Long non‑coding RNA FEZF1‑AS1 facilitates non‑small cell lung cancer progression via the ITGA11/miR‑516b‑5p axis
- Authors:
- Published online on: October 29, 2020 https://doi.org/10.3892/ijo.2020.5142
- Pages: 1333-1347
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Copyright: © Song et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Lung cancer, as one of the most common types of cancer, causes more deaths than breast, prostate, colorectal and brain cancer combined in 2017 in the United States, with the 5-year relative survival rate being 5% (1). Non-small cell lung cancer (NSCLC) accounted for >80% of all lung cancer cases worldwide in 2016, which fall into several categories, including adenocarcinoma, squamous cell carcinoma and large cell carcinoma (2). The 5-year survival rate for metastatic NSCLC is <5%, and that for patients with early-stage NSCLC is <50% (3). Therefore, finding novel target biomarkers is urgently required.
Accumulating evidence has revealed that most RNA transcripts do not encode proteins in mammals (4,5). Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs of >200 nucleotides in length (6). lncRNAs used to be considered non-functional (7), but an increasing number of lncRNAs have now been reported to serve important roles in biological processes; for example, lncRNAs form extensive networks of ribonucleoprotein complexes with numerous chromatin regulators and then target enzymatic activities to appropriate locations in the genome (8,9). Rigorous evidence has suggested that certain lncRNAs exert important regulatory effects in carcinogenesis and the progression of prostate, hepatocellular and lung cancer, with potential roles in oncogenic or tumor-suppressive signaling pathways (4,10,11-13).
FEZ family zinc finger 1 antisense RNA 1 (FEZF1-AS1) is a lncRNA that is highly expressed in colorectal carcinoma (CRC), and its overexpression can promote the aggressive behavior of CRC cells, both in vitro and in vivo (14). In gastric cancer, high FEZF1-AS1 expression promotes cell proliferation (14). In addition, the inhibition of FEZF1-AS1 can suppress the activation of the Wnt/p-catenin signaling pathway (15). Additionally, FEZF1-AS1 expression is upregulated in lung adenocarcinoma and is mediated by FEZF1 (16). However, the mechanism of FEZF1-AS1 in NSCLC remains unclear. Although FEZF1-AS1 expression is upregulated in multiple types of cancer, there are different views on the location and mechanism of FEZF1-AS1 in the existing literature. He et al (17) reported that FEZF1-AS1 is located in the cytoplasm and nucleus in lung adenocarcinoma A549 and SPC-A1 cells, while in other studies FEZF1-AS1 has been reported to be mainly located in the cytoplasm in osteosarcoma, pancreatic ductal adenocarcinoma and myeloma (18-20). Therefore, further elucidation of the mechanism and location of FEZF1-AS1 in NSCLC is required.
In the present study, the association between the expression levels of FEZF1-AS1 in NSCLC tissues and overall survival (OS) was explored. In addition, the function of FEZF1-AS1 in NSCLC cells, the N6-methyladenosine (m6A) modification and the involvement of the FEZF1-AS1/integrin subunit all (ITGA11)/microRNA (miRNA/miR)-516b-5p axis in NSCLC were investigated. The present results may provide novel biomarkers for NSCLC.
Materials and methods
Patient and clinical data collection
The study protocol was approved by the Institutional Review Board of Hebei Medical University (Shijiazhuang, China). Frozen surgical tumor tissues and corresponding normal lung tissues (5 cm from the edge of the tumor) of 45 patients with NSCLC (including 17 squamous cell carcinoma, 27 adenocarcinoma and 1 atypical carcinoid) were obtained from The Fourth Hospital of Hebei Medical University (Shijiazhuang, China) between February 2009 and February 2018. The median age of the patients was 61 years (range, 44-73 years). All patients were diagnosed with NSCLC on histopathological evaluation, and were reviewed by experienced pathologists and staged according to the 8th edition lung cancer classification of the American Joint Committee of Cancer (21). EGFR was detected via PCR, and ALK was detected via fluorescence in situ hybridization (FISH) by the Department of Pathology of the Fourth Hospital of Hebei Medical University. PD-1 and PD-L1 were detected via immunohistochemistry (IHC) by the Department of Pathology of the Fourth Hospital of Hebei Medical University. PCR, FISH and IHC were performed according to routine hospital protocols. No patients received any other treatment prior to surgery, and all patients were followed up until February 2020. The smoking index was calculated as cigarettes/day x smoking time (years). Since a smoking index >400 indicated a high risk of lung cancer, patients were divided into never smokers (0, smoking index=0), light smokers (1, smoking index <400) and heavy smokers (2, smoking index >400). The study was conducted in accordance with the Declaration of Helsinki, and approval was obtained from the Research Ethics Committee of the Fourth Hospital of Hebei Medical University.
Cell culture
Human NSCLC H358, H1299, A549, H520, SK-MES-1, H1703 and normal human bronchial epithelial BEAS-2B cells were purchased from the China Infrastructure of Cell Line Resources. SK-MES-1 cells were cultured in Minimum Essential Medium (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gemini Bio Products), 10,000 U/ml penicillin and 10,000 μg/ml streptomycin (Thermo Fisher Scientific, Inc.). H520 cells were cultured in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gemini Bio Products), 10,000 U/ml penicillin and 10,000 μg/ml streptomycin (Thermo Fisher Scientific, Inc.). The other cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 10,000 U/ml penicillin and 10,000 μg/ml streptomycin. BEAS-2B cells were cultured in Bronchial Epithelial Cell Medium (ScienCell Research Laboratories, Inc.) with 1% cell growth supplements (cat. no. 3962; ScienCell Research Laboratories, Inc.) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Inc.). All cells were incubated at 37°C with 5% CO2.
RNA isolation and reverse transcription-quantitative PCR (RT-qPCR)
Total RNA from frozen tissues or cultured cells was extracted using TRIzol® reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA (500 ng) was reverse transcribed in a 10-μl reaction volume, using random primers according to the manufacturer's protocol of the ReverTra Ace™ qPCR RT Master mix with gDNA Remover kit (Toyobo Life Science). The primer sequences for lncRNA and mRNA expression are shown in Table I. qPCR was performed on the obtained cDNA using SYBR Green (Promega Corporation) following the manufacturer's protocol. PCR was performed using a Gene Amp PCR System MX3005P (Agilent Technologies, Inc.) with the following protocol: 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 15 sec and annealing/extension at 60°C for 1 min, and a final extension step at 72°C for 5 min. p-actin was used as the reference gene. Analysis of relative gene expression was performed using the 2−ΔΔCq method (22).
Small interfering RNA (siRNA) silencing and miRNA mimics
H1299 and H520 cells were transfected with siRNA (si-NC, si-FEZF1-AS1, si-ITGA11-1, si-ITGA11-2, si-ITGA11-3, si-METTL3, si-METTL14, si-YTHDF1 and si-YTHDF2; 40 pmol/well; Shanghai GenePharma Co., Ltd.) or miRNA mimics (NC mimics and miR-516b-5p mimics, miR-126a mimics, miR-29b mimics, miR-145 mimics, miR-486 mimics, miR-584 mimics; 100 nmol; Guangzhou RiboBio Co., Ltd.) using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) and incubated at 37°C with 5% CO2 for 48 h before subsequent experiments. The siRNA and miRNA mimic sequences are shown in Table II.
Plasmid transfection
The pcDNA-3.1-NC and pcDNA3.1-FEZF1-AS1 plasmids for FEZF1-AS1 overexpression were purchased from GenScript. The plasmids (1 μg/well) were transfected into H1299 and H520 cells using Lipofectamine 2000 according to the manufacturer's protocol.
Rescue assays
Cells were divided into 4 groups for transfection, which were NC, pcDNA3.1-FEZF1-AS1 only, miR-516b-5p mimics only and pcDNA3.1-FEZF1-AS1 plus miR-516b-5p mimics co-transfection, performed as aforementioned. Cells were harvested after 48 h of transfection.
Cell viability assay
Transfected cells (H1299 and H520) were seeded onto 96-well plates at a density of 1.5×104 cells/well after 8 h of transfection. The cell viability was evaluated every 24 h using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) assay, following the manufacturer's protocol.
Cell migration and invasion assay
H1299 and H520 cells were plated in their respective medium (RPMI-1640 and DMEM, respectively) with 1% FBS in the upper chamber of Transwell inserts (8.0-μm pores; Corning, Inc.). For the migration assays, the cells (5×104) were suspended in 200 μl RPMI-1640 medium or DMEM in the upper chamber. For the invasion assays, the cells (2×105) were suspended in 200 μl RPMI-1640 medium or DMEM, and placed into the upper chamber (precoated with Matrigel at 37°C for 1 h; Merck KGaA). The bottom chamber contained medium with 10% FBS. Following incubation at 37°C for 48 h, the cells that had migrated or invaded through the membrane were fixed with 4% formaldehyde for 30 min at room temperature, stained with 0.5% crystal violet for 8 min at room temperature and imaged using a light microscope at a magnification of ×400.
Cell cycle analysis
The cells were collected by centrifugation at 160 × g at 4°C for 5 min and washed twice with cold PBS. For cell cycle analysis, the cells were fixed overnight in 70% ethanol at 4°C. Following centrifugation for 5 min at 160 × g at 4°C, the pellet was stained with 50 μg/ml propidium iodide (BD Biosciences) in 0.1% Triton X-100, 100 μg/ml RNase A (Thermo Fisher Scientific, Inc.) and 0.02 mg/ml EDTA. Cell suspensions were analyzed via flow cytometry using a FACSCalibur system (BD Biosciences). The cell cycle distribution was analyzed using Flow Jo 7.6 software (FlowJo LLC).
Coding potential tool
The coding potential of FEZF1-AS1 was calculated using the Coding-Potential Assessment Tool (lilab.research.bcm.edu/cpat/).
m6A prediction and analysis
The sequence-based RNA adenosine methylation site predictor (SRAMP) software (http://www.cuilab.cn/sramp) was used for m6A prediction and analysis (23). Treatment with 3-Deazaadenosine can cause tumor cells to be in an RNA demethylation state (24). Therefore, H1299 cells were incubated with 10 μM 3-Deazaadenosine at 37°C for 4 or 24 h.
Subcellular fractionation
The Minute Cytoplasmic and Nuclear Extraction kit (Invent Biotechnologies, Inc.) was used to extract cytoplasmic and nuclear fractions from NSCLC cell lines, following the manufacturer's protocol. The RNA was extracted using TRIzol reagent, as aforementioned. Subsequently, RT-qPCR was performed as aforementioned to evaluate the expression levels of each fraction of nuclear control transcript [metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)], cytoplasmic control transcript (GAPDH) and FEZF1-AS1.
Western blot analysis
Total protein was extracted from cell lines using freshly prepared RIPA lysis buffer (150 mM NaCL, 1% NP-40, 0.1% SDS, 2 μg/ml Aprotinin, 2 μg/ml Leupeptin, 1 mM PMSF, 1.5 mM EDTA, 1 mM NaVanadate) and protein concentration was determined using a BCA assay. A total of 40 μg protein/lane was separated via 15% SDS-PAGE and transferred electrophoretically to nitrocellulose membranes. To evaluate protein expression, the blots were blocked with 5% skimmed milk in TBS with 0.05% Tween-20 at 37°C for 1.5 h and incubated at 4°C overnight with a primary rabbit monoclonal antibody against ITGA11 (1:1,000; cat. no. ab198826; Abcam) and an antibody against p-actin (1:10,000; cat. no. ac009; ABclonal Biotech Co., Ltd.) used as a control. The blots were then probed with HRP-conjugated goat anti-rabbit (1:5,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) or anti-mouse (1:8,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) secondary antibodies at 37°C for 1.5 h, visualized using enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) and scanned using ImageQuant LAS 4010 Imaging System (GE Healthcare Life Sciences).
RNA-binding protein immunoprecipitation (RIP)
RIP experiments were performed using the Magna RIP RNA-binding protein immunoprecipitation kit (EMD Millipore) and anti-argonaute 2 (Ago2) antibody (5 μg; cat. no. ab32381; Abcam), according to the manufacturer's protocol. Purified, co-precipitated RNAs were subjected to RT-qPCR analysis, as aforementioned.
RNA pulldown assay with biotinylated miRNA
H1299 cells were transfected with biotinylated miRNAs (30 nM; Table II) as aforementioned, and harvested 48 h after transfection. The cell lysates were incubated at 4°C for 6 h with M-280 streptavidin magnetic beads (Thermo Fisher Scientific, Inc.) as previously described (25). The bound RNAs were purified using TRIzol reagent for further RT-qPCR analysis as aforementioned.
Luciferase reporter assay
To create a 3′-untranslated region (UTR) luciferase reporter construct of ITGA11, the sequences from putative miR-516b-5p binding sites were synthesized and cloned into the pmirGLO vector (Promega Corporation). The following wild-type (WT) and mutated (MUT) primers were used to construct the 3′-UTR of ITGA11: WT forward, 5′-ACTCGAGcccgagcaatggcgcctgctccctccagaatggaactcaagctg gttAGATC-3′ and WT reverse, 5′-CGCGTGAGCTCgggctcg ttaccgcggacgagggaggtcttaccttgagttcgaccaaT-3′; MUT forward, 5′-ACTCGAGcccgacaatggcgcctgctccagaactcatggaactcaagctggttAGATC-3′ and MUT reverse, 5′-CGCGT GAGCT Cgggctcg ttaccgcggacgaggtcttgagtaccttgagttcgaccaaT-3′. The amplified fragment was cloned into the pmirGLO luciferase reporter vector at the Mlul and XbaI sites. Briefly, cancer cells (5×104 per well) were seeded in a 24-well plate the day before transfection, and then co-transfected using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) with the firefly luciferase-3′-UTR (pmirGLO or pmirGLO-ITGA11; 500 ng), along with miR-516b-5p or control mimics (Guangzhou RiboBio Co., Ltd.). After 48 h, luciferase activity was measured using the Luc-Pair™ Duo-Luciferase HS assay kit (GeneCopoeia, Inc.) and normalized to the Renilla luciferase activity. All experiments were repeated at least three times.
Microarray analysis
Arraystar Human LncRNA Microarray V4.0 (Kangchen BioTech Co., Ltd.) was used to screen the global profiling of human lncRNAs and protein-coding transcripts. The samples included eight frozen tissues of NSCLCs (four samples of squamous cell carcinoma and four of adenocarcinoma) and paired normal lung tissues (GSE137445) (26). The total RNA was extracted from the cells or tissues as aforementioned. Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technologies, Inc.) with minor modifications. Briefly, mRNA was purified from total RNA after removal of ribosomal RNA (mRNA-ONLY™ Eukaryotic mRNA Isolation kit; Epicentre; Illumina, Inc.). Subsequently, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′bias utilizing a random priming method (Arraystar Flash RNA Labeling kit; Arraystar, Inc.). The labeled cRNAs were purified using an RNeasy Mini kit (Qiagen, Inc.). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000 (Thermo Fisher Scientific, Inc.). A total of 1 μg of each labeled cRNA was fragmented by adding 5 μl 10X blocking agent and 1 μl 25X fragmentation buffer; the mixture was heated at 60°C for 30 min, and finally 25 μl 2X GE hybridization buffer was added to dilute the labeled cRNA. A total of 50 μl hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 h at 65°C in an Agilent Hybridization Oven (Agilent Technologies, Inc.). The hybridized arrays were washed, fixed and scanned using the Agilent DNA Microarray Scanner (part no. G2505C; Agilent Technologies, Inc.). Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies, Inc.). The thresholds used to screen the upregulated or downregulated lncRNAs were fold-change (FC) >2.0 and P<0.05. In addition, the competing endogenous (ce)RNA network of FEZF1-AS1 was analyzed by integrating the Arraystar Human mRNA microarray data and miRNA bioinformatic analysis software (TargetScan v7.2; http://www.microrna.org) by Kangchen BioTech Co., Ltd..
Statistical analysis
All data are presented as the mean ± SEM. All experiments were repeated >3 times. SPSS 22.0 (IBM Corp.) was used for statistical analysis. The association between the expression levels of FEZF1-AS1, ITGA11 and miR-516b-5p, and the clinicopathological variables of patients with NSCLC was determined using the χ2 test. Paired t-test was used to examine the expression levels of FEZF1-AS1, ITGA11 and miR-516b-5p in normal versus tumor tissue samples. Unpaired t-test was utilized to examine the differences between two groups in vitro. Significant differences among multiple groups were investigated using one-way ANOVA followed by Bonferroni's post hoc test. Kaplan-Meier analysis with log-rank test was used to assess the overall survival rate. The correlation between FEZF1-AS1, ITGA11 and miR-516b-5p expression in NSCLC tissues was analyzed using Spearman's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.
Results
FEZF1-AS1 is upregulated in NSCLC tissues and is associated with a poor prognosis
To identify the regulatory networks of mRNA and lncRNAs in NSCLC, eight pairs of NSCLC and non-tumorous adjacent tissues, including four pairs of adenocarcinoma and four pairs of squamous cell carcinoma, were analyzed using the Arraystar Human lncRNA microarray. A total of 1,157 lncRNAs were found to be significantly differentially expressed, of which 505 were upregulated and 652 were downregulated by >2-fold (Fig. 1A). With the further restriction of FC≥5 and P<0.05, FEZF1-AS1 was one of the most upregulated lncRNAs (Fig. 1B), suggesting the potential crucial role of FEZF1-AS1 in NSCLC tumorigenesis and development. The coding potential of FEZF1-AS1 was calculated using the Coding-Potential Assessment Tool (lilab.research.bcm.edu/cpat/), revealing that the possible open reading frame of FEZF1-AS1 was very short, with a very low coding probability (Fig. 1C).
To investigate the expression levels of FEZF1-AS1 in NSCLC, RT-qPCR analysis was performed using the total RNA extracted from 45 pairs of NSCLC tissues and their matched non-neoplastic counterparts. The results revealed that FEZF1-AS1 expression was significantly upregulated in NSCLC samples compared with that in the corresponding normal tissues (Fig. 1D). To determine the association between FEZF1-AS1 and clinicopathological features, the 45 patients were divided into the high (FC>9.00; n=23) or low (FC≤9.00; n=22) FEZF1-AS1 expression groups according to the median of FEZF1-AS1 expression (Fig. 1E). Kaplan-Meier analysis demonstrated that high FEZF1-AS1 expression was significantly associated with a poor OS rate (P=0.045; Fig. 1F). However, there were no significant associations between FEZF1-AS1 expression and other important clinicopathological features, such as lymph node metastasis and clinical staging in patients with NSCLC (all P>0.05; Tables III and SI).
 | Table IIIAssociation between low (n=22) and high (n=23) FEZF1-AS1 expression and clinicopathological features in patients with non-small cell lung cancer. |
FEZF1-AS1 silencing inhibits lung cancer cell proliferation, migration and invasion
The function of FEZF1-AS1 was investigated in NSCLC cell lines. Compared with BEAS-2B cells, FEZF1-AS1 expression was significantly upregulated in H358, H1299, H520 and SK-MES-1 NSCLC cells (Fig. 2A). In order to elucidate the mechanism of FEZF1-AS1 activity in oncogenesis, the expression levels of FEZF1-AS1 were further examined in H1299 cells by cytoplasmic and nuclear extraction, revealing that FEZF1-AS1 was mostly located in the cytoplasmic fraction (Fig. 2B). To investigate the biological function of FEZF1-AS1, knockdown efficiency of si-FEZF1-AS1 was measured using RT-qPCR in H1299 and H520 cells (Fig. 2C). The CCK-8 assay revealed that FEZF1-AS1-knowckdown significantly decreased the proliferative capacity of NSCLC cells after ≥72 h (Fig. 2D). Additionally, it was observed that FEZF1-AS1 silencing induced G2/M arrest (Figs. 2E and S1A), and Transwell assays demonstrated that migration and invasion were significantly inhibited following FEZF1-AS1 silencing (Fig. 2F and G).
High FEZF1-AS1 expression in NSCLC cells is regulated by m6A methylation
To reveal the motif of the high expression levels of FEZF1-AS1 in NSCLC, m6A prediction and analysis software SRAMP was used to predict the full-length sequence (2,653 bp) of FEZF1-AS1. As presented in Fig. 3A and B, seven m6A modified sites were predicted, suggesting that FEZF1-AS1 expression may be regulated by m6A modification.
m6A is the most widely used mRNA modification method in mammals; this modification can be added, deleted and preferentially combined with m6A through three regulators, including 'writers' that generate m6A, 'erasers' that exhibit demethylation activity and 'readers' that decode the m6A modification (27). On the basis of this prediction, specific siRNAs targeting the 'readers' [methyltransferase-like 3 (METTL3) and METTL14] and 'writers' [YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and YTHDF2] were transfected into H1299 cells to examine their effects on FEZF1-AS1 expression. The efficacy of siRNA interference against METTL3, METTL14, YTHDF1 and YTHDF2 was analyzed via RT-qPCR (Fig. 3C). Compared with the control group, downregulating these four m6A modified molecules inhibited the expression levels of FEZF1-AS1 (Fig. 3D). Subsequently, H1299 cells were treated with 3-Deazaadenosine, an RNA demethylation drug, at a concentration of 10 μM. FEZF1-AS1 expression was detected in the cells collected at 4 and 24 h following administration. Compared with in the control group, FEZF1-AS1 expression was significantly decreased in the 4-h group and reversed in the 24-h group (Fig. 3E). The present results suggested that the high expression levels of FEZF1-AS1 in NSCLC cells was positively regulated by m6A methylation.
ITGA11 is a potential target of FEZF1-AS1 through miRNAs in NSCLC cells
FEZF1-AS1 was mostly located in the cytoplasm. It has been demonstrated that lncRNAs in the cytoplasm can interact with miRNAs as ceRNAs (7). Ago2 is a crucial factor in miRNA biogenesis (28). An RIP experiment was performed using an Ago2 antibody to investigate whether FEZF1-AS1 may bind to miRNAs. As expected, FEZF1-AS1 was detected in Ago2 immunoprecipitates from control cells, indicating that the RNA could indeed bind to miRNAs (Fig. 4A).
lncRNAs can regulate miRNAs to influence the expression levels of mRNAs as ceRNAs (8). Combined with the lncRNA microarray and miRNA bioinformatic analysis using TargetScan software programs results (data not shown), mRNA-ceRNA analysis was performed to identify target genes. Based on the gene frequency, a total of 12 mRNAs (ITGA11, RHOV, ONECUT2, SOX4, MARK4, UBN1, COL1A1, PRKAB2, PIP5K1A, CDK6, SKA3 and XKR9) were selected for further validation. Following the knockdown of FEZF1-AS1, ITGA11 was the most downregulated mRNA, as revealed using RT-qPCR (Fig. 4B). Compared with BEAS-2B cells, the expression levels of ITGA11 were significantly increased in H358, H1299, H520 and SK-MES-1 cells (Fig. 4C). Additionally, western blot analysis confirmed that ITGA11 protein levels were decreased following FEZF1-AS1 knockdown in H1299 and H520 cells (Fig. 4D). Subsequently, the role of FEZF1-AS1 overexpression on ITGA11 expression was further investigated. As shown in Fig. 4E-G, FEZF1-AS1 overexpression significantly upregulated ITGA11 expression both at the mRNA and protein levels in H1299 and H520 cells. Therefore, it was concluded that FEZF1-AS1 was able to upregulate ITGA11 expression.
FEZF1-AS1 can compete with miR-516b-5p for direct binding to ITGA11 in NSCLC cells
Based on the present ceRNA networks results and gene ontology analysis in the GSE137445 array dataset, three miRNAs (miR-486, miR-516b-5p and miR-584-3p) binding to both FEZF1-AS1 and ITGA11 were identified. Furthermore, three other miRNAs (miR-126a, miR-29b and miR-145) targeting ITGA11 were selected based on previous reports (29-31). After miRNA overexpression, miR-516b-5p significantly downregulated FEZF1-AS1 expression (Fig. 5A). Proof of transfection was performed for all miRNA mimics (data not shown). Additionally, it was verified that ITGA11 expression was negatively regulated by miR-516b-5p at both the transcriptional (Fig. 5B) and translational (Fig. 5D) levels, as measured using RT-qPCR and western blot analysis, respectively, after verifying that miR-516b-5p was successfully overexpressed in H1299 and H520 cells (Fig. S1C). Compared with BEAS-2B cells, the expression levels of miR-516b-5p were significantly decreased in H358, H1299, H520, A549 and SK-MES-1 cells (Fig. 5C). Subsequently, it was examined whether miR-516b-5p-mediated ITGA11 regulation occurred through direct targeting of miRNA-binding sites in the ITGA11 sequence. According to the bioinformatics analysis using TargetScan software, the construction diagram of the miR-516b-5p binding site reporter gene in the ITGA11 3′-UTR region (WT and MUT) is shown in Fig. 5E. ITGA11 was subcloned into the pmirGLO dual-luciferase reporter vector, and luciferase assays were performed in H1299 cells by inducing miR-516b-5p overexpression using miR-516b-5p mimics. As shown in Fig. 5F, co-transfection with pmirGLO-ITGA11-WT and miR-516b-5p mimics demonstrated a significant decrease in luciferase reporter activity compared with the negative control (NC) group (P<0.05). This repressive effect was abolished by directed mutagenesis of the miR-516b-5p binding seed region in ITGA11.
To assess whether miR-516b-5p interacted with FEZF1-AS1, the biotin-avidin pulldown system was used, revealing that FEZF1-AS1 co-precipitated miR-516b-5p (Fig. 5G). For the rescue assay, FEZF1-AS1 and miR-516b-5p were overexpressed either alone or together using transfection. Compared with the NC group, the results revealed that overexpression of miR-516b markedly suppressed ITGA11 expression, whereas overexpression of FEZF1-AS1 partly abolished the silencing effect of miR-516b-5p on ITGA11, suggesting that ITGA11 was regulated by miR-516b-5p and FEZF1-AS1 (Fig. 5H). Therefore, FEZF1-AS1 may function as a ceRNA, regulating miR-516b-5p and its target gene ITGA11 in NSCLC.
Inhibiting ITGA11 or overexpressing miR-516b-5p inhibits cell proliferation and invasion in NSCLC
ITGA11 expression was examined in 45 pairs of NSCLC tissues and their matched non-neoplastic counterparts. It was identified that ITGA11 expression was significantly upregulated in the NSCLC samples compared with that in the corresponding normal tissues (Fig. 6A). To determine the association between ITGA11 and clinicopathological features, the 45 patients were divided into high (FC>6.96; n=23) or low (FC<6.96; n=22) ITGA11 expression groups according to the median of ITGA11 expression. However, there were no significant associations between ITGA11 expression and clinicopathological features, such as lymph node metastasis and clinical staging in patients with NSCLC (all P>0.05; Table IV).
| Table IVAssociation between low (n=22) and high (n=23) ITGA11 or miR-516b-5p expression and clinicopathological features in patients with non-small cell lung cancer. |
To examine the role of ITGA11 in cell proliferation, ITGA11 expression was knocked down in H1299 and H520 cells (Fig. 6B), revealing that the proliferative capacities of these cells were significantly decreased after >72 h (Fig. 6C). Transwell assays revealed that the knockdown of ITGA11 significantly decreased the migratory and invasive abilities of NSCLC cells (Fig. 6E and F). However, ITGA11-knockdown did not influence the cell cycle (Figs. 6D and S1B).
Compared with surrounding healthy tissues, miR-516b-5p expression was significantly inhibited in tumor tissues compared with in normal tissues (Fig. 7A). To determine the association between miR-516b-5p expression and clinicopathological features, the 45 patients were divided into high (FC>0.29; n=23) or low miR-516b-5p expression groups (FC <0.29; n=22) according to the median of miR-51b-5p expression. Smoking history (P=0.014) and tumor size (P=0.009) were associated with miR-516b-5p expression using a χ2 test in patients with NSCLC (Table IV). However, due to the small sample size of NSCLC tissues, a statistical association between miR-516b-5p expression and OS was not identified (data not shown), nor a correlation between FEZF1-AS1, ITGA11 and miR516b-5p expression (data not shown). The CCK-8 assay revealed that overexpression of miR-516b-5p significantly inhibited NSCLC cell proliferation after >72 h (Fig. 7B). Additionally, in the Transwell assay, the migratory and invasive abilities of tumor cells were inhibited in the miR-516b-5p mimic group (Fig. 7C and D) compared with in the control group. The current data indicated that inhibiting ITGA11 or overexpressing miR-516b-5p inhibited cell proliferation and invasion in NSCLC.
Discussion
NSCLC accounts for the vast majority of lung cancer cases (2). In past years, numerous studies regarding non-coding RNAs, including lncRNAs, have been conducted (4,6-9). lncRNA FEZF1-AS1 is the antisense RNA of FEZF1. In various types of cancer, FEZF1-AS1 expression in cancer tissues is significantly higher compared with that in normal tissues, and high FEZF1-AS1 expression indicates a poor survival in patients with colon, gastric and cervical cancer (15,16,32). The present data revealed that FEZF1-AS1 was highly expressed in NSCLC tissues, compared with normal tissues, and was associated with a poor prognosis in patients with NSCLC. FEZF1-AS1 downregulation in NSCLC cells inhibited cell proliferation, migration and invasion, and arrested the cell cycle at the G2/M phase, in accordance with findings previously reported on the function of FEZF1-AS1 in NSCLC and breast cancer cell lines (18,33).
Subsequently, the mechanism underlying the high FEZF1-AS1 expression in NSCLC was explored, and m6A modification was identified. m6A is the most widely used mRNA modification method in mammals (27). The modification can be added, deleted and preferentially combined with m6A through three regulators of 'writers', 'erasers' and 'readers' (27). The m6A modification is involved in a variety of mRNA processes, including mRNA translation and decay (34). In addition to mRNA, non-coding RNAs are also regulated by m6A, and miRNAs and lncRNAs are important classes of non-coding RNAs (35). It has been reported in several studies that the expression levels of lncRNAs are regulated by m6A modification. For example, the change of lncRNA MALAT1 cut by m6A 'reading' molecules is associated with the progression of cancer (36,37). The downregulation of METTL3 in the 'writing' module can damage the lncRNA Xist-mediated gene silencing (38). However, to the best of our knowledge, whether FEZF1-AS1 expression is regulated by m6A modification has not been reported. In the present study, knockdown of 'readers' (METTL3 and METTL14) and 'writers' (YTHDF1 and YTHDF2) resulted in the decrease of FEZF1-AS1 expression. Treatment with 3-Deazaadenosine causes tumor cells to be in an RNA-demethylation state (24). Following treatment with 3-Deazaadenosine in the present study, FEZF1-AS1 expression was significantly decreased. The current results demonstrated that m6A modification may have an important regulatory effect on FEZF1-AS1 expression.
To investigate the mechanism of FEZF1-AS1 in NSCLC, mRNAs that could be regulated by FEZF1-AS1 were screened, and ITGA11, a member of the integrin family, was selected. Integrins are transmembrane receptors that mediate the connection between cells and their external environment (39). These proteins are heterodimers formed by two subunits, the α (120-185 kD) and β (90-110 kD) subunits (40). In cells, ITGA11 is involved in collagen-mediated biological processes, such as cell migration, collagen deposition and collagen recombination (41). ITGA11 is highly expressed in esophageal squamous cell carcinoma, head and neck squamous carcinoma, prostate cancer and breast cancer, and is closely associated with the migration and invasion of tumor cells (42-44). In lung adenocarcinoma A549, H460 and H520 cell lines, tumor growth was significantly higher in A549+WT, compared with A549+Knockout (KO) tumors (45). Re-expression of human a11 cDNA in KO cells rescued the tumor growth rate to a rate that was comparable with that of the A549+WT tumors (45). In patients with NSCLC, ITGA11 expression is significantly upregulated and is associated with a poor OS (46). In the present study, both the migration and invasion of NSCLC cells were inhibited following ITGA11-knockdown. This was likely due to the function of integrin in connecting cells to collagen in the extracellular matrix, but this hypothesis needs to be further confirmed.
miR-516b-5p was selected and validated in the present study as a possible binding miRNA to both FEZF1-AS1 and ITGA11. Low miR-516 expression can significantly improve OS in patients with lung squamous cell carcinoma (47). In the present study, miR-516b-5p expression in NSCLC tissues was lower compared with in non-neoplastic tissues, and the proliferation, migration and invasion of cells was inhibited following miR-516b-5p upregulation. In the present study, both ITGA11 and miR-516b-5p were involved in cell proliferation, migration and invasion, which is consistent with the biological role in tumor progression of FEZF1-AS1. Following FEZF1-AS1-knockdown, ITGA11 expression was decreased at both the RNA and protein levels. Additionally, ITGA11 expression was decreased following miR-516b overexpression. Therefore, both FEZF1-AS1 and miR-516b may affect ITGA11 expression. Through RIP and RNA pull-down assays, together with the effects of miR-516b-5p upregulation on FEZF1-AS1, it was concluded that miR-516b-5p and FEZF1-AS1 may share a binding site. The rescue assay further confirmed this regulatory axis (Fig. 7E).
Future studies should explore the effects of silencing both ITGA11 and miR-516b-5p, or of overexpressing FEZF1-AS1 and silencing ITGA11, and to observe phenotypic changes in proliferation and invasion. In addition to the involvement of miR-516b-5p and ITGA11, other FEZF1-AS1-regulated genes may also contribute to the pro-tumorigenic function of FEZF1-AS1. Future studies are warranted to comprehensively explore the role of FEZF1-AS1 in NSCLC pathogenesis.
In conclusion, the present study identified a novel lncRNA, FEZF1-AS1, associated with a poor prognosis in patients with NSCLC. The current results suggested that FEZF1-AS1 was an oncogenic regulator that promoted cell proliferation and invasion. It induced competitive binding with miR-516b-5p, resulting in the upregulation of ITGA11 expression. Therefore, the FEZF1-AS1/miR-516b-5p/ITGA11 axis may be a valuable target for NSCLC prognosis and treatment.
Supplementary Data
Acknowledgments
Not applicable.
Funding
The present study was supported by Hebei Postgraduate Innovation Funding Project (grant no. CXZZBS2017111).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
LX designed the study. HeS, HL, XD and ML contributed to cell transfection, RT-qPCR and western blotting. HeS contributed to RIP, RNA pull-down and luciferase assays. HL contributed to data analysis. YL and XZ collected the clinical data and follow-up data. HaS performed the flow cytometric and data analysis. HeS and LX participated in writing and revising the manuscript. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
The research protocol conformed to the principles outlined in the Declaration of Helsinki. All patients provided written informed consent and the protocol of the study was approved by the Research Ethics Committee of the Fourth Hospital of Hebei Medical University.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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