Fetal bovine serum (FBS), Dulbecco's phosphate-buffered saline (DPBS), TRIzol^® reagent, Lipofectamine^® 2000, Cellfectin II Reagent, OPTI-MEM medium, and mirVana™ quantitative polymerase chain reaction (qPCR) miRNA Detection kit were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Standard SYBR-Green reverse transcription (RT)-PCR kit was purchased from Takara Bio. Inc. (Otsu, Japan). Quick-Change Site-Directed Mutagenesis kit was purchased from Agilent Technologies, Inc. (La Jolla, CA, USA). PsiCHECK™-2 vector and Dual-Luciferase Reporter Assay system were purchased from Promega Corporation (Madison, WI, USA). Transwell chambers (24-well) were purchased from Chemicon; EMD Millipore (Billerica, MD, USA). Bicinchoninic acid (BCA) Protein Assay kit and Enhanced chemiluminescence (ECL) Western Blotting kit were purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA). All antibodies were purchased from Abcam (Cambridge, MA, USA).

The present study was approved by the Ethics Committee of Central South University (Changsha, China). Written informed consent was obtained from each patient. The NSCLC patients (n=16) included 10 men and 6 women aged between 45 and 73 years old (mean age, 56.4 years). Patients were recruited between April 2013 and October 2013. All patients received neither radiation therapy nor chemotherapy prior to surgical resection. A total of 16 primary NSCLC tissues, and their matched adjacent normal tissues, were collected at the Department of Cardiothoracic Surgery, The Third Xiangya Hospital of Central South University (Changsha, China). The histomorphology of all samples was confirmed by the Department of Pathology, The Third Xiangya Hospital of Central South University. Tissues were immediately snap-frozen in liquid nitrogen following surgical removal and stored at −80°C prior to use.

The H460, SK-MES-1, A549 and SPC-A1 human NSCLC cell lines, and the BEAS-2B normal human lung epithelial cell line were purchased from the Cell Bank of Central South University (Changsha, China). All cells were cultured in Dulbecco's modified Eagles' medium (DMEM; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS at 37°C in an atmosphere containing 5% CO2.

Tissues were homogenized and total RNA was extracted from the tissues and cells using TRIzol^® reagent, according to the manufacturer's protocol. Takara PrimeScript™ RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) was used to perform the RT prior to mRNA and miRNA detection, according to the manufacturer's instruction. The relative expression levels of miR-138 were determined by RT-qPCR using mirVana™ qPCR microRNA Detection kit, according to the manufacturer's protocol. An Applied Biosystems^® 7500 thermal cycler, (Thermo Fisher Scientific, Inc.) was used and the reaction conditions were as follows: 95°C for 10 min, 40 cycles at 95°C for 15 sec and 60°C for 30 sec. For the PCR reaction, 1 µl cDNA solution, 10 µl PCR master mix, 2 µl of primers and 7 µl H₂O were mixed to obtain a final reaction volume of 20 µl. Specific primer sets for miR-138 and U6 were obtained from Genecopoeia (Rockville, MD, USA). The mRNA expression levels of LIMK1 were detected by RT-qPCR using the standard SYBR-Green RT-PCR kit, according to the manufacturer's protocol. The specific primer pairs (purchased from Sangon Biotech Co., Ltd., Shanghai, China) were as follows: LIMK1, sense 5′-CAAGGGACTGGTTATGGTGGC-3′, antisense 5′-CCCCGTCACCGATAAAGGTC-3′; and β-actin (used as an internal control), sense 5′-AGGGGCCGGACTCGTCATACT-3′, and antisense 5′-GGCGGCACCACCATGTACCCT-3′. The relative expression levels of LIMK1 mRNA or miR-138 were quantified using GraphPad Prism 4.0 software (GraphPad Software, Inc., San Diego, CA, USA), and the 2-ΔΔCq method (16).

Cells were lysed in cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). The BCA Protein Assay kit was used to determine protein concentration. Protein samples (50 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to a polvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.). The PVDF membrane was blocked with 5% nonfat dried milk in PBS for 4 h. Subsequently, the PVDF membrane was incubated with mouse anti-LIMK1 monoclonal antibody (1:200; cat no. ab117623), mouse anti-cofilin monoclonal antibody (1:200; cat no. ab54532), rabbit anti-phosphorylated-cofilin monoclonal antibody (1:200; cat no. ab47281) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:50; ab8245) at room temperature for 3 h. The membrane was washed three times with PBS (5 min/wash) and then incubated with rabbit anti-mouse secondary antibody (1:10,000; cat no. ab6728) or goat anti-rabbit secondary antibody (1:10,000 cat no. ab6721). Following a further three 5 min washes with DPBS, an ECL Western Blotting kit was used to detect the immune complexes on the PVDF membrane. Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to analyze relative protein expression levels, represented as the density ratio vs. GAPDH. GAPDH was used as an internal reference.

LIMK1 plasmid, miR-138 mimics and a miR-138 inhibitor were generated by Nlunbio (Changsha, China). Blank pcDNA3.1(+) vector and scramble miRNA mimic were purchased from Nlunbio. Lipofectamine^® 2000 was used to perform transfection, according to the manufacturer's protocol. Briefly, LIMK1 plasmid, miRNA mimics or miR-138 inhibitor and Lipofectamine^® 2000 were diluted with serum-free medium. The diluted Lipofectamine^® 2000 was added to the diluted plasmid or miRNA mimics, and incubated for 20 min at room temperature. Subsequently, the mixture was added to the H460 cell suspension. The H460 cells were then incubated at 37°C in an atmosphere containing 5% CO2 for 6 h. Finally, the medium in each well was replaced with the normal serum-containing medium, and the cells were cultured for 24 h prior to the following assays.

The normal and mutant (mut) 3′-UTRs of LIMK1 were constructed by PCR, and were then inserted into the multiple cloning site of the psiCHECK™-2 vector. For the luciferase reporter assay, 2×104 H460 cells were cultured to 50–60% confluence in a 24-well plate. In each well, medium was replaced with 300 µl OPTI-MEM medium. The H460 cells were then co-transfected with psiCHECK™-2-LIMK1-3′-UTR or psiCHECK™-2-LIMK1-mut 3′-UTR vector (both purchased from Nlunbio) plus 50 nM miR-138 mimics using Cellfectin II reagent, according to the manufacturer's protocol. Cells were incubated with the transfection reagent/DNA complex for 5 h, and the medium was then replaced with fresh complete medium. A Dual-Luciferase Reporter Assay system was used to determine the luciferase activities 48 h post-transfection using a Lucetta™ Luminometer, (cat no. AAL-1001; Lonza Group, Ltd., Basel, Switzerland). Renilla luciferase activity was normalized to firefly luciferase activity.

Cell migratory capability was estimated using a wound healing assay. Briefly, H460 cells were cultured to confluence. Wounds (~1 mm) were created in the cell monolayer using a plastic scriber, and cells were washed and incubated in serum-free medium. A total of 24 h after wound generation, the cells were incubated in medium supplemented with 10% FBS. Cultures at 0 and 36 h were fixed and observed under a CX23 Microscope (Olympus Corporation, Tokyo, Japan).

The invasive ability of H460 cells was determined in 24-well Transwell chambers, which contained a layer of Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). For each group, a cell suspension (1×10⁵ cells/ml) was added to the upper chamber, whereas DMEM containing 10% FBS was added to the lower chamber. Following a 24 h incubation, non-invading cells and the Matrigel on the interior of the inserts was removed using a cotton-tipped swab. Invasive cells on the lower surface of the membrane were stained with gentian violet, rinsed with water, and air-dried. Five fields were randomly selected and the cell number was counted under a CX23 Microscope (Olympus Corporation).

The results are presented as the mean ± standard deviation of at least three independent experiments. Statistical analysis was performed using SPSS 17 software (SPSS Inc., Chicago, IL, USA). Statistical analysis of differences between groups was performed by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

To determine the role of miR-138 and LIMK1 in NSCLC, the present study examined the expression levels of miR-138 in human NSCLC tissues and matched adjacent normal tissues using RT-qPCR. As shown in Fig. 1A, miR-138 was significantly downregulated in NSCLC tissues compared with in the matched adjacent normal tissues. The mRNA expression levels of LIMK1 were also examined by RT-qPCR. As shown in Fig. 1B, the mRNA expression levels of LIMK1 were increased in NSCLC tissues, as compared with in the matched adjacent normal tissues. These results indicate that miR-138 is downregulated, whereas LIMK1 is upregulated in NSCLC tissues.

The present study further determined the expression levels of miR-138 in the following human NSCLC cell lines: H460, SK-MES-1, A549 and SPC-A1. The BEAS-2B normal human lung epithelial cell line was used as a control. As shown in Fig. 2A, the expression levels of miR-138 were significantly reduced in the NSCLC cell lines compared with in BEAS-2B cells. Consistent with results of the tissue analysis, the mRNA and protein expression levels of LIMK1 were also increased in NSCLC cell lines compared with in BEAS-2B normal human lung epithelial cells (Fig. 2B and C). Since H460 cells exhibited the most significant changes in miR-138 and LIMK1 expression among these NSCLC cell lines, this cell line was used for subsequent experiments.

Bioinformatic prediction using Targetscan online software version 3.1 (targetscan.org) suggested that LIMK1 may be a direct target of miR-138. Therefore, the present study performed a luciferase reporter assay to confirm this relationship in NSCLC cells. As shown in Fig. 3A, luciferase activity was significantly reduced in H460 NSCLC cells co-transfected with miR-138 mimics and wild type LIMK1 3′-UTR. Conversely, luciferase activity was unchanged in the other groups compared with the control group (Fig. 3A). These results indicate that LIMK1 may be a direct target of miR-138 in NSCLC cells.

Accordingly, the present study examined the role of miR-138 in the regulation of LIMK1 expression in NSCLC cells. Post-transfection with miR-138 mimics or a miR-138 inhibitor, the expression levels of miR-138 were detected. As shown in Fig. 3B, H460 cells transfected with miR-138 mimics exhibited a significant increase in miR-138 expression, whereas transfection with the miR-138 inhibitor markedly suppressed miR-138 expression, as compared with the control group. These results indicate that the transfection was successful. Since miRs can negatively regulate the expression of their target genes at the post-transcriptional level, the present study subsequently detected the protein expression levels of LIMK1 in each group. As shown in Fig. 3C, the protein expression levels of LIMK1 were reduced in H460 NSCLC cells post-transfection with miR-138 mimics, but were increased following miR-138 knockdown compared with in the control group. These findings further confirm that miR-138 may negatively regulate the expression of LIMK1 via directly binding to the 3′-UTR of its mRNA in H460 NSCLC cells.

The roles of LIMK1 and miR-138 in NSCLC cell migration and invasion were further studied. As well as modulating miR-138 expression in H460 cells, the present study also transfected H460 cells with a LIMK1 plasmid, in order to upregulate its expression. As shown in Fig. 4A, transfection with the LIMK1 plasmid markedly enhanced the protein expression levels of LIMK1 compared with in the control group. Subsequently, the roles of miR-138 and LIMK1 in the invasion of H460 NSCLC cells were investigated. Overexpression of miR-138 inhibited H460 NSCLC cell invasion, whereas overexpression of LIMK1 markedly promoted the invasion of H460 cells (Fig. 4B). In addition, overexpression of miR-138 led to a significant reduction in the migration of H460 NSCLC cells. Conversely, overexpression of LIMK1 significantly promoted the migration of H460 NSCLC cells (Fig. 4C). These results suggest that the inhibitory effects of miR-138 on H460 NSCLC cell invasion and migration may be caused by direct inhibition of LIMK1 expression.

It has been suggested that cofilin signaling, downstream of LIMK1, has an important role in mediating the migration and invasion of cancer cells. Therefore, the present study further examined the activity of this signaling pathway in NSCLC cells transfected with miR-138 mimics or a miR-138 inhibitor. As shown in Fig. 5, overexpression of miR-138 significantly inhibited the activity of cofilin, whereas inhibition of miR-138 promoted its activity in H460 NSCLC cells. These findings suggest that the inhibitory effects of miR-138 on H460 NSCLC cell migration and invasion may be caused by suppression of LIMK1/cofilin signaling activity.