OC cell lines SKOV3, HOC1, H0-8910, OVAC and the IOSE386 normal ovarian epithelial cell line were purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China) and maintained in RPMI-1640 medium (HyClone) containing 10% fetal bovine serum (FBS; Invitrogen Life Technologies, Grand Island, NY, USA) in a humidified atmosphere of 5% CO₂ at 37°C.

The present study was approved by the Ethics Committee of the Central South University, Changsha, China. The tissue microarray (TC0217; Auragene, Nanjing China) covering 80 case OC samples of different TNM stages (T1=15, T2=10, T3=25, T4=15 and TNF undefined=5) and 10 normal ovarian tissues were processed for immunohistochemistry. Tissue microarray blocks were incubated with anti-Limk1 antibody (Abcam, Cambridge, MA, USA) or normal rabbit IgG as the negative control. Immunostaining was performed using the Moticam 3000 System with diaminobenzidine (Zhongshan Jinqiao Co., Beijing, China). Image-Pro Plus software was used to quantify the mean density of Limk1 staining, according to the manufacturer’s instructions.

Total RNA was extracted from the cells with TRIzol (Invitrogen Life Technologies). The relative expression level of miR-138 was determined by quantitative RT-PCR (RT-qPCR) using a mirVana™ real-time RT-PCR microRNA detection kit (Ambion, Austin, TX, USA) following the manufacturer’s instructions. Specific primer sets for miR-138 and U6 (internal reference) were obtained from GeneCopoeia, Guangzhou, China. The expression of Limk1 mRNA was detected by RT-qPCR using the standard SYBR-Green RT-PCR kit (Takara, Shiga, Japan) following the manufacturer’s instructions. The specific primer pairs used were: Limk1, sense, 5′-ACCTCAACTCCCACAACT-3′ and antisense, 5′-TCTCGCACAGGACGATCC-3′; with β-actin as an internal control, sense, 5′-AGGGGCCGGACTCGTCAT ACT-3′ and antisense, 5′-GGCGGCACCACCATGTACCCT-3′. The relative expression of Limk1 mRNA or miR-138 was quantified using the GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA, USA) and the 2^−ΔΔCt method.

Cells were solubilized in cold RIPA lysis buffer. After that, proteins were separated with 8% SDS-PAGE, and then transferred to a PVDF membrane. The membranes were blocked in 5% non-fat dried milk in PBST for 3 h and then incubated overnight with relative specific primary antibodies (Abzoom, Dallas, TX, USA) with β-actin as a control. After incubation with the appropriate secondary antibody, immune complexes were detected using an ECL kit (Auragene Bioscience Inc., Changsha, China).

The normal and mutated 3′-UTR of Limk1 were constructed by PCR, and then inserted into the MCS in the psiCHECK™-2 vector (Promega, Madison, WI, USA). For the luciferase reporter assay, 2×10⁴ SKOV3 cells were cultured to ~50–60% confluence in a 24-well plate. For each well, the medium was replaced by 300 μl OPTI-MEM medium (Invitrogen, Carlsbad, CA, USA). The cells were co-transfected with psiCHECK™-2-Limk1-3′-UTR or psiCHECK™-2-Limk1-mut 3′-UTR vector plus 50 nM miR-138 mimics or 100 mM miR-138 inhibitor using Cellfectin^® II reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. The cells were incubated with transfection reagent/DNA complex for 5 h, and then refreshed with fresh complete medium. A dual-luciferase reporter assay system (Promega) was used to determine the luciferase activities 48 h after co-transfection. Renilla luciferase activity was normalized to firefly luciferase activity.

An MTT assay was used to measure cell proliferation. Cells in exponential growth were plated at a final concentration of 2,000 cells/well in 96-well plates. Cell viability was evaluated by MTT assay after 24, 48, 72 and 96 h of seeding. The optical density at 570 nm (OD570) of each well was measured with an ELISA reader (ELx800 type; BioTek Instruments, Winooski, VT, USA). The experiment was repeated three times.

The invasive ability of OC cells was studied in 24-well Transwell chambers (Chemicon International, Temecula, CA, USA), which has a layer of Matrigel. For each group, 200 μl of cell suspension (1×10⁶ cells/ml) was added in triplicate wells. After 24-h incubation, the number of cells that had migrated through the filter into the lower wells was determined by gentian violet assay.

Cell migratory ability was estimated using a wound healing assay. In brief, cells were cultured to a confluence of ?. Wounds of ~1 mm width were created with a plastic scriber, and the cells were washed and incubated in a serum-free medium. After wounding for 24 h, the cells were incubated in a medium including 10% fetal bovine serum. Cultures at 0, 24 and 48 h were fixed and observed under a microscope.

To generate Limk1 or cofilin knockout stable cell lines, Limk1- and cofilin-Talen plasmid pair were constructed. A Talen kit was used to knock out the Limk1 or cofilin genes in SKOV3 and HO-8910 cells according to the manufacturer’s instructions. Briefly, the plasmids pCMV-NLS-N-terminal-L1-C-terminal-Fok1(L)-IRES-PURO-pA and pCMV-NLS-N-terminal-R1-C-terminal-Fok1(R)-pA were against the Talen recognition module from the 5′- and 3′-ends. The Talen sequences of Limk1 were: left arm, GACGCTACTTTGTTGCACC and right arm, CGCTTCCTTCCTCTCCCA. The Talen sequences of cofilin were: left arm, GACGCTACTTTGTTGCACC and right arm, GAAGACTTACGCACCT. Sequencing was then performed to determine the sequences of the two plasmids. Three sequencing primers were used as follows: 5′-CTCCCC TTCAGCTGGACAC-3′; 5′-AGCTGGGCCACGATTGAC-3′; 5′-GGGAGCACCCCTCAACCTGAC-3′. After confirming that the sequences of these two plasmid pairs were correct, the two pairs of plasmids were co-transfected into SKOV3 and HO-8910 cells using Lipofectamine 2000 (Invitrogen, Life Technologies), respectively, according to the manufacturer’s instructions. The mRNA level of Limk1 and cofilin was subsequently assessed to confirm the efficiency of Talen-mediated Limk1 or cofilin KO using RT-qPCR. For the miR-138 functional analysis, the cells were transfected with the pre- or anti-miR-138 to generate gain-of-function or loss-of-function cell models, respectively. Transfection was performed according to the manufacturer’s instructions.

Statistical analysis was performed by SPSS 17 software. The results are expressed as the mean ± SD of three independent experiments. For each experiment, the statistical analysis was repeated three times. Statistical analysis of differences was performed by one-way analysis of variance (ANOVA) or two-way ANOVA. The null hypothesis was rejected at the 0.01 level.

RT-qPCR was performed to detect the miR-138 and Limk1 mRNA levels in the IOSE386 normal ovarian epithelial cell line and SKOV3, HOC1, HO-8910 and OVAC OC cell lines. The relative expression of miR-138 was significantly lower in the four OC cell lines compared to the control IOSE386 cells. By contrast, the levels of Limk1 mRNAs significantly increased in OC cell lines as compared to the control (Fig. 1A and B). Additionally, the Limk1 protein was confirmed to be higher in OC cells as compared to the control (Fig. 1C). The results suggested a negative relationship between miR-138 and Limk1 in OC cells. To evaluate the correlation between Limk1 expression and the progression of OC, immunohistochemical analysis of tissue microarrays was performed. The expression of Limk1 in OC tissues was clearly increased compared to normal ovarian tissues. Furthermore, the expression of Limk1 was gradually elevated with the malignant progression of OC (Fig. 1D). This suggested that increased Limk1 may contribute to the malignant progression of OC.

A luciferase reporter assay was performed to determine whether Limk1 was a direct target of miR-138 in OC SKOV3 and HO-8910 cells, based on the bioinformatic prediction that the binding site for miR-138 at the 3′-UTR of Limk1 mRNA was strong and conserved. Data of the luciferase reporter assay showed that the activity of luciferase was significantly decreased only after transfection with 3′-UTR of the Limk1 gene in the miR-138 treated cells. However, in the other groups, the activity of luciferase showed no difference (Fig. 2A and B). These findings suggested that Limk1 is a direct target of miR-138 in OC cells.

The effects of miR-138 overexpression or downregulation on the mRNA level of Limk1 in SKOV3 and HO-8910 cells were investigated. Overexpression of miR-138 significantly inhibited the mRNA level of Limk1 (Fig. 2C and D). However, the inhibition of miR-138 upregulated the mRNA level of Limk1 in both cells (Fig. 2E and F). These findings suggested that the mRNA level of Limk1 was negatively regulated by miR-138 at a post-transcriptional level.

To obtain loss-of-function of Limk1, Talen technology was used to construct Limk1 knockout stale cell lines. SKOV3 and HO-8910 cells were transfected with a Talen-Limk1 plasmid pair. The expression of Limk1 in the two cell lines was almost undetectable (Fig. 3A), suggesting that Limk1 was successfully knocked out in the two cell lines. Moreover, it was found that the knockout of Limk1 reduced the cell invasive and migratory abilities of SKOV3 and HO-8910 cells transfected with the Talen-Limk1 plasmid pair (Fig. 3B–D), suggesting that Limk1 promoted the invasion and migration of OC cells. These results suggested that Limk1 plays a promotional role in OC growth and metastasis.

To clarify the underlying molecular mechanisms of miR-138 mediating OC cell viability and metastasis, western blot analysis was performed to detect the proliferation and apoptosis-associated protein. It was found that Limk1 and the proliferation marker PCNA were decreased, whereas the anti-apoptotic marker Bcl-2 was upregulated in SKOV3 and HO-8910 cells transfected with pre-miR-138 (Fig. 4A). Anti-miR-138 increased Limk1 and PCNA expression but decreased Bcl-2 compared to the control (Fig. 4B). The results suggested that miR-138 loss mediated Limk1 upregulation and subsequently promoted PCNA expression and reduced Bcl-2 expression, which plays an important role in cell growth. The activation of cofilin signaling is a classic pathway mediating cell metastasis. To clarify whether miR-138-mediated metastasis was involved in Limk1/cofilin signaling, cofilin was knocked out by Talen technology in the two OC cells to obtain Talen-cofilin stable cell lines. Western blot analysis revealed that anti-miR-138 enhanced the expression levels of Limk1, p-Limk1 and p-cofilin. By contrast, in Talen-cofilin cells, anti-miR-138 was able to increase the expression of Limk1 and p-Limk1 but did not phosphorylate cofilin because of the cofilin knockout (Fig. 4C).

To examine the functional role of miR-138 in OC cells, gain-of-function and loss-of-function cell models were established by transfecting with pre- and anti-miR-138, respectively, in SKOV3 and HO-8910 cell lines. It was confirmed that the overexpression of miR-138 reduced cell viability, while the downregulation of miR-138 increased cell viability in the two OC cells (Fig. 5A and B). Moreover, the invasive and migratory abilities of SKOV3 and HO-8910 cells transfected with pre-miR-138 were reduced. Inversely, cells transfected with anti-miR-138 caused increased cell invasion and migration (Fig. 5C–F). These results suggested that miR-138 served as a suppressor in OC, which was completely opposite to Limk1. Limk1 was also found to be a target of miR-138 in OC cells. Further functional analysis demonstrated that the knockout of cofilin attenuated the anti-miR-138 promotion of invasion and migration of OC cells (Fig. 5C–F), suggesting that miR-138 functioning in OC is partly involved in activation of the cofilin signaling pathway.