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Introduction

Lung cancer is one of the most common type of cancer and the leading cause of cancer-associated death worldwide, which accounts for >1.6 million cases and >1.3 million mortalities due to this disease (1). Non-small cell lung cancer (NSCLC), which accounts for >80% of lung cancer cases, is comprised of three histological subtypes: Squamous-cell carcinoma, adenocarcinoma and large-cell carcinoma. At present, the primary therapeutic treatments for NSCLC patients are comprehensive, including surgery resection, chemotherapy and radiotherapy (2). Unfortunately, despite recent progress in the diagnosis and treatment of cancer, the five-year survival rate for patients with NSCLC remains at 11% (3). Metastasis is a major cause of mortality among patients with NSCLC (4). Therefore, fully understanding the mechanisms underlying the carcinogenesis and progression of NSCLC would facilitate the development of novel therapeutic targets and improve the prognosis.

Emerging evidences has suggested that microRNAs (miRNAs) are deregulated in NSCLC and serve important roles in NSCLC initiation and development (5–7). miRNAs represent a large family of non-coding and small RNA molecules of ~20–23 nucleotides in length (8). They can negative regulate the expression of their target messenger RNAs (mRNAs) via interacting with the 3′ untranslated regions (3′UTRs) of genes, and therefore enhancing mRNA degradation or translational suppression (9,10). There are ~1881 precursor and 2588 mature miRNAs in the human genome which are estimated to regulate the expression of >60% of coding-genes (11). Through these mechanisms, miRNAs are involved in regulation of diverse bio-behaviors including cell proliferation, cell cycle, apoptosis, metastasis and chemotherapy resistance, which are important in developmental processes and tumorigenesis of cancers (12–14). It has previously been reported that miRNAs whose expression are dysregulated in human cancers can function as oncogenes or tumor suppressors, primarily depending on the characteristic of their target genes (15). For example, the expression level of miR-370 is reduced in NSCLC, and upregulation of miR-370 inhibits cell proliferation and induces apoptosis via directly targeting oncogene tumor necrosis factor receptor associated factor 4 (16). In contract, miR-575 is upregulated in NSCLC, and targets the tumor suppressor gene BH3-like motif-containing cell death inducer to promote cell proliferation, migration and invasion in vitro (17).

The present study revealed that miR-454 was downregulated in NSCLC tissues and cell lines. Low miR-454 expression was significantly associated with aggressive clinicopathological features in NSCLC. Functional experiments indicated that miR-454 overexpression suppressed proliferation, migration and invasion of NSCLC cells in vitro. Furthermore, signal transducer and activator of transcription-3 (STAT3) was demonstrated to be a direct target gene of miR-454. These results suggested that miR-454 served as a tumor suppressor in NSCLC via directly targeting STAT3.

Materials and methods

Tissue samples

Tissue samples, including NSCLC and adjacent normal tissues, were obtained from 67 NSCLC patients who underwent surgery at Department of Thoracic Surgery, Yantaishan Hospital (Yantai, China) between 2012 and 2014. The adjacent normal tissues were obtained 5 cm from the edge of tumor tissues. None of these NSCLC patients received chemotherapy or radiotherapy prior to surgery. The Ethical Committee of Yantaishan Hospital approved this study, and written informed consent was obtained from all subjects. Patient characteristics are presented in Table I.

Table I. Correlations between miR-454 expression and clinicopathological features in NSCLC.

Table I.

Correlations between miR-454 expression and clinicopathological features in NSCLC.

		miR-454 expression
Clinicopathological features	Cases	High	Low	P-value
Gender				0.376
Male	32	13	19
Female	35	18	17
Age (years)				0.434
<55	29	15	14
≥55	38	16	22
Smoke				0.318
Yes	45	19	26
No	22	12	10
Differentiation				0.773
I–II	30	15	15
III–IV	37	16	21
TNM stage				0.023
I–II	29	18	11
III–IV	38	13	25
Lymph node metastasis				0.001
No	29	20	9
Yes	38	11	27

[i] miR-454, microRNA-454; NSCLC, non-small cell lung cancer.

Cell lines and culture conditions

The A549, SPC-A-1, H520 and H1299 NSCLC cell lines and the BEAS-2B human lung epithelial cell were obtained from the China Center for Type Culture Collection (Wuhan, China). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 µM glutamine, and 1% penicillin and streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in humidified air with 5% CO2 at 37°C.

Transient transfection

Both oligonucleotide miR-454 mimics, negative control (NC), STAT3 small interfering (si)RNA and NC siRNA were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The STAT3 siRNA sequence was 5′-CAUCUGCCUAGAUCGGCUA-3′ and the NC siRNA sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. NSCLC cells were seeded into 6-well plates (5×105 cells/well) and transfected with oligonucleotides using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The transfection efficiency was assessed by using reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

RT-qPCR

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.). For miR-454 expression, reverse transcription was performed using TaqMan MicroRNA Reserve Transcription kit, followed by qPCR with a Taqman MicroRNA Assay kit (both from Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 60 sec. To quantify STAT3 mRNA expression, cDNA was synthesized using a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) and qPCR was performed with a SYBR-Green PCR Master Mix (Takara Biotechnology Co., Ltd). The thermocycling conditions were as follows: 95°C for 10 min, then 40 cycles of 95°C for 15 sec and 60°C for 1 min. PCR was performed on an Applied Biosystems Real-Time 7500 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). U6 and GADPH served as internal controls for miR-454 and STAT3 mRNA expression, respectively. The primers were designed as follows: miR-454 forward, 5′-ACCCTATCAATATTGTCTCTGC-3′ and reverse, 5′-GCGAGCACAGAATTAATACGAC-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; STAT3 forward, 5′-TTGCCAGTTGTGGTGATC-3′ and reverse, 5′-AGACCCAGAAGGAGCCGC-3′; and GAPDH forward, 5′-CATCTTCTTTTGCGTCGCCA-3′ and reverse, 5′-TCGCCCCACTTGATTTTGG-3′. The relative fold expressions were determined with the 2−ΔΔCq method (18).

Cell Counting Kit-8 (CCK-8) assay

Cell proliferation was measured using a CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Transfected (miRNA/siRNA) cells were cultured into 96-well plates at a density of 2,000 cells/well. CCK-8 assay was performed every 24 h for 96 h following the manufacturer's protocol. CCK-8 (10 µl) solution was added and cells were cultured for 2 h in humidified air with 5% CO2 at 37°C. Absorbance was then measured at a wavelength of 450 nm using a microplate reader. All experiments were performed in triple.

In vitro migration and invasion assay

In vitro migration and invasion assays were performed with 24-well Transwell chambers (8-mm pore size; BD Biosciences). For the invasion assay, Transwell chambers were pre-coated with Matrigel (BD Biosciences). For both assays, 5×104 transfected cells in 100 µl FBS-free culture medium were injected into the upper chamber, while 500 µl culture medium containing 20% FBS was added into the lower chambers as a chemoattractant. A total of 48 h after transfection in humidified air with 5% CO2 at 37°C, the Transwell chambers were fixed with 95% methanol and stained with 0.1% crystal violet. After washing, migrated and invaded cells were counted and imaged under an inverted microscope at ×200 magnification (IX71; Olympus Corporation).

Bioinformatics analysis

Bioinformatics analysis was performed to predict the putative targets of miR-454 using TargetScan (version 6.0; www.targetscan.org).

Luciferase reporter assay

Luciferase reporter plasmids, pmirGLO-STAT3-3′UTR wild-type (Wt) and pmirGLO-STAT3-3′UTR mutant (Mut), were synthesized by Shanghai GenePharma. For the luciferase reporter assay, HEK293T cells (China Center for Type Culture Collection) were transfected with miR-454 mimics or NC together with pmirGLO-STAT3-3′UTR Wt or pmirGLO-STAT3-3′UTR Mut using Lipofectamine 2000. A total of 48 h after co-transfection, luciferase activities were measured using the Dual-Luciferase Reporter Assay system (Promega Corporation, Madison, WI, USA). Renilla luciferase activity was used for normalization. This assay was performed in triplicate and repeated three times.

Western blotting

A total of 72 h after transfection, cells were washed with PBS (Gibco; Thermo Fisher Scientific, Inc.) and lysed in radioimmunoprecipitation assay buffer supplemented with freshly added protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). Following centrifugation at 16,000 × g and 4°C for 10 min, protein concentration was determined using a Bicinchoninic Acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (30 µg) were loaded onto a 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and subsequently blocked with 5% non-fat milk in TBS containing 0.1% Tween-20 (TBST). After incubation with primary antibodies at 4°C overnight, the membranes were washed with TBST three times and then probed with a corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000; cat no. sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at room temperature. The protein bands were visualized using an Enhanced Chemiluminescence kit (EMD Millipore, Billerica, MA, USA), and the relative expression of STAT3 was normalized to the level of GADPH. The primary antibodies used in this study were mouse anti-human STAT3 monoclonal (1:1,000; cat no. sc-77441) and mouse anti-human monoclonal GADPH (1:1,000; cat no. sc-166574) (both from Santa Cruz Biotechnology, Inc.).

Statistical analysis

Data are presented as the mean ± standard deviation. SPSS version 19.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis and a Student's t-test or one-way analysis of variance, with a Student-Newman-Keuls post hoc test for multiple comparisons, were performed. P<0.05 was considered to indicate a statistically significant difference.

Results

Low levels of miR-454 in NSCLC tissues and cell lines

To investigate the potential roles of miR-454 in NSCLC progression, miR-454 expression in NSCLC and adjacent normal tissues was assessed. As presented in Fig. 1A, miR-454 was downregulated in NSCLC tissues compared with adjacent normal tissues. The expression of miR-454 in NSCLC cell lines (A549, SPC-A-1, H520 and H1299) and the human lung epithelial cell (BEAS-2B) was also detected; the expression levels of miR-454 were significantly lower in NSCLC cell lines than in the BEAS-2B cell line (Fig. 1B).

Figure 1. Low expression levels of miR-454 in NSCLC tissues and cell lines. (A) Relative expression of miR-454 in NSCLC and adjacent normal tissues. Data are presented as box plots and a Student's t-test was used to determine the significant differences between groups. The top of the box indicates the upper quartile, while the bottom of the box indicates the lower quartile. The central line in the box indicates the median and the whiskers indicate the minimum and maximum. (B) miR-454 expression in NSCLC cell lines and the NEAS02B human lung epithelial cell line was measured by reverse transcription-quantitative polymerase chain reaction. Data are expressed as the mean ± standard deviation. *P<0.05. NSCLC, non-small cell lung cancer; miR-454, microRNA-454.

Correlations between miR-454 expression and clinicopathological features in NSCLC

The correlations between miR-454 expression and clinicopathological features in NSCLC are presented in Table I. The results of the statistical analysis revealed that miR-454 expression in NSCLC was significantly correlated with TNM stage (P=0.023) and lymph node metastasis (P=0.001), but no correlation was observed between miR-454 expression and gender (P=0.376), age (P=0.434), smoking (P=0.318) and differentiation (P=0.773).

miR-454 inhibits proliferation, migration and invasion of NSCLC cells

A549 and H1299 cells, which express relatively low levels of endogenous miR-454, were transfected with miR-454 mimics or NC. The transfection efficiency was assessed by RT-qPCR, and miR-454 was demonstrated to be markedly overexpressed in both A549 and H1299 cells following transfection with miR-454 mimics (Fig. 2A).

Figure 2. Ectopic miR-454 inhibits A549 and H1299 cell proliferation, migration and invasion. (A) A549 and H1299 cells were transfected with miR-454 mimics or NC, and the expression level of miR-454 was determined by reverse transcription-quantitative polymerase chain reaction. The effects of miR-454 on cell proliferation, migration and invasion were evaluated by (B) CCK-8 and (C) in vitro migration and invasion assays, respectively. Data are expressed as the mean ± standard deviation. *P<0.05. NSCLC, non-small cell lung cancer; miR-454, microRNA-454; NC, negative control.

Following this, the effects of miR-454 overexpression on the proliferation, migration and migration of A549 and H1299 cells was assessed using CCK-8 and in vitro migration and invasion assays, respectively. As presented in Fig. 2B, A549 and H1299 cells transfected with miR-454 mimics exhibited reduced proliferation. Furthermore, the number of migrated and invaded cells was significantly lower in A549 and H1299 cells transfected with miR-454 mimics (Fig. 2C). Collectively, these results indicated that miR-454 serves as a tumor suppressor in NSCLC via inhibiting tumor cell proliferation, migration and invasion.

STAT3 is a direct target of miR-454 in NSCLC

To identify potential target genes of miR-454, bioinformatics analysis was performed with TargetScan (http://www.targetscan.org/). The analysis of TargetScan indicated that STAT3 is a putative target of miR-454 (Fig. 3A). Following this, a luciferase reporter assay was performed to investigate whether the 3′UTR of STAT3 could be directly targeted by miR-454. HEK293T cells were co-transfected with miR-454 mimics or NC, and pmirGLO-STAT3-3′UTR Wt or pmirGLO-STAT3-3′UTR Mut. The results demonstrated that overexpression of miR-454 resulted in decreased luciferase activities in pmirGLO-STAT3-3′UTR Wt, but not pmirGLO-STAT3-3′UTR Mut (Fig. 3B).

Figure 3. miR-454 negatively regulates STAT3 expression by directly binding to its 3′UTR. (A) The potential miR-454 seed region at the 3′UTR of STAT3 was predicted by bioinformatics analysis with TargetScan software. (B) Luciferase reporter plasmids were co-transfected with miR-454 mimics or NC in HEK293T cells. Luciferase activities were measured 48 h after transfection. Data are expressed as the mean ± standard deviation. STAT3 (C) mRNA and (D) protein expression levels in A549 and H1299 cells transfected with miR-454 mimics or NC were detected by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. Data are expressed as the mean ± standard deviation. *P<0.05. NSCLC, non-small cell lung cancer; miR-454, microRNA-454; NC, negative control; UTR, untranslated region; STAT3, signal transducers and activators of transcription 3; Wt, wild-type; Mut, mutant.

Following this, the regulatory effects of miR-454 overexpression on STAT3 expression were investigated. RT-qPCR and western blotting were performed to detect STAT3 mRNA and protein expression levels in A549 and H1299 transfected with miR-454 mimics or NC, respectively. As presented in Fig. 3C, upregulation of miR-454 attenuated STAT3 mRNA expression in A549 and H1299 cells. The results of western blot analysis revealed that STAT3 protein expression was reduced by miR-454 overexpression in A549 and H1299 cells (Fig. 3D). These results demonstrated that STAT3 is a direct target of miR-454 in NSCLC.

Downregulation of STAT3 may simulate the miR-454-induced tumor suppressive roles in NSCLC

To investigate the roles of STAT3 in NSCLC, A549 and H1299 cells were transfected with STAT3 siRNA or NC siRNA. The transfection efficiency was determined using RT-qPCR and western blotting, and it was demonstrated that STAT3 was significantly downregulated in A549 and H1299 cells at both the mRNA (Fig. 4A) and protein (Fig. 4B) level. In addition, decreased cell proliferation (Fig. 4C) was accompanied by decreased cell migration and invasion (Fig. 4D) in A549 and H1299 cells transfected with STAT3 siRNA. Therefore, it was concluded that the tumor suppressive roles of miR-454 in NSCLC cells was a consequence of reduced STAT3 expression in NSCLC.

Figure 4. Downregulation of STAT3 inhibits A549 and H1299 cell proliferation, migration and invasion. A549 and H1299 cells were injected with STAT3 siRNA or NC siRNA, and the (A) mRNA and (B) protein expression levels of STAT3 were determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The effects of STAT3 underexpression on cell proliferation, migration and invasion were evaluated by (C) Cell Counting kit-8 and (D) in vitro migration and invasion assays, respectively. Data are expressed as the mean ± standard deviation. NSCLC, non-small cell lung cancer; miR-454, microRNA-454; NC, negative control; STAT3, signal transducers and activators of transcription 3; siRNA, small interfering RNA.

Discussion

Previous studies have revealed that a great deal of miRNAs are dysregulated in NSCLC, and part of these miRNAs have been demonstrated to be correlated with particular clinicopathological factors of NSCLC (19–21). Notably, accumulated evidence has verified that miRNAs are involved in NSCLC carcinogenesis and progression via regulation of cell growth, apoptosis, angiogenesis, migration and invasion (22–24). Therefore, it is beneficial to investigate the expression, functions and corresponding targets of deregulated miRNAs in NSCLC, which could provide therapeutic targets to improve the prognosis of this disease. In the present study, it was demonstrated that miR-454 is downregulated in NSCLC. In addition, reduced miR-454 expression was significantly correlated with aggressive clinicopathological features. miR-454 overexpression suppressed cell proliferation, migration and invasion of NSCLC. In addition, STAT3 was identified as a direct target of miR-454. To the best of our knowledge, our current study is the first to investigate the expression, clinical significance and biological roles of miR-454 in NSCLC.

The aberrant expression of miR-454 has been reported in various kinds of cancers. Sun et al (25) demonstrated that miR-454 is obviously upregulated in uveal melanoma. In colorectal cancer, the expression level of miR-454 was higher in tumor tissues and cell lines than the matched tumor adjacent tissues and normal colonic cell line, respectively (26). Zhou et al (27) reported that miR-454 was upregulated in hepatocellular carcinoma, and upregulation of miR-454 was associated with a low 5-year overall survival. Multivariate analysis indicated that miR-454 overexpression was an independent prognostic factor for 5-year overall survival and disease-free survival (27). However, a study by Niu et al (28) demonstrated that expression of miR-454 was lower in osteosarcoma tissue specimens and cell lines. Fang et al (29) revealed that miR-454 was significantly downregulated in glioblastoma tissue samples and cells. These studies suggested that miR-454 expression is deregulated in cancers, and may contribute to tumorigenesis and tumor progression.

The biological functions of miR-454 have been studied in many kinds of human cancer. For example, upregulation of miR-454 suppresses osteosarcoma cell proliferation and invasion via negative regulation of c-Met (28). miR-454 targets stromal cell derived factor-1 to regulate the growth of pancreatic ductal adenocarcinoma cells (30). Downregulation of miR-454 represses hepatocellular carcinoma cell proliferation, invasion and epithelial mesenchymal transition by directly targeting chromodomain helicase DNA binding protein 5 (31). miR-454 serves as an oncogene in uveal melanoma via promotion of cell growth, colony formation, invasion and induction of the cell cycle (25). Liang et al (26) demonstrated that miR-454 enhances cell proliferation in colorectal cancer via blockade of ubiquitin carboxyl-terminal hydrolase CYLD (26). In glioblastoma, miR-454 overexpression suppresses cell growth by downregulation of 3-phosphoinositide-dependent protein kinase 1 (29). These findings suggested that miR-454 may represent an efficient therapeutic target in human cancers.

Furthermore, STAT3, an oncogene, was identified as a direct target of miR-454. STAT3 is a signal mediator that can be activated by various kinds of cytokines, growth factors and interferons (32). Mounting studies have demonstrated that STAT3 is constitutively activated in dozens of human cancers, including prostate (33), breast (34), head and neck (35) and lung (36). In the present study, STAT3 mRNA and protein expression levels were upregulated in NSCLC. Furthermore, high STAT3 expression was associated with tumor differentiation, clinical stage and lymph node metastasis of NSCLC patients. NSCLC patients with high STAT3 expression had a poorer prognosis compared with patients with low STAT3 expression. In a previous study, multivariate analysis revealed that high STAT3 protein expression was an independent prognostic factor for NSCLC patients (37). A functional study by Zhu et al (38) revealed that antisense oligonucleotides targeting STAT3 inhibit NSCLC cell proliferation and induce apoptosis (37). Siveen et al (39) reported that STAT3 inhibits apoptosis and induces cell proliferation, angiogenesis, invasion and metastasis in various human cancers (39). Given the importance and role of STAT3 in multiple oncogenic signaling pathways, STAT3 may be validated as a therapeutic target for patients with NSCLC.

In conclusion, the current study provided evidence that miR-454 may function as a tumor suppressor in NSCLC, partly by regulating STAT3 expression. Therefore, regulating miR-454 expression represents a potential strategy for the treatment of NSCLC patients.

References