Introduction
Lung cancer is the leading cause of cancer-associated mortality, with an increasing incidence worldwide (1). Despite improvements in diagnostic imaging, surgery, radiotherapy and chemotherapy, the overall survival rate of patients with lung cancer remains poor, with only 14% of patients surviving 5 years from the date of diagnosis (1). As non-small cell lung cancer (NSCLC) is the most common type of lung cancer, the development of effective therapeutic targets for NSCLC is urgently required (1).
The development and progression of NSCLC are associated with the dysregulation of oncogenes or tumor suppressors, including microRNAs (miRNAs) (2). miRNAs are 18–25 nucleotide, non-coding RNAs, which can result in the inhibition of gene expression at the post-transcriptional level, through direct binding to the 3′-untranslational region (UTR) of mRNAs (3). In addition, the dysregulation of miRNAs has been reported to be associated with the development and progression of NSCLC, including miRNA (miR)-7 (4,5). miR-7 has been demonstrated to act as a tumor suppressor in NSCLC, through targeting B-cell lymphoma (BCL)-2 and PA28γ (6,7). As one miRNA can directly bind to several target mRNAs, whether other target genes of miR-7 exist in NSCLC remains to be fully elucidate.
Paired box 6 (Pax6) has been demonstrated as a highly conserved transcription factor during embryogenesis, and is involved in the development and function of the central nervous system, endocrine glands, eyes and pancreas (8–12). The role of Pax6 in the development and progression of cancer been gradually revealed (13,14). Zhao et al reported that shRNA-induced PAX6 downregulation notably suppressed proliferation and cell cycle progression in NCSLC cells. They further demonstrated that the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways are involved in Pax6-mediated cell cycle progression in NCSLC cells (15).
The present study aimed to examine the role of miR-7 in the regulation of NSCLC in vitro. In addition, as a previous study demonstrated a target association between miR-7 and Pax6 in colon cancer (16), the present study also investigated whether Pax6 is involved in the regulatory effect of miR-7 on NSCLC cells.
Materials and methods
Agents
TRIzol reagent, fetal bovine serum (FBS) Lipofectamine 2000 and the mirVana™ qRT-PCR miRNA Detection kit were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Mouse anti-Pax6, -total ERK, -phosphorylated (p)-ERK, -p38 MAPK and -GAPDH primary antibodies, and rabbit anti-mouse secondary antibody were purchased from Abcam (Cambridge, UK). Bioinformatics analysis was conducted to predicate the putative target genes of miR-7 using the Targetscan online software (http://www.targetscan.org/). The Quick-Change Site-Directed Mutagenesis kit was purchased from Stratagene (La Jolla, CA, USA). The PsiCHECK™2 vector was purchased from Promega Corporation (Madison, WI, USA). The enhanced chemiluminescence (ECL) kit was purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA).
Cell lines and cell culture
The A549, H460, SK-MES-1 and SPC-A1 human NSCLC cell lines, and normal BEAS-2B 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 Eagle's medium (DMEM; Invitrogen Life Technologies) supplemented with 10% FBS at 37°C with 5% CO2.
RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from the cells using TRIzol reagent, according to the manufacturer's instructions. The relative expression level of miR-7 was determined by RT-qPCR using a mirVana™ qRT-PCR miRNA Detection kit, according to the manufacturer's instruction. Specific primer sets for miR-7 and U6 (internal reference) were obtained from GeneCopoeia, Inc. (Maryland Rockville, MD, USA). The PCR cycling conditions were as follows: 95°C for 10 min, denaturation at 95°C for 15 sec and an annealing/elongation step at 60°C for 1 min for 40 cycles. The data were analyzed with SDS relative quantification software version 2.2.2 (Applied Biosystems, Foster City, CA, USA). The 2-ΔΔCt method was used for the relative quantification of the differences in expression levels of each target.
Western blotting
The cells (107) were solubilized in cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The proteins (60 µg) were separated with 12% SDS-PAGE (Beijing Biolab Science and Technology Co., Ltd., Beijing, China), and transferred onto a polyvinylidene difluoride (PVDF) membrane (Invitrogen Life Technologies), which was then incubated with Tris-buffered saline with Tween 20 (TBST; Fanke Biotech Co., Ltd., Shanghai, China) containing 5% milk at room temperature for 3 h. The PVDF membrane was then incubated with primary antibodies at room temperature for 3 h. The primary antibodies were as follows: Monoclonal mouse anti-human PAX6 (1:100, cat. no. ab78545), monoclonal mouse anti-human ERK (1:100, cat. no. ab119933), polyclonal rabbit anti-human p-ERK (1:200, cat. no. ab131438), monoclonal mouse anti-human p38 MAPK, (1:100, cat. no. ab31828) and monoclonal rabbit anti-human p-p38 MAPK, (1:100, cat. no. EPR16587). This was followed by incubation with rabbit anti-mouse IgG, (cat. no. ab46540) and mouse anti-rabbit IgG (cat. no. ab99700) secondary antibodies at room temperature for 40 min. All antibodies were purchased from Abcam. Chemiluminescent detection was performed using an ECL kit. The relative protein expression was analyzed using Image-Pro plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), and presented as the density ratio, vs. GAPDH.
Transfection
The plasmid of Pax6, scramble miRNA mimics, miR-7 mimics and miR-7 inhibitor were constructed by Nlunbio (Changsha, China). Lipofectamine 2000 was used to perform transfection, according to the manufacture's instruction. Briefly, the plasmid or miRNA mimics and Lipofectamine 2000 were diluted with 100 nM serum-free medium, respectively. The diluted Lipofectamine 2000 was added into the diluted plasmid or miRNA mimics, respectively, and incubated for 20 min at room temperature, following which they were added to the cell suspension. The cells (105 cells/ml), which were suspended in DMEM were then incubated at 37°C, 5% CO2 for 6 h. Following incubation, the medium in each well was replaced with normal serum-containing medium, and the cells were cultured for 24 h prior to performing the subsequent assays. The successful transfection was confirmed by detecting the expression levels of PAX6 or miR-7, respectively.
Dual luciferase reporter assays
A Quick-Change Site-Directed Mutagenesis kit was used to generate a mutant 3′-UTR of Pax6, according to the manufacturer's instructions. The wild-type or mutant 3′-UTR of Pax6 were inserted into the psiCHECK™2 vector, respectively. A549 cells (105 cells/well), which were cultured to ~70% confluence, were the transfected with either the psiCHECK™2-Pax6-3′-UTR or psiCHECK™2-mutant Pax6 -3′-UTR vector, with or without 100 nM miR-7 mimic, respectively. Following transfection for 48 h, the luciferase activities were determined using an LD400 luminometer (Beckman Coulter, Fullerton, CA, USA). The activity of Renilla luciferase was normalized to that of firefly luciferase.
Cell proliferation assay
An MTT assay was performed to measure cell proliferation. Cells in the exponential growth were plated, at a final concentration of 2,000 cells per well, into 96-well plates. The viability of the cells were evaluated using an MTT assay (20 µl MTT) 24, 48, 72 and 96 h after seeding. The optical density (OD) at 570 nm of each well was measured using an ELISA reader (ELX-800; BioTek Instruments, Inc., Winooski, VT, USA).
Cell invasion assay
The invasive abilities of the A549 cells were determined using 24-well Transwell chambers (Chemicon, Temecula, CA, USA), which contained a layer of Matrigel. For each group, the cell suspension was added to the upper chamber, and DMEM, containing 10% FBS, was added to the lower chamber. Following incubation for 24 h at 37°C, the non-invading cells and the matrix gel on the interior of the inserts were removed using a cotton-tipped swab. The invasive cells on the lower surface of the membrane were stained with gentian violet, followed by rinsing with water and air drying. Subsequently, the number of cells were counted in five randomly-selected fields under an inverted microscope (IX71; Olympus, Tokyo, Japan).
Statistical analysis
The results are expressed 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 was performed using one-way analysis of variance. P<0.01 was considered to indicate a statistically significant difference.
Results
miR-7 is downregulated, while Pax6 is upregulated in NSCLC cell lines
To determine the role of miR-7 in NSCLC, the present study first examined the expression level of miR-7 in the A549, H460, SK-MES-1 and SPC-A1 human NSCLC cell lines, and BEAS-2B normal human lung epithelial cell line. As shown in Fig. 1A, the expression level of miR-133a was significantly reduced in the NSCLC cell lines, compared with the BEAS-2B normal human lung epithelial cells. Subsequently, the expression level of Pax6 was determined by performing RT-qPCR. As shown in Fig. 1B, the mRNA level of Pax6 was upregulated in the NSCLC cell lines, compared with the BEAS-2B normal human lung epithelial cells. Accordingly, these data demonstrated that miR-7 was downregulated, while Pax6 was upregulated in the NSCLC cell lines.
As the A549 cells exhibited the most marked changes in expression levels of miR-7 and Pax6 among the four NSCLC cell lines, when compared with the BEAS-2B normal human lung epithelial cells (Fig. 1B), the A549 cell line was selected for examination in the subsequent experiments.
miR-7 negatively regulates the protein expression of its target, Pax6, in A549 NSCLC cells
Based on the above data, the regulatory association between miR-7 and Pax6 in A549 NSCLC cells were further investigated. Following transfection with miR-7 mimics or inhibitor, the level of miR-7 in the A549 NSCLC cells was examined, and the data revealed that the transfection had been successful (Fig. 2A). The protein level of Pax6 in each group was then determined by performing western blotting, which demonstrated that, in the A549 NSCLC cells transfected with miR-7 mimics, the protein level of Pax6 was reduced (Fig. 2B). By contrast, inhibition of miR-7 led to an increase in the protein expression of Pax6 in the A549 NSCLC cells (Fig. 2B). Based on the bioinformatical prediction that the putative seed sequences for miR-7 at the 3′UTR of Pax6 is conserved (Fig. 2C), the present study performed a luciferase reporter assay to determine whether Pax6 was a target of miR-7. The resulting data indicated that the luciferase activity was reduced only in the A549 NSCLC cells that were co-transfected with the miR-7 mimics and wild-type Pax6 3′UTR. In the remaining groups, the luciferase activity was unchanged (Fig. 2D). These findings indicated that Pax6 was a direct target of miR-7 in the A549 NSCLC cells.
Overexpression of miR-7 inhibits NSCLC A549 cell proliferation by targeting Pax6
To further investigate the effect of Pax6 and miR-7 on NSCLC cell proliferation, an MTT assay was performed. As shown in Fig. 3, in the miR-7-overexpressed A549 cells, the cell proliferation was downregulated, whereas in the Pax6-overexpressed A549 cells, the cell proliferation rate was upregulated. In addition, upregulation of Pax6 attenuated the inhibitory effect of miR-7 overexpression on A549 cell proliferation. These data suggested that overexpression of miR-7 inhibited A549 cell proliferation by targeting Pax6.
Overexpression of miR-7 inhibits NSCLC A549 cell invasion through inhibition of Pax6
The present study subsequently further investigated the roles of miR-7 and Pax6 in the regulation of A549 NSCLC cell invasion. The findings demonstrated that upregulation of miR-7 inhibited A549 NSCLC cell invasion, whereas in the Pax6-overexpressed A549 NSCLC cells, cell invasion was upregulated. Furthermore, restoration of the expression of Pax6 reversed the suppressive effect of miR-7 overexpression on NSCLC A549 cell proliferation. These data suggested that overexpression of miR-7 inhibited NSCLC cell invasion through the inhibition of Pax6 (Fig. 4).
Activities of the ERK and p38 MAPK pathways are mediated by miR-7 and Pax6 in A549 NSCLC cells
The activities of the ERK and p38 MAPK signaling pathways were determined following the overexpression of Pax6 or miR-7 in the A549 human NSCLC cells. As shown in Fig. 5, the phosphorylated protein levels of ERK and p38 MAPK were suppressed in the miR-7-overexpressed A549 cells, but were increased in the Pax6-overexpressed A549 cells. These data indicated that the activities of the ERK and p38 MAPK signalingpathways were downregulated by the overexpression of miR-7, but upregulated by the overexpression of Pax6 in A549 NSCLC cells.
Discussion
In the present study, the roles of miR-7 and Pax6, as well as their associations, were investigated in NSCLC cells. The results demonstrated that miR-7 was downregulated, while Pax6 upregulated in the NSCLC cell lines. Further investigation identified Pax6 as a target of miR-7 in the A549 NSCLC cells, and the protein expression of Pax6 was negatively mediated by miR-7. Overexpression of miR-7 significantly inhibited A549 cell proliferation and invasion, which was reversed by the upregulation of Pax6. In addition, the ERK and p38 MAPK signaling pathways were downregulated in miR-7-overexpressed A549 cells, but activated in Pax6-overexpressed A549 cells.
It has been demonstrated that miR-7 acts as a tumor suppressor in various types of cancer. Reduced levels of miR-7 have been linked to the development of cancer and metastasis (17). Zhou et al reported that miR-7 inhibits tumor metastasis and reverses epithelial-mesenchymal transition through inhibition of AKT and ERK signaling in epithelial ovarian cancer (18). Xie et al demonstrated that miR-7 inhibits gastric cancer cell invasion and metastasis by suppressing the expression of epidermal growth factor receptor (19). In adddition, miR-7 has been reported to inhibit colorectal cancer cell proliferation and induce apoptosis (20). The role of miR-7 in NSCLC has also been reported. Xiong et al revealed that miR-7 has a suppressive effect in NSCLC by targeting PA28γ (6). In addition miR-7 has been observed to inhibit the growth of A549 NSCLC cells through targeting BCL-2 (7). As a single miRNA has multiple targets, and one gene can be regulated by various miRNAs (21), the presents study aimed to identify other targets of miR-7 in NSCLC cells, and demonstrated that the inhibition of Pax6 was involved in miR-7-overexpression-induced downregulation of A549 NSCLC cell proliferation and invasion.
As a transcription factor, Pax6 mediates the expression of various genes. In addition, the expression of Pax6 has been reported to be upregulated in pancreatic cancer and retinoblastoma, but downregulated in glioma and prostate cancer, suggesting that Pax6 may have different roles in different types of cancer (13,22–24). In the present study, the results suggested that, as a target of miR-7, Pax6 acts as an oncogene in NSCLC through promoting NSCLC cell proliferation and invasion. Previously, Zhao et al investigated the role of Pax6 in NSCLC cell proliferation and reported that inhibition of the expression of Pax6 suppresses cell growth and colony formation in A549 and H1299 NSCLC cells (15). In addition, the percentage of NSCLC cells in the G1-phase is increased following a reduction in the expression of Pax6, indicating that the inhibition of Pax6 induces cell cycle arrest at the G1 phase (15). It was also demonstrated that the activitites of the ERK and p38 MAPK signaling pathways were suppressed in Pax6 knockdown cells (15), consistent with the data obtained the present study, that overexpression of Pax6 induced upregulation in the activity of ERK and p38 MAPK signaling. It has been well established that the ERK and p38 MAPK signaling pathways are involved in the development and progression of various types of cancer, including NSCLC, and targeting components of the ERK and MAPK signaling pathways offers an attractive strategy in the development of novel therapeutic approaches to treat NSCLC (25,26).
In addition, the present study demonstrated for the first time, to the best of our knowledge, that miR-7/Pax6 is involved in the regulation of NSCLC cell invasion. The role of Pax6 in cancer cell invasion has also been demonstrated in several other types of cancers, including glioma and colon cancer (27). Cheng et al reported that Pax6 inhibits cell proliferation and invasion in glioma cells (27), and, Liu et al suggested that brain fatty acid-binding protein 7 is a target gene of Pax6, and is involved in the regulation of glioma cell invasion (28). In glioblastoma cells, Pax6 has been reported to suppress cell invasion, at least in part, through inhibiting the protein expression of matrix metalloproteinase (MMP) 2 (29). Huang et al also demonstrated that MMP2 and MMP9 are downstream effectors of Pax7 in the regulation of glioblastoma cell invasion (13).
In conclusion, the present study demonstrated that miR-7 inhibited NSCLC cell proliferation and invasion through the direct targeting of Pax6, suggesting that Pax6 and miR-7 may serve as potential therapeutic targets in the treatment of NSCLC.