MicroRNA‑718 serves a tumor‑suppressive role in non‑small cell lung cancer by directly targeting CCNB1.

MicroRNA‑718 (miR‑718) serves crucial roles in tumorigenesis and in the progression of a number of cancers. However, the expression profile, specific functions and mechanisms of action of miR‑718 in non‑small cell lung cancer (NSCLC) are still elusive. The aims of the present study were to quantify the expression of miR‑718, determine its biological roles and elucidate the molecular mechanisms responsible for its activities in NSCLC cells. Reverse transcription‑quantitative PCR was carried out to assess miR‑718 expression in NSCLC tissue samples and cell lines. The Cell Counting Kit‑8 assay, flow cytometry, cell migration and invasion assays, and a tumor xenograft experiment were performed to evaluate the effects of miR‑718 overexpression on the malignant biological behaviors of NSCLC cells. miR‑718 expression was demonstrated to be significantly decreased in NSCLC tissue samples and cell lines. This reduced expression was significantly associated with tumor, node, metastasis stage, tumor size, lymph node metastasis and poor overall survival among patients with NSCLC. Exogenous miR‑718 expression suppressed NSCLC cell proliferation, migration and invasion, and promoted apoptosis in vitro; whereas it hindered tumor growth in vivo. Experiments to elucidate the mechanisms involved revealed that miR‑718 functions by directly targeting cyclin B1 (CCNB1) mRNA. CCNB1 expression was found to be upregulated in NSCLC and inversely correlated with miR‑718 levels. CCNB1 depletion had effects similar to those of miR‑718 overexpression in NSCLC cells. Furthermore, restoration of CCNB1 expression attenuated the tumor‑suppressive effects of miR‑718 overexpression in NSCLC cells. These results indicated that miR‑718 suppressed NSCLC progression in vitro and in vivo by directly targeting CCNB1 mRNA, which may indicate a potential target for the diagnosis and treatment of this fatal disease.

Introduction

Lung cancer is a malignant cancer that ranks third among cancers in terms of incidence and is a leading cause of cancer-related deaths globally (1). Worldwide, ~1.8 million people are diagnosed with lung cancer, and ~1.6 million patients die of lung cancer each year (2). Lung cancer is subdivided into two main histopathological types: Non-small cell lung cancer (NSCLC) and small cell lung cancer (3). NSCLC, which includes squamous cell carcinoma and adenocarcinoma, accounts for ~85% of all lung cancer cases (4). Diagnostic and therapeutic techniques have improved considerably in recent years; however, long-term clinical outcomes among patients with NSCLC are still poor, with a 5-year survival rate of <15% (5,6). The poor prognosis of patients with NSCLC is mainly attributed to diagnostic delay as well as tumor invasion, metastasis and recurrence (7,8). Accordingly, elucidation of the NSCLC pathogenesis may facilitate the development of novel and more effective therapies for patients with this disease.

MicroRNAs (miRNAs) are a class of non-coding, single-stranded short RNA molecules 18-22 nucleotides long (9). miRNAs negatively regulate gene expression by completely or incompletely interacting with the 3′-untranslated region (UTR) of their target mRNAs, thereby causing translational suppression and/or mRNA degradation (10). To date, over 1,881 human miRNAs have been identified according to miRBase (Release 21; http://www.mirbase.org). These molecules have been proposed to regulate the expression of >30% of all protein-coding genes (11). Numerous studies have revealed that various miRNAs are abnormally expressed in NSCLC and contribute to the aggressive phenotype of NSCLC cells by affecting a wide range of biological processes (12-14). Hence, miRNA-based targeted therapy may be a promising therapeutic strategy against NSCLC.

In our pre-experiment, the expression levels of several miRNAs which had not been studied in NSCLC, including miR-671, miR-718, miR-767 and miR-791, were assessed. miRNA (miR)-718 expression was observed to be low in NSCLC. miR-718 is aberrantly expressed in multiple cancers (15-18) and serves crucial roles in carcinogenesis and cancer progression. However, the expression profile, specific functions and mechanisms of action of miR-718 in NSCLC are still unclear. In the present study, the expression of miR-718 in NSCLC tissue samples and cell lines was measured. Cell proliferation, apoptosis, migration and invasion in vitro, as well as tumor growth in vivo were analyzed to determine whether miR-718 overexpression influenced the oncogenicity of NSCLC cells. Furthermore, the mechanisms by which miR-718 exerts its tumor-suppressive actions in NSCLC were elucidated in detail.

Materials and methods

Patient samples

A total of 54 pairs of NSCLC tissue samples and adjacent normal tissue samples were collected from patients with NSCLC (29 males, 25 females; age, 47-75 years) who had undergone surgical resection at Jilin Province Tumor Hospital (Changchun, China; Table I) between May 2011 to March 2014. All tissue specimens were immediately frozen in liquid nitrogen and then transferred to a −80°C freezer for storage until subsequent analysis. The patients who had received preoperative chemotherapy, radiotherapy, or other anticancer treatments were excluded from the present study. All experimental protocols were approved by the Ethics Committee of Jilin Province Tumor Hospital and all the experiments were conducted in accordance with the Declaration of Helsinki. In addition, written informed consent was obtained from all patients prior to enrolment in the present study.

Table I

Association between miR-718 expression and clinicopathological features of 54 patients with NSCLC.

Clinicopathological feature	miR-718 expression		P-value
	Low	High
Sex			0.275
Male	17	12
Female	10	15
Age (years)			0.786
<60	13	15
≥60	14	12
Tumor size (cm)			0.010
<3	12	22
≥3	15	5
Histological grade			0.766
Well/moderate	18	20
Poor	9	7
TNM stage			0.012
I-II	6	16
III-IV	21	11
Lymph node metastasis			0.028
Negative	8	17
Positive	19	10

miR-718, microRNA-718; TNM, tumor-node-metastasis.

Cell lines and cultures

NSCLC cell lines (H522, H460, H1299, A549 and SK-MES-1) and a non-tumorigenic bronchial-epithelium BEAS2B cell line, which served as the control, were bought from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich, Merck KGaA) were used for cell culture. All the cells were kept at 37°C in a humidified atmosphere supplied with 5% CO2 until use for subsequent experiments.

Cell transfection

miR-718 agomir (agomir-718) and negative control (NC) agomir (agomir-NC) were chemically synthe-sized by Shanghai GenePharma Co., Ltd. The small interfering RNA (siRNA) targeting cyclin B1 (si-CCNB1) and a negative control siRNA (si-NC) were from Guangzhou RiboBio Co., Ltd. CCNB1 overexpression vector pcDNA3.1-CCNB1 (pc-CCNB1) obtained from OriGene Technologies, Inc. was used to restore CCNB1 expression, the empty pcDNA3.1 plasmid was used as a control. Agomir (50 nM), siRNA (100 pmol) or overexpression plasmid (4 µg) were transfected into cells using the Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The cells used for transfection were seeded into 6-well plates at a density of 8×105 cells/well. After 8 h transfection, the cell culture medium was replaced with DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. Reverse transcription-quantitative PCR (RT-qPCR), flow cytometric analysis and cell migration and invasion assays were conducted at 48 h after transfection. Cell Counting Kit-8 (CCK-8) assay and western blotting were performed at 24 and 72 h post-transfection, respectively.

RNA preparation and RT-qPCR

TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for total RNA isolation from the tissues (100 mg) and cells (1.5×106). To quantify miR-718 expression, total RNA was transcribed into cDNA with the miScript Reverse Transcription kit (Qiagen GmbH) as follows: 37°C for 60 min and 95°C for 5 min. qPCR was conducted using the miScript SYBR-Green PCR kit (Qiagen GmbH) and all reactions were performed on an ABI Prism 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following thermocycling conditions: 95°C for 2 min, followed by 40 cycles of 95°C for 10 sec, 55°C for 30 sec and 72°C for 30 sec. Small nuclear RNA U6 served as an internal control and for normalization of miR-718 expression. The primers were as follows: miR-718, forward 5′-CAG TGC GTG TCG TGG AGT-3′, reverse 5′-CAG TGC GTG TCG TGG AGT-3′; U6, forward 5′-GCT TCG GCA GCA CAT ATA CTA AAA T-3′, reverse 5′-CGC TTC ACG AAT TTG CGT GTC AT-3′. For the detection of CCNB1 expression, cDNA was synthesized with the PrimeScript RT-reagent kit (Takara Bio, Inc.); the temperature protocols for reverse transcription were as follows: 37°C for 15 min and 85°C for 5 sec. qPCR using performed using the SYBR Premix Ex Taq™ kit (Takara Bio, Inc.) and an ABI Prism 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following thermocycling conditions: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. The expression of CCNB1 mRNA was normalized to the internal reference gene GAPDH. The 2−ΔΔCq method was used to analyze the relative gene expression (19). CCNB1, forward 5′-TTG GGG ACA TTG GTA ACA AAG TC-3′, reverse 5′-ATA GGC TCA GGC GAA AGT TTT T-3′; and GAPDH, forward 5′-GGA TTT GGT CGT ATT GGG-3′, reverse 5′-GTG GCT GGG GCT CTA CTT C-3′.

CCK-8 assay

CCK-8 assay was performed to determine cellular proliferation according to the manufacturers protocol. Transfected cells were collected 24 h post-transfection and seeded (2×103 cells/well) in 96-well plates in triplicate. The CCK-8 assay was conducted at four time points (0, 24, 48 and 72 h after seeding) to determine cell proliferation. Briefly, transfected cells were incubated with 10 µl of the CCK-8 solution (Dojindo Molecular Technologies, Inc.) at 37°C for 2 h. The absorbance was measured at a 450 nm wavelength on a microplate reader (Molecular Devices, LLC).

Flow cytometric detection of apoptosis

Transfected cells were collected by treatment with trypsin containing no EDTA, rinsed thrice with ice-cold PBS and subjected to the detection of apoptosis using the Annexin V-FITC Apoptosis Detection kit (BioLegend, Inc.). Briefly, 2.0×105 cells were resuspended in 100 µl of binding buffer and then double-stained with 5 µl of Annexin V-FITC and 5 µl of the propidium iodide solution. After incubation for 20 min at room temperature in the dark, the stained cells were analyzed on a flow cytometer (FACScan™; BD Biosciences). Data was analyzed with CellQuest™ software version 5.1 (BD Biosciences).

Cell migration and invasion assays

The migratory capacity of the transfected cells was evaluated by means of filter Transwell chamber inserts with 8 µm pore size (Corning, Inc.). To be precise, the transfected cells were washed thrice with PBS and resuspended in FBS-free DMEM. The cell concentration was adjusted to 5×105 cells/ml. A total of 200 µl of each cell suspension was plated in the upper chambers, and the lower chambers were filled with 500 µl of DMEM supplemented with 20% FBS. After incubation for 24 h at 37°C, the cells that had not migrated were gently removed with a cotton swab, whereas the migratory cells that passed through the 8 µm pores were fixed in 100% methanol at room temperature for 30 min and stained with 0.1% crystal violet at room temperature for 30 min. The invasive ability of the transfected cells was evaluated in a similar manner, but using Transwell inserts precoated with Matrigel (BD Biosciences). Finally, images of the migratory and invading cells were captured using an Olympus light microscope (×200 magnification; Olympus Corporation).

Tumor xenograft experiment

A total of eight male BALB/c nude mice (weight, 20 g; age, 4-6 weeks) were obtained from the Core Animal Facility of Nanjing Medical University and were maintained under specific pathogen-free conditions (25°C; 50% humidity; 10-h light/14-h dark cycle) and ad libitum access to food and water. H522 cells transfected with either agomir-718 or agomir-NC were collected after 24 h of incubation, resuspended in 0.2 ml of PBS, and subcutaneously injected into the dorsal region of each nude mouse. From day 12 post-injection, the width and length of the resultant subcutaneous tumors were measured every 4 days. Tumor volume was calculated according to the following formula: 0.5× tumor length x tumor width2. All mice were sacrificed 4 weeks after the cell injection, and tumor xenografts were carefully excised, weighed and stored for further use. All the animal experimental procedures were approved by the Animal Research Ethics Committee of Jilin Province Tumor Hospital and conducted following the Animal Protection Law of the People's Republic of China-2009.

miR-718 target prediction

miRanda (http://www.microrna.org/microrna/home.do) and TargetScan (http://www.targetscan.org), were used to predict the target genes of miR-718.

Luciferase reporter assay

The 3′-UTR fragments of CCNB1 containing the wild-type (wt) miR-718-binding site or a mutant (mut) miR-718-binding site were amplified by GenePharma and cloned into the pmirGLO luciferase reporter vector (Promega Corporation). The generated luciferase reporter plasmids were designated as wt-CCNB1-3′-UTR and mut-CCNB1-3′-UTR, respectively. Cells were seeded into 24-well plates at a density of 1.0×105 cells/well and transfected with either agomir-718 (25 nM) or agomir-NC (25 nM) in combination with either wt-CCNB1-3′-UTR (0.8 µg) or mut-CCNB1-3′-UTR (0.8 µg) using the Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h of incubation at 37°C, the activity of luciferase was determined using a Dual-Luciferase Reporter Assay System (Promega Corporation). The firefly luciferase activity was normalized to Renilla luciferase activity.

Western blot analysis

Tissues (100 mg; homogenized tissues by grinding in liquid nitrogen) and cultured cells (1.5×106 cells) were lysed with RIPA buffer (Beyotime Institute of Biotechnology) to isolate total protein. The concentration of total protein was measured with the Bicinchoninic Acid Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein were separated by 10% SDS-PAGE, electrotransferred onto PVDF membranes, and blocked at room temperature for 2 h with 5% fat-free milk diluted with Tris-buffered saline containing 0.1% Tween-20 (TBST). Subsequently, the membranes were incubated at 4°C overnight with the following primary antibodies: Mouse anti-human CCNB1 antibody (cat. no. sc-7393; 1:1,000; Santa Cruz Biotechnology, Inc.) and mouse anti-human GAPDH antibody (cat. no. sc-69778; 1:1,000; Santa Cruz Biotechnology, Inc.). Following three rinses with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (cat. no. 516102; 1:5,000; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. The Amersham ECL Western Blotting Detection kit (GE Healthcare Life Sciences) was used for protein signal detection. GAPDH served as the loading control and for normalization of protein expression. Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.) was used for densitometric analysis.

Statistical analysis

All data are presented as the mean ± SD. Significant differences between two groups were examined by Student's t-test, and differences among multiple groups were evaluated by one-way ANOVA followed by the Student-Newman-Keuls post hoc multiple-comparison test. The correlation between miR-718 and CCNB1 expression levels was assessed by Spearman's correlation analysis. Survival analysis was performed using the Kaplan-Meier survival curve and logrank test. All statistical analyses were conducted using SPSS 17.0 software (SPSS Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results

miR-718 expression is decreased in NSCLC and is associated with poor prognosis

miR-718 expression levels in 54 pairs of NSCLC and adjacent normal tissues were determined by RT-qPCR. The data demonstrated that miR-718 expression was significantly lower in NSCLC tissue samples compare with the corresponding normal tissue samples (Fig. 1A; P<0.01). miR-718 expression was also decreased in the NSCLC cell lines (H522, H460, H1299, A549 and SK-MES-1) compared with that observed in the non-tumorigenic bronchial-epithelium BEAS-2B cell line (Fig. 1B; P<0.05). H522 and H460 cells notably expressed lower miR-718 levels compared with H1299, A549 and SK-MES-1 cells and were therefore chosen for subsequent experiments.

Figure 1

Lower miR-718 levels are expressed in both NSCLC tissue samples and cell lines. (A) Relative miR-718 expression levels were evaluated in 54 pairs of NSCLC tissue samples and adjacent normal tissues by RT-qPCR analysis. *P<0.05 vs. Normal. (B) RT-qPCR was performed to determine relative miR-718 expression in five NSCLC cell lines (H522, H460, H1299, A549 and SK-MES-1); the non-tumorigenic bronchial-epithelium cell line BEAS-2B served as the control. *P<0.05 vs. BEAS-2B. (C) Kaplan-Meier survival curve and logrank test were used to examine the relationship between miR-718 expression and overall survival among patients with NSCLC. P=0.0004. miR-718, microRNA-718; NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription-quantitative PCR.

To further explore the clinical value of miR-718 among patients with NSCLC, the 54 cases of NSCLC were classified into either miR-718 low-expression group (n=27) or miR-718 high-expression group (n=27), using the median value of miR-718 among the NSCLC tissue samples as a cutoff point. The analysis indicated that patients in the miR-718 low-expression group had a larger tumor size (P=0.010), a more advanced tumor-node-metastasis (TNM) stage (P=0.012) and a higher frequency of lymph node metastasis (P=0.028) (Table I). In addition, patients in the low miR-718 expression group were revealed to have shorter overall survival compared with patients in the high miR-718 expression group (Fig. 1C). These data suggested that miR-718 underexpression may be implicated in the malignancy of NSCLC.

miR-718 upregulation exerts an inhibitory effect on the proliferation, migration and invasion of NSCLC cells in vitro

Having demonstrated the low expression of miR-718 in NSCLC, its potential effects on the malignant characteristics of NSCLC cells were explored. To this end, agomir-718 was transfected into H522 and H460 cells to increase endogenous miR-718 expression, which was validated by RT-qPCR analysis (Fig. 2A). The CCK-8 assay was conducted to examine the influence of miR-718 on NSCLC cell proliferation. Increased miR-718 expression significantly suppressed the proliferative ability of H522 and H460 cells in comparison with the cells treated with agomir-NC at 48 and 72 h (Fig. 2B). Inhibition of cell proliferation is often accompanied by an increase in apoptosis. As expected, transfection with agomir-718 resulted in a significant increase in the total (early + late) apoptotic rate of H522 and H460 cells (Fig. 2C). Exogenous miR-718 expression led to a significant decrease in the migratory and invasive abilities of H522 and H460 cells (Fig. 2D), as revealed by cell migration and invasion assays. These results suggested that upregulation of miR-718 may act as a tumor suppressor in vitro, as indicated by the inhibition of proliferation, migration and invasion in transfected cells, as well as the increased apoptotic rates of NSCLC cells.

Figure 2

Exogenous miR-718 expression suppresses the proliferation, migration and invasion, and increases apoptosis of H522 and H460 cells. (A) The expression levels of miR-718 in H522 and H460 cells after transfection with either agomir-718 or agomir-NC were determined by reverse transcription-quantitative PCR. *P<0.05 vs. agomir-NC. (B) The Cell Counting Kit-8 assay was conducted to evaluate the proliferation of H522 and H460 cells that were transfected with either agomir-718 or agomir-NC. *P<0.05 vs. agomir-NC. (C) The proportion of apoptotic H522 and H460 cells with miR-718 overexpression was quantified by flow-cytometric analysis. *P<0.05 vs. agomir-NC. (D) Cell migration and invasion assays were performed on miR-718-overexpressing H522 and H460 cells (×200 magnification). *P<0.05 vs. agomir-NC. miR-718, microRNA-718; NC, negative control.

CCNB1 is a direct target of miR-718 in NSCLC cells

To elucidate how miR-718 may affect the progression of NSCLC in vitro, bioinformatics analysis was conducted to predict candidate targets of miR-718. Among these candidates, CCNB1 (Fig. 3A) was selected for further study since this gene has been reported to have crucial roles in NSCLC tumorigenesis and tumor development (20-26). To corroborate this target, the luciferase reporter assay was performed to determine whether miR-718 is able to directly interact with the 3′-UTR of CCNB1 mRNA in NSCLC cells. The luciferase activity of wt-CCNB1-3′-UTR was significantly decreased miR-718-expressing H522 and H460 cells, whereas the luciferase activity of mut-CCNB1-3′-UTR remained unaffected in cells transfected with agomir-NC (Fig. 3B). Furthermore, to examine whether miR-718 is able to regulate CCNB1 expression, RT-qPCR and western blotting were performed to assess CCNB1 mRNA and protein levels in H522 and H460 cells after agomir-718 or agomir-NC transfection. Results revealed that the mRNA (Fig. 3C) and protein (Fig. 3D) levels of CCNB1 were significantly decreased in the miR-718-overexpressing H522 and H460 cells. These results confirmed CCNB1 as a direct target of miR-718 in NSCLC cells.

Figure 3

CCNB1 is a direct target of miR-718 in NSCLC cells. (A) The wt miR-718-binding site in the 3′-UTR of CCNB1 was predicted by bioinfor-matics analysis. The mut binding sequences are also shown. (B) Either agomir-718 or agomir-NC was co-transfected with either wt-CCNB1-3′-UTR or mut-CCNB1-3′-UTR into H522 and H460 cells. The luciferase reporter assay was then performed to confirm the binding of miR-718 to the CCNB1 mRNA 3′-UTR in NSCLC cells. *P<0.05 vs. agomir-NC. (C) Reverse transcription-quantitative PCR and (D) western blotting were performed to evaluate the expression levels of CCNB1 mRNA and protein in H522 and H460 cells transfected with either agomir-718 or agomir-NC. *P<0.05 vs. agomir-NC. CCNB1, cyclin B1; miR-718, microRNA-718; mut, mutant; NSCLC, non-small cell lung cancer; NC, negative control; UTR, untranslated region; wt, wild-type.

CCNB1 mRNA expression is upregulated in NSCLC and inversely correlated with miR-718 levels

To assess the correlation between miR-718 and CCNB1 levels in NSCLC, the expression of CCNB1 in the 54 pairs of NSCLC tissue samples and adjacent normal tissue samples was measured by RT-qPCR. The relative expression of CCNB1 in NSCLC tissue samples was significantly higher compared with that in the adjacent normal tissues (Fig. 4A). In addition, an inverse correlation between miR-718 and CCNB1 mRNA expression levels in the 54 NSCLC tissues was confirmed by Spearman's correlation analysis (Fig. 4B). Furthermore, the NSCLC tissue samples in the miR-718 high-expression group were demonstrated to express significantly lower levels of CCNB1 mRNA (Fig. 4C) and protein (Fig. 4D and E) compared with those in the miR-718 low-expression group.

Figure 4

An inverse correlation was noted between miR-718 and CCNB1 mRNA expression among the 54 NSCLC tissue samples. (A) Total RNA was isolated from the 54 pairs of NSCLC tissue samples and adjacent normal tissue samples and then subjected to reverse transcription-quantitative PCR for the quantitation of CCNB1 mRNA expression. *P<0.05 vs. Normal. (B) The correlation between miR-718 and CCNB1 mRNA expression levels was investigated among the 54 NSCLC tissue samples by Spearman's correlation analysis. (C) mRNA and (D and E) protein expression levels of CCNB1 in the miR-718 low-expression and miR-718 high-expression groups. *P<0.05 vs. miR-718 low. CCNB1, cyclin B1; miR-718, microRNA-718; NSCLC, non-small cell lung cancer.

Decreased CCNB1 expression exerts effects similar to those of miR-718 overexpression in NSCLC cells

Having confirmed CCNB1 as a direct target gene of miR-718, the functions of CCNB1 in NSCLC cells were subsequently explored. A loss-of-function experiment was performed on H522 and H460 cells, in which cells were transfected with either si-CCNB1 or si-NC. The efficient silencing of CCNB1 expression in H522 and H460 cells was confirmed by western blotting (Fig. 5A). The knockdown of CCNB1 significantly inhibited the proliferation at 48 and 72 h (Fig. 5B) and increased the early + late apoptosis (Fig. 5C) of H522 and H460 cells, as revealed by the CCK-8 assay and flow-cytometric analysis, respectively. In addition, the migratory and invasive abilities of the CCNB1-deficient H522 and H460 cells were demonstrated to be significantly reduced in comparison with si-NC-transfected H522 and H460 cells (Fig. 5D). These observations suggested that suppression of CCNB1 imitated the effects of miR-718 overexpression in NSCLC cells, which indicated that CCNB1 downregulation may be a downstream mediator of the actions of miR-718 in NSCLC cells.

Figure 5

Effects of CCNB1 knockdown on the proliferation, apoptosis migration, and invasion of H522 and H460 cells. (A) Either si-CCNB1 or si-NC was transfected into H522 and H460 cells, and the decrease in CCNB1 protein expression was validated by western blotting. *P<0.05 vs. si-NC. (B and C) Effects of the CCNB1 knockdown on the (B) proliferation and (C) apoptosis of H522 and H460 cells were assessed by the Cell Counting Kit-8 assay and flow cytometry, respectively. *P<0.05 vs. si-NC. (D) Cell migration and invasion assays were conducted to evaluate the migratory and invasive abilities of H522 and H460 cells after either si-CCNB1 or si-NC transfection (×200 magnification). *P<0.05 vs. si-NC. CCNB1, cyclin B1; NC, negative control; si, small interfering RNA.

CCNB1 overexpression neutralizes the influence of miR-718 overexpression on the malignant phenotype of NSCLC cells

Based on the aforementioned results, the possibility of CCNB1 downregulation being responsible for the effects of miR-718 on the proliferation, migration and invasion of NSCLC cells was evaluated. H522 and H460 cells were co-transfected with agomir-718 and either CCNB1 overexpression plasmid pc-CCNB1 or the empty pcDNA3.1 vector. First, pc-CCNB1 or pcDNA3.1 was successfully introduced into H522 and H460 cells, confirmed by RT-qPCR analysis (Fig. 6A). The decrease in CCNB1 protein expression caused by miR-718 overexpression was almost completely reversed in H522 and H460 cells after co-transfection with pc-CCNB1 (Fig. 6B). Subsequently, CCK-8 assay, flow cytometric analysis, and cell migration and invasion assays were performed on H522 and H460 cells treated as described above. It was observed that the restoration of CCNB1 expression reversed the miR-718 overexpression-induced effects on proliferation (Fig. 6C), early + late apoptosis (Fig. 6D), migration and invasion (Fig. 6E) of H522 and H460 cells. Collectively, these results suggested that miR-718 was able to suppress the proliferation, migration and invasion of NSCLC cells, at least partly by decreasing CCNB1 expression.

Figure 6

CCNB1 overexpression reverses the effects of miR-718 overexpression on the malignant characteristics of H522 and H460 cells. (A) Reverse transcription-quantitative PCR was performed to determine CCNB1 mRNA expression in H522 and H460 cells after transfection with pc-CCNB1 or pcDNA3.1. *P<0.05 vs. pcDNA3.1. (B) Western blotting was used to measure CCNB1 protein expression in H522 and H460 cells after co-transfection of agomir-718 and either pc-CCNB1 overexpression vector or pcDNA3.1 empty vector. *P<0.05 vs. agomir-NC group; #P<0.05 vs. agomir-718 + pcNDA3.1. (C) Proliferation and (D) apoptosis of the aforementioned cells were investigated using the Cell Counting Kit-8 assay and flow-cytometric analysis, respectively. *P<0.05 vs. agomir-NC; #P<0.05 vs. agomir-718 + pcNDA3.1. (E) Cell migration and invasion assays were performed to evaluate the migratory and invasive abilities of H522 and H460 cells following co-transfection of agomir-718 and either pc-CCNB1 or pcDNA3.1 (×200 magnification). *P<0.05 vs. agomir-NC; #P<0.05 vs. agomir-718 + pcNDA3.1 group. CCNB1, cyclin B1; miR-718, microRNA-718; NC, negative control.

miR-718 overexpression decreases NSCLC tumor growth in vivo

Tumor xenograft experiments performed to evaluate the tumor-suppressive actions of miR-718 on NSCLC tumor growth in vivo. The weights (Fig. 7A), sizes (Fig. 7B) and volumes (Fig. 7C) of the tumor xenografts were significantly lower in the agomir-718 group compared with the agomir-NC group. After the tumor xenografts were excised, RT-qPCR was performed to measure the expression levels of miR-718. The results demonstrated that miR-718 expression levels were significantly higher in the tumor xenografts derived from agomir-718-transfected H522 cells compared with agomir-NC cells (Fig. 7D). Furthermore, the expression levels of CCNB1 mRNA (Fig. 7E; P<0.05) and protein (Fig. 7F and G; P<0.05) were significantly decreased in the tumor xenografts of the agomir-718 group compared with the agomir-NC group. These results indicated that the proliferation of NSCLC cells in vivo was hindered by miR-718 overexpression and this suppressive effect was potentially mediated by CCNB1 downregulation.

Figure 7

miR-718 overexpression hinders tumor growth in vivo. (A) Weights of tumor xenografts in the agomir-718 group and agomir-NC group. *P<0.05 vs. agomir-NC. (B) Representative images of tumor xenografts in the agomir-718 group and agomir-NC group. (C) The average volume of the tumor xenografts was assessed in the agomir-718 and agomir-NC groups. *P<0.05 vs. agomir-NC. (D and E) Reverse transcription-quantitative PCR was performed to quantify the expression of (D) miR-718 and (E) CCNB1 mRNA in the tumor xenografts derived from agomir-718-transfected or agomir-NC-transfected H522 cells. *P<0.05 vs. agomir-NC. (F and G) CCNB1 protein expression levels in tumor xenografts were measured by western blotting. *P<0.05 vs. agomir-NC. CCNB1, cyclin B1; miR-718, microRNA-718; NC, negative control.

Discussion

Dysregulation of miRNAs has been frequently reported in the past several decades (27-29). Differential miRNA expression may serve a crucial role in the oncogenicity of NSCLC by affecting a series of biological behaviors (30-32). Therefore, further exploration of cancer-related miRNAs in NSCLC may reveal potential targets for the diagnosis, prevention and treatment of NSCLC. In the present study, miR-718 expression was measured in NSCLC tissues and cell lines, and its clinical significance was examined among patients with NSCLC. In addition, the influence of miR-718 on the malignant characteristics of NSCLC cells in vitro and in vivo was evaluated.

miR-718 is known to be upregulated in gastric cancer tissues (15). Patients with gastric cancer and miR-718 overexpression have a poorer prognosis than patients with low miR-718 expression (15). miR-718 has been identified as a biomarker to predict an unfavorable prognosis among patients with gastric cancer (15). Conversely, miR-718 under-expression has been observed in ovarian cancer (16), papillary thyroid cancer (17) and hepatocellular carcinoma (18). These conflicting data prompted the evaluation of the expression status of miR-718 in NSCLC. Results from the present study demonstrated that miR-718 expression is decreased in NSCLC tissues and cell lines. miR-718 underexpression in NSCLC tissue samples were significantly associated with tumor size, TNM stage and lymph node metastasis in patients with NSCLC. In addition, patients with NSCLC with low miR-718 expression had shorter overall survival. These results suggested that miR-718 may be a novel biomarker for NSCLC diagnosis and the prediction of clinical outcomes in patients with NSCLC.

miR-718 exerts oncogenic functions in gastric cancer progression by promoting cell proliferation and invasion (15). The opposite effects are observed in ovarian cancer (16), papillary thyroid cancer (17) and hepatocellular carcinoma (18), where miR-718 is validated as a tumor-suppressive miRNA. For example, ectopic miR-718 expression suppresses the growth of ovarian cancer in vitro and in vivo (16). Exogenous miR-718 expression restricts cell proliferation, metastasis and glucose metabolism in papillary thyroid cancer (17). In hepatocellular carcinoma, recovery of miR-718 expression decreases the ability of colony formation, cell viability, migration and invasion (18). Nevertheless, the functions of miR-718 in the malignant progression of NSCLC, to the best of our knowledge, have not yet been explored. Results from the present study revealed that increased miR-718 expression reduced NSCLC cell proliferation, migration and invasion, and promoted apoptosis in vitro, while impairing tumor growth in vivo. These observations suggested that miR-718 is a potential target for anticancer therapy of patients with NSCLC.

Several genes, including PTEN homolog, vascular endothelial growth factor, 3-phosphoinositide-dependent protein kinase 1 and early growth response protein 3 have been identified as direct targets of miR-718 in (15-18). In the present study, CCNB1, a key initiator of mitosis, was identified as a direct and functional target of miR-718 in NSCLC. CCNB1 is overexpressed in NSCLC, and its high expression is closely correlated with tumor type, tumor differentiation, vascular invasion and the male sex (20,21). Patients with NSCLC featuring high CCNB1 expression have a shorter overall survival compared with patients with low CCNB1 expression (20-22). Notably, multivariate analysis has identified CCNB1 expression as a prognostic factor for patients with NSCLC (21,22). Functionally, CCNB1 exerts oncogenic actions and is implicated in the control of multiple cancer-related behaviors of NSCLC cells (23-26). In the present study, it was revealed that miR-718 directly targets CCNB1 mRNA and decreases CCNB1 expression in NSCLC, thereby inhibiting cell proliferation, migration and invasion, and increasing apoptosis in vitro and suppressing tumor growth in vivo. Accordingly, the knockdown of CCNB1 through miR-718 overexpression may be an effective approach for the treatment of NSCLC.

Three limitations are included in the present study. Firstly, endogenous miR-718 expression was not knocked down and subsequently the effects of miR-718 silencing in NSCLC progression were not explored. Loss-of-function assays would be able to further demonstrate the tumor-suppressive roles of miR-718 in the oncogenicity of NSCLC, and therefore may aid in our understanding of the mechanisms of action of miR-718 in the context of NSCLC. Secondly, the influence of miR-718 on the NSCLC cell cycle was not examined. Lastly, the correlation between miR-718 and CCNB1 protein expression in NSCLC tissues was not analyzed. Further studies may be able to resolve these limitations.

In summary, data from the present study indicated that miR-718 may exert its tumor-suppressive effects on the malignant biological behaviors of NSCLC cells by directly targeting CCNB1 mRNA and thereby downregulating CCNB1 expression. These results may offer new insights into the molecular pathogenesis of NSCLC and, thus, may facilitate the validation of miR-718 as a therapeutic target for managing this fatal disease.