Figure 1.
miR-7 expression is inversely correlated with EOC metastasis.
(A) The relative expression of miR-7 in 17-paired EOC tissues from omentum or peritoneum metastases and primary EOC tissues was detected by qRT-PCR. The U6 small nuclear RNA was used as an internal control and the fold change was calculated by the ΔΔCt method (B) The expression level of miR-7 in one pair of low and high metastatic EOC cell lines. (C) The expression level of miR-7 in seven EOC cell lines. (*P<0.05. **P<0.01).
Figure 2.
miR-7 downregulates EGFR in EOC cells.
(A) The protein expression of EGFR in 17-paired EOC tissues from omentum or peritoneum metastases and primary EOC tissues was examined by western blot. (B) The protein expression of EGFR in HO-8910 and HO-8910pm cell lines was examined by western blotting. (C) The expression level of miR-7 in HO-8910pm and ES-2 cells transfected with miR-7 or NC was analyzed by qRT-PCR. (D) The expression level of EGFR mRNA in HO-8910pm and ES-2 cells transfected with miR-7 or NC was analyzed by qRT-PCR. (E) The expression of EGFR protein was analyzed by western blotting in HO-8910pm and ES-2 cells transfected with miR-7 or NC, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. (*P<0.05. **P<0.01).
Figure 3.
miR-7 suppresses EOC cell invasion and migration in vitro.
(A) Transwell migration and invasion assays using HO-8910pm cells transfected with miR-7 or negative control (NC). Representative images are shown on the left, and the quantification of 6 randomly selected fields is shown on the right. (B) Transwell migration and invasion assays using ES-2 cells transfected with miR-7 or NC. Representative images are shown on the left, and the quantification of 6 randomly selected fields is shown on the right. The values shown are expressed as the means ± SD. (*P<0.05.**P<0.01).
Figure 4.
Overexpression of miR-7 reverses EMT in EOC cell.
(A) Phase-contrast images of ES-2 cells infected with the miR-7 or NC. (B) The protein expression of E-cadherin, CK-18, β-catenin and Vimentin in HO-8910pm and ES-2 cells transfected with miR-7 or NC were examined by western blotting, GAPDH was used as an internal control. (*P<0.05. **P<0.01).
Figure 5.
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).
Figure 6.
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).
Figure 7.
miR-7 reverses EMT through EGFR/AKT and EGFR/ERK1/2 pathway.
(A) HO-8910pm and ES-2 cells were transfected with EGFR siRNA or NC, the protein expression of E-cadherin, CK-18, β-catenin and vimentin were explored by western blotting. (B) HO-8910pm and ES-2 cells were treated with LY294002 (20 umol/l) or U0126 (10 umol/l), AKT and ERK1/2 phosphorylation were analyzed by western blotting. (C) HO-8910pm and ES-2 cells were treated with LY294002 (20 umol/l) or U0126 (10 umol/l), the protein expression of CK-18, β-catenin and vimentin were explored by western blotting. GAPDH was used as an internal control (*P<0.05. **P<0.01).
Figure 8.
miR-7 and EGFR are inersely expressed in EOC tissues.
(A) Expression of miR-7 in primary EOC and its matched metastatic tissue by CISH. (B) Expression of EGFR in primary EOC and its matched metastatic tissue by IHC. (*P<0.05. **P<0.01).
Table 1.
miR-7 expression in EOC tissues and matched metastases.
Table 2.
EGFR expression in EOC tissues and matched metastases.
Table 3.
Correlation between the expression of EGFR protein and miR-7 in 25 pairs of EOC and their matched metastases.