The present study was approved by the Ethics Committee of Chengdu Women and Children's Central Hospital (Chengdu, China). A total of 52 pairs of cervical cancer tissues and adjacent normal tissues were collected from patients with primary cervical cancer at Chengdu Women and Children's Central Hospital between March 2010 and September 2012. Written informed consent was obtained from all participants. None of the patients had received chemotherapy or radiotherapy prior to surgery. The cervical cancer tissues and adjacent normal tissues were rapidly frozen in liquid nitrogen shortly following surgical resection and stored until use. The clinical characteristics of the patients are summarized in Table I.

A normal human cervix epithelial Ect1/E6E7 cell line and cervical cancer SiHa, HeLa, C-33A and CaSki cell lines were purchased from the American Type Culture Collection. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) containing 10% foetal bovine serum (Thermo Fisher Scientific, Inc.) at 37°C in an incubator with 5% CO₂. Cell transfection was performed with 100 nM of negative control (NC) small interfering RNA (siRNA; cat. no. 4390843; Thermo Fisher Scientific, Inc.), NCK1-AS1 siRNA (cat. no. 4392420; Thermo Fisher Scientific, Inc.), NC inhibitor (cat. no. 4464076; Thermo Fisher Scientific, Inc.) or miR-134 inhibitor (cat. no. 4464084; Thermo Fisher Scientific, Inc.) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The following experiments were conducted 48 h after transfection.

Total RNA was extracted from tissues and cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA (1 µg) was then reverse transcribed into cDNA using the miScript Reverse Transcription kit (Qiagen GmbH), according to the manufacturer's protocol. Then, qPCR was performed using cDNA with a SYBR^® Green Real-Time PCR Master Mix (Thermo Fisher Scientific, Inc.), following the manufacturer's protocol. The qPCR thermocycler conditions were as follows: 95°C for 3 min, followed by 45 cycles of 95°C for 15 sec, 60°C for 15 sec and 72°C for 15 sec. GAPDH and U6 were used as internal references. The 2^−ΔΔCq method was used to analyse the relative gene expression (19). The following primer sequences were used: GAPDH forward, 5′-CTGGGCTACACTGAGCACC-3′ and reverse, 5′-AAGTGGTCGTTGAGGGCAATG-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The primers for NCK1-AS1 (cat. no. Hs00606619_CE) and miR-134 (cat. no. Hs00500260_CE) were purchased from Thermo Fisher Scientific, Inc.

The transfected HeLa and SiHa cells (3×10³ cells/well) were seeded into 96-well plates and cultured at 37°C with 5% CO₂ for 0, 24, 48 and 72 h. Then, CCK-8 solution (Dojindo Molecular Technologies) was added, and cells were incubated at 37°C for 2 h. Subsequently, the absorbance value was determined at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

The transfected HeLa and SiHa cells were seeded into 24-well plates and cultured at 37°C with 5% CO₂ to 100% confluence. Then, the cells were scratched with a sterile 200 µl pipette tip and washed twice with PBS. Cells in DMEM were then incubated at 37°C with 5% CO₂ for 24 h. Images were captured at 0 and 24 h using a light microscope (magnification, ×40).

The target miRNAs of NCK1-AS1 were predicted using RNAhybrid 2.12 software (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/). miR-134 was predicted to have binding sites in the NCK1-AS1 gene. To confirm this prediction, wild-type (WT) and mutant (Mut) NCK1-AS1 sequences were produced by Shanghai GenePharma Co., Ltd., and inserted into the pGL3 luciferase vector (Promega Corporation). HeLa and SiHa cells were transfected with miR-134 mimics or miR-NC, together with WT or Mut pGL3-NCK1-AS1 reporter plasmids, using Lipofectamine^® 2000 according to the manufacturer's protocol. At 48 h after transfection, luciferase activities were determined using a Dual Luciferase Reporter Assay kit (Promega Corporation) in accordance with the manufacturer's protocol. Renilla luciferase activity was normalized to that of firefly luciferase.

Transfected cells were lysed in cold radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.). The protein concentration was determined using the BCA Protein Assay kit (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. Proteins (50 µg/lane) were separated with 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.). The membranes were incubated in 5% non-fat dried milk (Yili Group) in PBS at 4°C overnight. Following incubation with rabbit anti-human matrix metalloproteinase (MMP)-2 antibody (1:500; cat. no. ab37150; Abcam), rabbit anti-human MMP-9 antibody (1:200; cat. no. ab73734; Abcam) or rabbit anti-human GAPDH antibody (1:500; cat. no. ab9485; Abcam) at room temperature for 3 h, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. ab6721; Abcam) at room temperature for 1 h. An ECL Western Blotting kit (Thermo Fisher Scientific, Inc.) was used to detect the immune complexes. Image-Pro Plus software 6.0 (Media Cybernetics, Inc.) was used to analyse the relative protein expression.

Statistical analysis was performed using SPSS 19.0 software (IBM Corp.). The data are expressed as the mean ± standard deviation. An unpaired Student's t-test was used to analyse differences between two groups. A one-way analysis of variance and Turkey's post-hoc test was used for multiple comparisons. The χ² test was performed to analyse the association between NCK1-AS1 expression and the clinical features of patients. A Kaplan-Meier survival curve and a log-rank test were applied for survival analysis. Spearman's correlation analysis was conducted to evaluate the correlation between NCK1-AS1 and miR-134 expression in cervical cancer tissues. P<0.05 was considered to indicate a statistically significant difference.

In the present study, NCK1-AS1 expression was first analysed in several human cervical cancer cell lines and normal cells using the qPCR analysis. As demonstrated in Fig. 1A, NCK1-AS1 was significantly upregulated in cervical cancer cells compared with the NCK1-AS1 levels in Ect1/E6E7 cells. To additionally confirm the upregulation of NCK1-AS1 in cervical cancer, a total of 52 paired cervical cancer tissues and adjacent normal tissues were analysed. The qPCR analysis was then performed to examine NCK1-AS1 expression. As indicated in Fig. 1B, the expression levels of NCK1-AS1 were increased in cervical cancer tissues compared with the expression levels in adjacent normal tissues. Then, the clinical significance of NCK1-AS1 expression in cervical cancer was studied. Patients were then divided into high and low NCK1-AS1 expression groups, using its mean expression value as the cut-off (2.44). As indicated in Table I, a high expression NCK1-AS1 level was significantly associated with positive lymph node metastasis and advanced clinical stage. In addition, it was identified that the patients with cervical cancer with high NCK1-AS1 expression exhibited a shorter survival times compared with patients with low NCK1-AS1 expression levels (Fig. 1C). These data suggest that NCK1-AS1 may be involved in the malignant progression of cervical cancer.

The function of NCK1-AS1 in cervical cancer was studied using HeLa and SiHa cells. These 2 cell lines were transfected with NCK1-AS1 siRNA to knock down the expression of NCK1-AS1. Transfection with NC siRNA was performed as a control. qPCR data indicated that the expression of NCK1-AS1 was significantly downregulated following transfection with NCK1-AS1 siRNA compared with the expression in the NC siRNA group (Fig. 2A and B). The CCK-8 and wound healing assays additionally indicated that the knockdown of NCK1-AS1 significantly decreased the proliferation and migration of HeLa and SiHa cells (Fig. 2C-F), indicating that NCK1-AS1 may serve a promotive role in cervical cancer cell proliferation and migration. In addition, it was observed that silencing of NCK1-AS1 significantly decreased the protein expression of MMP-2 and MMP-9, 2 important markers for tumor cell migration and invasion (Fig. 2G and H).

Bioinformatic analysis was then performed to study the potential NCK1-AS1-miR interactions. As indicated in Fig. 3A, miR-134 had a potential binding site in NCK1-AS1. To clarify this prediction, a luciferase reporter gene assay was performed using cervical cancer cells. The data demonstrated that transfection with miR-134 mimics led to a significant decrease in the luciferase activity of the WT NCK1-AS1 luciferase reporter gene plasmid but did not affect the luciferase activity of the Mut NCK1-AS1 luciferase reporter gene plasmid (Fig. 3B and C). In addition, it was identified that silencing of NCK1-AS1 expression caused a significant upregulation of miR-134 in cervical cancer cells (Fig. 3D and E). These results suggested that NCK1-AS1 may directly target miR-134 in cervical cancer cells.

It was identified that the expression levels of miR-134 were significantly decreased in cervical cancer cells compared with the expression levels in Ect1/E6E7 cells (Fig. 4A). Consistently, miR-134 expression levels were also significantly downregulated in cervical cancer tissues compared with the expression levels in adjacent tissues (Fig. 4B). Notably, an inverse correlation between miR-134 and NCK1-AS1 expression was observed in cervical cancer tissues (Fig. 4C). Therefore, the upregulation of NCK1-AS1 may contribute to the downregulation of miR-134 in cervical cancer.

Based on the aforementioned data, we hypothesized that miR-134 may be involved in the NCK1-AS1-mediated proliferation and migration of cervical cancer cells. To confirm this hypothesis, HeLa and SiHa cells were co-transfected with NCK1-AS1 siRNA together with an NC inhibitor or a miR-134 inhibitor. As indicated in Fig. 5A, miR-134 was significantly downregulated in the siNCK1-AS1 + anti-miR-134 group compared with the miR-134 levels in the siNCK1-AS1 + anti-NC group. The CCK-8 and wound healing assays additionally demonstrated that the proliferation and migration of cervical cancer cells were significantly enhanced in the siNCK1-AS1 + anti-miR-134 group compared with the siNCK1-AS1 + anti-NC group (Fig. 5B-E), indicating that the knockdown of miR-134 impaired the inhibitory effects on cervical cancer cell proliferation and migration induced by NCK1-AS1 downregulation. Taken together, these data suggested that miR-134 was involved in the NCK1-AS1-mediated proliferation and migration of cervical cancer cells.