Ovarian cancer cell lines HO8910, A2780, SKOV3, and COV434 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), human ovarian surface epithelium HOSE cells were purchased from Pricells (Wuhan, China) and cultured in DMEM medium containing 10% fetal bovine serum (FBS). HO8910, A2780, SKOV3 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% antibiotic-antimycotic solution (100 U/ml penicillin and 100 μg/ml streptomycin). COV434 cell were cultured in McCoys 5A culture media. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂. Transfection was carried out by Lipofectamine 2000 reagent (Invitrogen). The plasmids of miR-382 mimics (product no. HmiR-AN0480), miR-382 inhibitor (product no. HmiR-AN0480-SN-10), miRNA NC mimics (product no. CmiR-AN0001) and miRNA NC inhibitor (product no. CmiR-AN0001-SN) were purchased from Fulen Gen (Guangzhou, China). The plasmid of pCDNA3.1-ROR1 was constructed by AuraGene (Changsha, China).

Prediction of miR-382 target sites was performed by the online software MicroRNA.org - Targets and Expression (http://www.microrna.org/microrna/home.do). The related function of the target was also considered.

Total RNA was extracted by TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) methods, and was reverse transcribed as cDNA by using reverse transcription system (Fermentans, Canada). The primers were designed using Primer 5.0, and the forward and reverse primer sequences are showed in Table I. The quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green qPCR (Toyobo, Japan) for studying the quantitative expression of various genes (ROR1, E-cadherin, vimentin, N-cadherin and Snail) according to the manufacturer's protocol. Three repetitions were tested for qRT-PCR. The relative expressions of miR-382 and mRNAs were normalized to those of internal reference U6 or β-actin, and were calculated by the 2^−ΔΔCt method.

Approximately 5×10³ cells per well were plated into 96-well plates (Costar, USA). After culturing for 24, 48, or 72 h, the cells were treated with fresh serum-free medium and 10 μl/well MTT (Biosharp, Hefei, China) solution (10 mg/ml in PBS) according to the manufacturer's protocol. DMSO 100 μl was added to every well after the incubation for 4 h. Following incubation at 37°C for 10 min, the absorbance was measured by microplate reader (Thermo, USA) at 570 nm at room temperature.

Cells were trypsinized and suspended in medium including 0.3% agar and 10% serum, then were plated onto a bottom layer with 0.6% agar. The cells were plated at 300 cells/well into 6-well plates (Costar). The number of colonies were counted after 14 days by Giemsa staining (Solarbio, Beijing, China) via the equation colony forming efficiency = (number of colonies/number of inoculated cells) × 100%.

Cells (closely 1×10⁵ cells) were seeded into 12-well plates (Costar), and were incubated at 37°C until cells reached a confluence of ≥90%. A scratch was generated by use of a sterile 10-μl pipette tip, and this time was considered as 0 h for each experiment. Then, at 48 h, photographic images were acquired with an inverted microscope (Motic, Xiamen, China). Thus, migration distance = the gap of 0 h - the gap of 48 h the gaps of 0 h, so the migration distances are presented a reverse relationship with the gap of 48 h (Figs. 3 and 7).

Invasion assay were performed in 24-well plates with 8-μm pore size chamber inserts (BD, Frankin Lakes, NJ, USA). Approximately 1×10⁵ cells/well were resuspended in 200 μl of medium without fetal bovine serum (FBS; Gibco), and seeded on the upper chamber with the Matrigel-coated membrane. In addition, 500 μl medium supplemented with 10% FBS was added into the lower chamber. After 24-h incubation at 37°C and 5% CO₂, the membranes were stained with 0.1% crystal violet. The cell numbers were counted via an inverted microscope (Motic). Each assay was performed three independent times.

Protein extracts from SKOV3 and COV434 cells were prepared using RIPA lysis buffer (AuraGene) according to the manufacturer's protocol. The protein concentration was confirmed in accordance with the Bradford protein assay reagent (Beyotime Biotechnology, Suzhou, China), and bovine serum albumin was utilized as a standard. Western blot analysis was subsequently carried out to evaluate the levels of ROR1 (1:200, BM0049, ABZOOM), E-cadherin (1:200, YT1453, Immunoway, Changsha, China), vimentin (1:1,000, YT4881, Immunoway), N-cadherin (1:1,000, ab18203, Abcam, China) and Snail (1:200, ab180714, Abcam). Equal amounts of lysate were resolved via SDS-PAGE, and transferred to a PVDF membrane (Millipore, Bedford, MA, USA) through a semidry transfer method. The PVDF membrane was blocked with 5% non-fat milk in TBST buffer for 2 h at room temperature, and then was incubated overnight with primary antibodies, and then was incubated for 1 h using a suitable secondary antibody (1:2,000, Jackson, USA). Electrochemiluminescence was performed with a Gel Documentation and Analysis System.

Luciferase assays were performed in SKOV3 and COV434 cells. SKOV3 and COV434 cells were transfected with each of the plasmids [empty vector (MOCK), ROR1 UTR wild-type (WT) and ROR1 UTR mutant (MUT), as a miR-382 binding site] together with miR-382 mimics, miR-382 inhibitor, and negative control RNA in 24-well plates. Two days after transfection, cells were harvested and lysed. Dual luciferase reporter gene assay kit (BioVision, Milpitas, CA, USA) was used to determine the luciferase activities on a luminometer (Roche, Basel, Switzerland). Renilla luciferase activity was normalized to firefly luciferase activity.

All statistical tests were conducted with SPSS 17.0 software. The variance between two groups and multiple groups were compared by Student's t-test and ANOVA, respectively. The data are expressed as mean ± standard deviation (SD). The p-values <0.05 were regarded as statistically significant.

To investigate the role of miR-382 in ovarian cancer, the expression of miR-382 was examined in tissues and cell lines by qRT-PCR assays. We found the expression of miR-382 was significantly downregulated in human ovarian cancer tissues compared to adjacent non-cancerous tissues (n=20) (Fig. 1A). Moreover, the expression of miR-382 in ovarian cancer cell lines (H08019, A2780, SKOV3 and COV434) was markedly less than in ovarian epithelial cells HOSE (Fig. 1B). These results indicate that miR-382 is downregulated in both human ovarian cancer tissues and cancer cell lines.

To better understand the role of miR-382 in ovarian cancer, we used retroviral vectors to establish ovarian cancer cell lines stably overexpressing or silencing miR-382. The expression levels of miR-382 in the SKOV3 and COV434 cell lines were examined by qRT-PCR (Fig. 2A and B). We also measured the growth-promoting effect of miR-382 on ovarian cancer cells by MTT and colony formation assays. MTT assays revealed that overexpression of miR-382 significantly suppressed proliferation of ovarian cancer cells and silencing miR-382 in ovarian cancer cells dramatically promoted proliferation (Fig. 2C and D). In colony formation assay, overexpression of miR-382 significantly inhibited the viability of indicated cells which formed less and smaller clones in SKOV3 (Fig. 2E) and COV434 cells (Fig. 2F). These findings suggest that miR-382 suppresses proliferation of ovarian cancer cells in vitro.

To evaluate the function of miR-382 in the migration and invasion efficiency of ovarian cancer cells, the scratch test and Transwell assays were performed. In scratch assay, overexpression of miR-382 significantly promoted wound healing of SKOV3 (Fig. 3A and B) and COV434 cells (Fig. 3C and D). Moreover, Transwell assay was used to evaluate the invasive ability of ovarian cancer cells. The results indicated that ectopic expression of miR-382 significantly decreased the invasion rate of SKOV3 (Fig. 3E and F) and COV434 cells (Fig. 3G and H).

EMT is taken for a key mechanism by which cancer cells acquire their migratory and invasive capabilities. To further investigate whether the inhibitory effect of miR-382 on migration and invasion was mediated by epithelial to mesenchymal transition (EMT), we examined the expression of several EMT markers by qRT-PCR and western blot assays. Both in mRNA and protein level as expected, miR-382 overexpression increased the expression level of epithelial marker (E-cadherin) and decreased the levels of mesenchymal markers in SKOV3 (Fig. 4A, C and D) and COV434 cells (Fig. 4B, C and E). Taken together, these findings suggest that miR-382 was able to impede invasion mediated by EMT in vitro.

Many miRNAs have been reported to function by binding a specific target. To examine whether miR-382 has a similar mechanism, prediction of miRNA target sites was performed by the online software MicroRNA.org - Targets and Expression (http://www.microrna.org/microrna/home.do), showing the ROR1-3′-UTR regions contain the miR-382 complementary sequence (Fig. 5A). In addition, it has been reported that ROR1 function is crucial in ovarian cancer. Thus, we focused on ROR1 as the primary candidate target of miR-382, and examined firstly the direct binding between miR-382 and ROR1, we constructed the vectors ROR1-3′-UTR and mut-ROR1-3′-UTR to observe their binding activity with miR-382. miR-382 overexpression consistently and significantly reduced the luciferase reporter activity by the ROR1-3′-UTR. However, ROR1-3′-UTR luciferase reporter activity was unaffected by point mutations in the miR-382-binding seed region (Fig. 5B and C). Collectively, these data suggest that miR-382 may inhibit ROR1 expression by targeting its 3′-UTR. As predicted, qRT-PCR and western blot assays showed that, at 48 h after transfection, the enhanced miR-382 in SKOV3 and COV434 cells significantly repressed ROR1 RNA and protein expression compared to cells transfected with a scrambled control. By comparison, downregulation of miR-382 by inhibitors in SKOV3 (Fig. 5D and F) and COV434 (Fig. 5E and G) cells led to a moderate increase in the ROR1 RNA and protein level. Together, these data provide strong evidence that ROR1 is a specific target of miR-382 in ovarian cancer cells.

To clarify the roles of ROR1 in ovarian cancer, the expression of ROR1 was investigated in tissues and cell lines. It was found that the mRNA level of ROR1 was upregulated in human ovarian cancer tissues (Fig. 6A), and was negatively correlated with the downregulation of miR-382 (Fig. 6B). The mRNA and protein levels of ROR1 in the human ovarian cancer cell lines HO8910, SKOV3 and COV434 were markedly higher than their expression in human ovarian epithelial HOSE cells (Fig. 6C). To evaluate the function of ROR1 that acted as a target of miR-382 in the migration and invasion efficiency of ovarian cancer cells, the scratch test and the Transwel assays were performed. The overexpression of ROR1 significantly increased cell migration, which was rescued by overexpression of miR-382 in both SKOV3 (Fig. 7A and B) and COV434 cells (Fig. 7C and D). Overexpression of ROR1 promoted the cell invasion, while this effect was reversed by overexpression of miR-382 in SKOV3 (Fig. 7E and F) and COV434 cells (Fig. 7G and H). Taken together, these findings suggested that miR-382 inhibited migration and invasion by directly targeting ROR1 in SKOV3 and COV434 cells.

The above observations suggested that miR-382 could inhibit ovarian cancer cell migration and invasion and exerted its function by directly targeting ROR1; we further investigated the mechanism by which miR-382 and ROR1 regulate the cell malignant phenotype. To determine whether the typical molecular alternations of EMT occurred, the RNA and protein levels of epithelial (E-cadherin), mesenchymal (vimentin and N-cadherin) markers and EMT-related transcription factor Snail were measured by qRT-PCR and western blot assays. ROR1 overexpression reduced the mRNA expression of the epithelial maker E-cadherin and increased the mesenchymal makers N-cadherin, vimentin and EMT-related transcription factor Snail, while the effect was reversed by overexpression of miR-382 in SKOV3 (Fig. 8A) and COV434 cells (Fig. 8B). In line with mRNA expression, ROR1 overexpression lowered the protein amounts of the epithelial maker E-cadherin and increased the expression of the mesenchymal makers N-cadherin, vimentin and EMT-related transcription factor Snail, which was reversed markedly by overexpression of miR-382 in SKOV3 (Fig. 8C) and COV434 cells (Fig. 8D). The above findings suggested that miR-382 and its target gene ROR1, could affect ovarian cancer cell migration and invasion by regulating of the EMT process.