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MicroRNA-148a is downregulated in gastric cancer, targets MMP7, and indicates tumor invasiveness and poor prognosis.

Sakamoto N, Naito Y, Oue N, Sentani K, Uraoka N, Zarni Oo H, Yanagihara K, Aoyagi K, Sasaki H, Yasui W - Cancer Sci. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology, Hiroshima University Institute of Biomedical and Health Sciences, Hiroshima, Japan.

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Effect of microRNA-148a (miR-148a) on MMP7 expression and gastric cancer (GC) cell invasiveness. (a) Western blot analysis of MMP7 in MKN-45 and MKN-74 cell lysates after treatment with negative control miRNA, pre-miR-148a, or MMP7-specific siRNA. A β-actin blot served as loading control. (b) Quantitative RT-PCR analysis of MMP7 in MKN-45 and MKN-74 cells after treatment with negative control miRNA, pre-miR-148a, or MMP7-specific siRNA. Results are mean ± SD of triplicate measurements. ***P < 0.001. (c) Schematic representation of miR-148a seed sequence and MMP7 3′-UTR, showing the putative miR-148a-binding site. The seed region is in bold text and the seed region is mutated in the reporter construct. (d) Luciferase activity of three GC cell lines cotransfected with reporter vector containing either control vector, wild-type (WT), or mutant miR-148a (Mut) 3-′UTR. Results are mean ± SD of triplicate measurements. ***P < 0.001. N.S., not significant. (e) Western blot analysis of MMP7 in MKN-45 cell lysates was carried out after stable transfection with the miR-148a construct, both miR-148a and MMP7 construct, or empty vector. β-Actin served as loading control. (f) Effect of miR-148a-stable transfection and restoration of MMP7 on cell invasion of MKN-45 cells. Boyden chambers were used for incubation of MKN-45 cells after stable transfection with miR-148a construct, both miR-148a and MMP7 construct, or empty vector. Results are mean ± SD of triplicate measurements. **P < 0.01; ***P < 0.001.
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fig03: Effect of microRNA-148a (miR-148a) on MMP7 expression and gastric cancer (GC) cell invasiveness. (a) Western blot analysis of MMP7 in MKN-45 and MKN-74 cell lysates after treatment with negative control miRNA, pre-miR-148a, or MMP7-specific siRNA. A β-actin blot served as loading control. (b) Quantitative RT-PCR analysis of MMP7 in MKN-45 and MKN-74 cells after treatment with negative control miRNA, pre-miR-148a, or MMP7-specific siRNA. Results are mean ± SD of triplicate measurements. ***P < 0.001. (c) Schematic representation of miR-148a seed sequence and MMP7 3′-UTR, showing the putative miR-148a-binding site. The seed region is in bold text and the seed region is mutated in the reporter construct. (d) Luciferase activity of three GC cell lines cotransfected with reporter vector containing either control vector, wild-type (WT), or mutant miR-148a (Mut) 3-′UTR. Results are mean ± SD of triplicate measurements. ***P < 0.001. N.S., not significant. (e) Western blot analysis of MMP7 in MKN-45 cell lysates was carried out after stable transfection with the miR-148a construct, both miR-148a and MMP7 construct, or empty vector. β-Actin served as loading control. (f) Effect of miR-148a-stable transfection and restoration of MMP7 on cell invasion of MKN-45 cells. Boyden chambers were used for incubation of MKN-45 cells after stable transfection with miR-148a construct, both miR-148a and MMP7 construct, or empty vector. Results are mean ± SD of triplicate measurements. **P < 0.01; ***P < 0.001.

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.


MicroRNA-148a is downregulated in gastric cancer, targets MMP7, and indicates tumor invasiveness and poor prognosis.

Sakamoto N, Naito Y, Oue N, Sentani K, Uraoka N, Zarni Oo H, Yanagihara K, Aoyagi K, Sasaki H, Yasui W - Cancer Sci. (2014)

Effect of microRNA-148a (miR-148a) on MMP7 expression and gastric cancer (GC) cell invasiveness. (a) Western blot analysis of MMP7 in MKN-45 and MKN-74 cell lysates after treatment with negative control miRNA, pre-miR-148a, or MMP7-specific siRNA. A β-actin blot served as loading control. (b) Quantitative RT-PCR analysis of MMP7 in MKN-45 and MKN-74 cells after treatment with negative control miRNA, pre-miR-148a, or MMP7-specific siRNA. Results are mean ± SD of triplicate measurements. ***P < 0.001. (c) Schematic representation of miR-148a seed sequence and MMP7 3′-UTR, showing the putative miR-148a-binding site. The seed region is in bold text and the seed region is mutated in the reporter construct. (d) Luciferase activity of three GC cell lines cotransfected with reporter vector containing either control vector, wild-type (WT), or mutant miR-148a (Mut) 3-′UTR. Results are mean ± SD of triplicate measurements. ***P < 0.001. N.S., not significant. (e) Western blot analysis of MMP7 in MKN-45 cell lysates was carried out after stable transfection with the miR-148a construct, both miR-148a and MMP7 construct, or empty vector. β-Actin served as loading control. (f) Effect of miR-148a-stable transfection and restoration of MMP7 on cell invasion of MKN-45 cells. Boyden chambers were used for incubation of MKN-45 cells after stable transfection with miR-148a construct, both miR-148a and MMP7 construct, or empty vector. Results are mean ± SD of triplicate measurements. **P < 0.01; ***P < 0.001.
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fig03: Effect of microRNA-148a (miR-148a) on MMP7 expression and gastric cancer (GC) cell invasiveness. (a) Western blot analysis of MMP7 in MKN-45 and MKN-74 cell lysates after treatment with negative control miRNA, pre-miR-148a, or MMP7-specific siRNA. A β-actin blot served as loading control. (b) Quantitative RT-PCR analysis of MMP7 in MKN-45 and MKN-74 cells after treatment with negative control miRNA, pre-miR-148a, or MMP7-specific siRNA. Results are mean ± SD of triplicate measurements. ***P < 0.001. (c) Schematic representation of miR-148a seed sequence and MMP7 3′-UTR, showing the putative miR-148a-binding site. The seed region is in bold text and the seed region is mutated in the reporter construct. (d) Luciferase activity of three GC cell lines cotransfected with reporter vector containing either control vector, wild-type (WT), or mutant miR-148a (Mut) 3-′UTR. Results are mean ± SD of triplicate measurements. ***P < 0.001. N.S., not significant. (e) Western blot analysis of MMP7 in MKN-45 cell lysates was carried out after stable transfection with the miR-148a construct, both miR-148a and MMP7 construct, or empty vector. β-Actin served as loading control. (f) Effect of miR-148a-stable transfection and restoration of MMP7 on cell invasion of MKN-45 cells. Boyden chambers were used for incubation of MKN-45 cells after stable transfection with miR-148a construct, both miR-148a and MMP7 construct, or empty vector. Results are mean ± SD of triplicate measurements. **P < 0.01; ***P < 0.001.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology, Hiroshima University Institute of Biomedical and Health Sciences, Hiroshima, Japan.

Show MeSH
Related in: MedlinePlus