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MicroRNA-7 inhibits tumor metastasis and reverses epithelial-mesenchymal transition through AKT/ERK1/2 inactivation by targeting EGFR in epithelial ovarian cancer.

Zhou X, Hu Y, Dai L, Wang Y, Zhou J, Wang W, Di W, Qiu L - PLoS ONE (2014)

Bottom Line: Overexpression of miR-7 markedly suppressed the capacities of cell invasion and migration and resulted in morphological changes from a mesenchymal phenotype to an epithelial-like phenotype in EOC.The pharmacological inhibition of PI3K-AKT and ERK1/2 both significantly enhanced CK-18 and β-catenin expression and suppressed vimentin expression, indicating that AKT and ERK1/2 pathways are required for miR-7 mediating EMT.Taken together, our results suggested that miR-7 inhibited tumor metastasis and reversed EMT through AKT and ERK1/2 pathway inactivation by reducing EGFR expression in EOC cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Gynecology and Obstetrics, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; Shanghai Key Laboratory of Gynecologic Oncology, Shanghai, China.

ABSTRACT
Epidermal growth factor receptor (EGFR) overexpression and activation result in increased proliferation and migration of solid tumors including ovarian cancer. In recent years, mounting evidence indicates that EGFR is a direct and functional target of miR-7. In this study, we found that miR-7 expression was significantly downregulated in highly metastatic epithelial ovarian cancer (EOC) cell lines and metastatic tissues, whereas the expression of, EGFR correlated positively with metastasis in both EOC patients and cell lines. Overexpression of miR-7 markedly suppressed the capacities of cell invasion and migration and resulted in morphological changes from a mesenchymal phenotype to an epithelial-like phenotype in EOC. In addition, overexpression of miR-7 upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, accompanied with EGFR inhibition and AKT/ERK1/2 inactivation. Similar to miR-7 transfection, silencing of EGFR with this siRNA in EOC cells also upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, and decreased phosphorylation of both Akt and ERK1/2, confirming that EGFR is a target of miR-7 in reversing EMT. The pharmacological inhibition of PI3K-AKT and ERK1/2 both significantly enhanced CK-18 and β-catenin expression and suppressed vimentin expression, indicating that AKT and ERK1/2 pathways are required for miR-7 mediating EMT. Finally, the expression of miR-7 and EGFR in primary EOC with matched metastasis tissues was explored. It was showed that miR-7 is inversely correlated with EGFR. Taken together, our results suggested that miR-7 inhibited tumor metastasis and reversed EMT through AKT and ERK1/2 pathway inactivation by reducing EGFR expression in EOC cell lines. Thus, miR-7 might be a potential prognostic marker and therapeutic target for ovarian cancer metastasis intervention.

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miR-7 suppresses AKT and ERK1/2 pathway activation dependent of its EGFR inhibition in EOC cells.(A) HO-8910pm and ES-2 cells were transfected with miR-7 or NC, EGFR, AKT and ERK1/2 phosphorylation were analyzed by western blotting. (B) HO-8910pm and ES-2 cells were transfected with EGFR siRNA or NC, EGFR, AKT and ERK1/2 phosphorylation were analyzed by western blotting. (*P<0.05. **P<0.01).
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pone-0096718-g006: miR-7 suppresses AKT and ERK1/2 pathway activation dependent of its EGFR inhibition in EOC cells.(A) HO-8910pm and ES-2 cells were transfected with miR-7 or NC, EGFR, AKT and ERK1/2 phosphorylation were analyzed by western blotting. (B) HO-8910pm and ES-2 cells were transfected with EGFR siRNA or NC, EGFR, AKT and ERK1/2 phosphorylation were analyzed by western blotting. (*P<0.05. **P<0.01).

Mentions: Previous studies showed that EGFR regulates AKT and ERK1/2 activity in ovarian cancer [15], [22]. Because miR-7 can post-transcriptionally inhibit the expression of EGFR, we hypothesized that miR-7 regulates AKT and ERK1/2 pathway activation. To investigate the effect of miR-7 on AKT and ERK1/2, we transfected HO-8910pm and ES-2 cells with miR-7 plasmid or miR-NC, and then examined phosphorylation of AKT and ERK1/2 by western blotting. Transfection with miR-7 suppressed phosphorylation of AKT at Ser473 and ERK1/2 at Thr202/Tyr204, however, miR-7 did not significantly alter AKT phosphorylation at Thr308 (Figure 6A). Since the activation of EGFR is playing major role in the metastasis of many cancers, we also detected its phosphorylation status after miR-7 transfection in HO-8910pm and ES-2 cells. However, in our studies, there was no EGFR phosphorylation pre and post miR-7 transfection, whereas miR-7 transfection did inhibit total EGFR protein expression (Figure 6A, Figure S3 (A-C)). As evidence that miR-7 has many target genes, we sought to determine whether miR-7 mediated AKT and ERK1/2 pathway activation is dependent of its EGFR inhibition. HO-8910pm and ES-2 cells were transfected with EGFR siRNA or the negative control. We showed that silencing of EGFR with this siRNA in ovarian cancer cells decreased phosphorylation of AKT at Ser473 and ERK1/2 at Thr202/Tyr204 on Western Blotting, but there was no significant change in phosphorylation of AKT at Thr308 (Figure 6B, Figure S3 (D-E)).


MicroRNA-7 inhibits tumor metastasis and reverses epithelial-mesenchymal transition through AKT/ERK1/2 inactivation by targeting EGFR in epithelial ovarian cancer.

Zhou X, Hu Y, Dai L, Wang Y, Zhou J, Wang W, Di W, Qiu L - PLoS ONE (2014)

miR-7 suppresses AKT and ERK1/2 pathway activation dependent of its EGFR inhibition in EOC cells.(A) HO-8910pm and ES-2 cells were transfected with miR-7 or NC, EGFR, AKT and ERK1/2 phosphorylation were analyzed by western blotting. (B) HO-8910pm and ES-2 cells were transfected with EGFR siRNA or NC, EGFR, AKT and ERK1/2 phosphorylation were analyzed by western blotting. (*P<0.05. **P<0.01).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016102&req=5

pone-0096718-g006: miR-7 suppresses AKT and ERK1/2 pathway activation dependent of its EGFR inhibition in EOC cells.(A) HO-8910pm and ES-2 cells were transfected with miR-7 or NC, EGFR, AKT and ERK1/2 phosphorylation were analyzed by western blotting. (B) HO-8910pm and ES-2 cells were transfected with EGFR siRNA or NC, EGFR, AKT and ERK1/2 phosphorylation were analyzed by western blotting. (*P<0.05. **P<0.01).
Mentions: Previous studies showed that EGFR regulates AKT and ERK1/2 activity in ovarian cancer [15], [22]. Because miR-7 can post-transcriptionally inhibit the expression of EGFR, we hypothesized that miR-7 regulates AKT and ERK1/2 pathway activation. To investigate the effect of miR-7 on AKT and ERK1/2, we transfected HO-8910pm and ES-2 cells with miR-7 plasmid or miR-NC, and then examined phosphorylation of AKT and ERK1/2 by western blotting. Transfection with miR-7 suppressed phosphorylation of AKT at Ser473 and ERK1/2 at Thr202/Tyr204, however, miR-7 did not significantly alter AKT phosphorylation at Thr308 (Figure 6A). Since the activation of EGFR is playing major role in the metastasis of many cancers, we also detected its phosphorylation status after miR-7 transfection in HO-8910pm and ES-2 cells. However, in our studies, there was no EGFR phosphorylation pre and post miR-7 transfection, whereas miR-7 transfection did inhibit total EGFR protein expression (Figure 6A, Figure S3 (A-C)). As evidence that miR-7 has many target genes, we sought to determine whether miR-7 mediated AKT and ERK1/2 pathway activation is dependent of its EGFR inhibition. HO-8910pm and ES-2 cells were transfected with EGFR siRNA or the negative control. We showed that silencing of EGFR with this siRNA in ovarian cancer cells decreased phosphorylation of AKT at Ser473 and ERK1/2 at Thr202/Tyr204 on Western Blotting, but there was no significant change in phosphorylation of AKT at Thr308 (Figure 6B, Figure S3 (D-E)).

Bottom Line: Overexpression of miR-7 markedly suppressed the capacities of cell invasion and migration and resulted in morphological changes from a mesenchymal phenotype to an epithelial-like phenotype in EOC.The pharmacological inhibition of PI3K-AKT and ERK1/2 both significantly enhanced CK-18 and β-catenin expression and suppressed vimentin expression, indicating that AKT and ERK1/2 pathways are required for miR-7 mediating EMT.Taken together, our results suggested that miR-7 inhibited tumor metastasis and reversed EMT through AKT and ERK1/2 pathway inactivation by reducing EGFR expression in EOC cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Gynecology and Obstetrics, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; Shanghai Key Laboratory of Gynecologic Oncology, Shanghai, China.

ABSTRACT
Epidermal growth factor receptor (EGFR) overexpression and activation result in increased proliferation and migration of solid tumors including ovarian cancer. In recent years, mounting evidence indicates that EGFR is a direct and functional target of miR-7. In this study, we found that miR-7 expression was significantly downregulated in highly metastatic epithelial ovarian cancer (EOC) cell lines and metastatic tissues, whereas the expression of, EGFR correlated positively with metastasis in both EOC patients and cell lines. Overexpression of miR-7 markedly suppressed the capacities of cell invasion and migration and resulted in morphological changes from a mesenchymal phenotype to an epithelial-like phenotype in EOC. In addition, overexpression of miR-7 upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, accompanied with EGFR inhibition and AKT/ERK1/2 inactivation. Similar to miR-7 transfection, silencing of EGFR with this siRNA in EOC cells also upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, and decreased phosphorylation of both Akt and ERK1/2, confirming that EGFR is a target of miR-7 in reversing EMT. The pharmacological inhibition of PI3K-AKT and ERK1/2 both significantly enhanced CK-18 and β-catenin expression and suppressed vimentin expression, indicating that AKT and ERK1/2 pathways are required for miR-7 mediating EMT. Finally, the expression of miR-7 and EGFR in primary EOC with matched metastasis tissues was explored. It was showed that miR-7 is inversely correlated with EGFR. Taken together, our results suggested that miR-7 inhibited tumor metastasis and reversed EMT through AKT and ERK1/2 pathway inactivation by reducing EGFR expression in EOC cell lines. Thus, miR-7 might be a potential prognostic marker and therapeutic target for ovarian cancer metastasis intervention.

Show MeSH
Related in: MedlinePlus