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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 downregulate EGFR by interaction with 3′UTR.(A) Diagram of EGFR 3′UTR reporter construct. (B) The wild type or mutant reporter plasmids were cotransfected with miR-7 or NC in ES-2. (*P<0.05. **P<0.01).
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pone-0096718-g005: miR-7 downregulate EGFR by interaction with 3′UTR.(A) Diagram of EGFR 3′UTR reporter construct. (B) The wild type or mutant reporter plasmids were cotransfected with miR-7 or NC in ES-2. (*P<0.05. **P<0.01).

Mentions: To understand the molecular mechanism by which miR-7 suppress EOC invasion and metastasis, we used different computational methods to search for miR-7 targets, such as Targetsan and pictar. These methods found 181 possible candidate genes. We were particularly interested in EGFR because of its positive roles in cancer cell invasion. Analysis of the 3′-UTR sequence of EGFR indentified three possible binding sites for miR-7. To determine whether EGFR is direct target of miR-7, we constructed its 3′-UTR fragments, in which wild-type and mutant binding sites were inserted into the region immediatedly downstream of the reporter gene (Figure 5A), luciferase reporter assays showed that miR-7 transfection caused a remarkable decrease in luciferase activity which contained wild-type 3′-UTR fragments binding sites (Figure 5B).


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 downregulate EGFR by interaction with 3′UTR.(A) Diagram of EGFR 3′UTR reporter construct. (B) The wild type or mutant reporter plasmids were cotransfected with miR-7 or NC in ES-2. (*P<0.05. **P<0.01).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096718-g005: miR-7 downregulate EGFR by interaction with 3′UTR.(A) Diagram of EGFR 3′UTR reporter construct. (B) The wild type or mutant reporter plasmids were cotransfected with miR-7 or NC in ES-2. (*P<0.05. **P<0.01).
Mentions: To understand the molecular mechanism by which miR-7 suppress EOC invasion and metastasis, we used different computational methods to search for miR-7 targets, such as Targetsan and pictar. These methods found 181 possible candidate genes. We were particularly interested in EGFR because of its positive roles in cancer cell invasion. Analysis of the 3′-UTR sequence of EGFR indentified three possible binding sites for miR-7. To determine whether EGFR is direct target of miR-7, we constructed its 3′-UTR fragments, in which wild-type and mutant binding sites were inserted into the region immediatedly downstream of the reporter gene (Figure 5A), luciferase reporter assays showed that miR-7 transfection caused a remarkable decrease in luciferase activity which contained wild-type 3′-UTR fragments binding sites (Figure 5B).

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