determined by qRT-PCR were consistent with the MMP7 protein levels determined by Western blotting (Fig. 3b). It was also found that upregulation of MMP7 was inversely associated with miR-148a expression in GC cases analyzed in Figure 1(b) (Table S2). In addition, we examined the effect of miR-148a deregulation on the expression of epithelial–mesenchymal transition-related molecules. However, no significant alterations of these molecules were detected in miR-148a deregulated cells (Fig. S4a). As shown in Figure 2(b), overexpression of miR-148a repressed invasion of MKN-1 cells. However, MMP7 expression was not detected in MKN-1 cells (data not shown). It was reported that ROCK1 is a direct target of miR-148a and could be involved in GC invasion.(22) We checked expression of ROCK1 in MKN-1 and the alteration of its expression in miR-148a-deregulated cells. Actually, we detected that expression of ROCK1 was downregulated by miR-148a overexpression in MKN-1 cells (Fig. S4b). Furthermore, to prove that MMP7 is a direct target of miR-148a, we used a 3′-UTR sequence of MMP7 cloned into a reporter vector downstream of the luciferase complementary DNA (Fig. 3c). Transfection of this construct into three GC cell lines with high endogenous miR-148a expression levels led to suppression of MMP7 reporter activity, and mutation of the miR-148a binding site abolished the inhibitory effect of miR-148a on reporter activity (Fig. 3d). Conversely, transfection of this construct into three GC cell lines with low endogenous miR-148a expression levels did not lead to suppression of MMP7 reporter activity. However, cotransfection of this construct with miR-148a precursor into these cells showed that miR-148a suppressed the MMP7 reporter activity (Fig. S5). These results imply that miR-148a downregulates MMP7 expression by directly targeting its 3′-UTR.
Affiliation: Department of Molecular Pathology, Hiroshima University Institute of Biomedical and Health Sciences, Hiroshima, Japan.