Long noncoding RNA NEAT1 promotes laryngeal squamous cell cancer through regulating miR-107/CDK6 pathway
© Wang et al. 2016
Received: 27 October 2015
Accepted: 21 January 2016
Published: 29 January 2016
Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) plays key role in the progression of some human cancers. However, the role of NEAT1 in human laryngeal squamous cell cancer (LSCC) is still unknown. We therefore investigated the expression and function of NEAT1 in LSCC.
NEAT1 level in LSCC and adjacent non-neoplastic tissues were detected by qRT-PCR. NEAT1 was knockdown in LSCC cells and cell proliferation, apoptosis and cell cycle were examined. The growth of xenografts with NEAT1 knockdown LSCC cells was analyzed.
NEAT1 level was significantly higher in LSCC than in corresponding adjacent non-neoplastic tissues, and patients with neck nodal metastasis or advanced clinical stage had higher NEAT1 expression. Moreover, siRNA mediated NEAT1 knockdown significantly inhibited the proliferation and induced apoptosis and cell cycle arrest at G1 phase in LSCC cells. The growth of LSCC xenografts was significantly suppressed by the injection of NEAT1 siRNA lentivirus. Furthermore, NEAT1 regulated CDK6 expression in LSCC cells which was mediated by miR-107.
NEAT1 plays an oncogenic role in the tumorigenesis of LSCC and may serve as a potential target for therapeutic intervention.
KeywordsLaryngeal squamous cell cancer Long noncoding RNA microRNA NEAT1 CDK6 miR-107
Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignancies in the head and neck . Despite the improvements in diagnostic and therapeutic modalities, the survival of laryngeal cancer patients is still poor over the past 20 years. Therefore, revealing the molecular mechanism underlying LSCC development is important for developing effective therapy. Recently, we and others have focused on the role of aberrant expression of noncoding RNAs in LSCC [2–6]. Noncoding RNAs, which do not encode protein, include short noncoding RNAs and long noncoding RNAs (lncRNAs). By binding to specific sequences usually in the 3′ untranslated region (3′UTR) to degrade the transcription of target genes, microRNAs have been demonstrated to play important role in multiple cancers including LSCC [2–7]. Long noncoding RNAs are a class of transcripts longer than 200 nucleotides. Recent studies have shown that lncRNAs are frequently dysregulated in cancer progression [8, 9]. Nuclear paraspeckle assembly transcript 1 (NEAT1) lncRNA is found to localize specifically to paraspeckles which has two isoforms: 3.7 kb NEAT1-1 and 23 kb NEAT1-2 . They share the same 5′-end but are processed alternatively at the 3′-termini. NEAT1 may serve as a core structural component because knockdown of NEAT1 led to the disintegration of paraspeckles . NEAT1 upregulation has been reported in human malignancies, including leukemia, hepatocellular carcinoma and lung cancers [11–13]. However, little is known about whether NEAT1 affects carcinogenesis and biological behavior of LSCC. Our preliminary experiments with microarray analysis showed that NEAT1 was upregulated in LSCC tissues (data not shown). In the present study, we further detected the expression of NEAT1 in LSCC by using real-time PCR and found that lncRNA NEAT1 was up-regulated and correlated with the progression of LSCC. Next, we investigated the function of NEAT1 in LSCC cells and found that NEAT1 knockdown inhibited the proliferation and invasion of LSCC cells. Furthermore, we demonstrated that NEAT1 upregulated cyclin-dependent kinase 6 (CDK6) through inhibiting the expression of miR-107. These results provide the evidence that NEAT1-miR-107-CDK6 regulatory network promotes LSCC.
Pairs of tumor tissues and adjacent non-cancerous matched tissues were obtained from 52 patients who underwent partial or total laryngectomy between August 2013 and December 2014 at the Department of Otorhinolaryngology, the Second Affiliated Hospital of Harbin Medical University, under an approved protocol of Harbin Medical University. The patients had not received any cancer therapy before admission. After surgery, the tissues obtained from patients were preserved in liquid nitrogen within 5 min of excision and then were transported frozen to the laboratory and stored at −80 °C.
Quantitative real-time PCR analysis
Total RNA was extracted by using Trizol reagent (Invitrogen, Carlsbad, CA, USA). 2 μg of total RNA were reversely transcribed to cDNA by using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed using Power SYBR Green RT-PCR Reagents (Applied Biosystems, Foster City, CA, USA). Primers were as follows: NEAT1 forward, 5′-CTTCCTCCCTTTAACTTATCCATTCAC-3′; reverse, 5′-CTCTTCCTCCACCATTACCAACAATAC-3′; miR-107 forward, 5′-ATGATGAGCAGCATTGTACAGG-3′; reverse, 5′-GCAGGGTCCGAGGTATTC-3′. All reactions were carried out on the Applied Biosystems 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Relative mRNA and lncRNA levels were normalized against β-actin and U6 snRNA, respectively, and calculated using the 2-ΔΔCt method.
Cell culture and virus transduction
Human LSCC cell line Hep-2 was cultured in Dulbecco’s modified Eagle’s medium containing 10 % fetal bovine serum (Gibco; Life Technologies, Carlsbad, CA) in a humidified incubator at 37 °C with 5 % CO2. SiRNAs of human NEAT1 lentivirus and vector harboring green fluorescent protein (GFP) and the control lentivirus (GFP-lentivirus) were constructed by Genechem (Shanghai, China). The siRNAs sequences targeting NEAT1 were as follows: 5′-GUGAGAAGUUGCUUAGAAACUUUCC-3′ (siRNA-1) and 5′-GATCCCTAAGCTGTAGAACAT-3′ (siRNA-2). The lentiviruses for the overexpression and knockdown of miR-107 were provided by Genechem (Shanghai, China). The lentiviruses were diluted in 0.2 mL complete medium at 107 transduction units (TU)/mL containing hexadimethrine bromide (Polybrene; 8 mg/mL) and were incubated with the cells for 1 h at 37 °C. Next, the cells were incubated with 0.3 mL fresh prepared Polybrene-Dulbecco’s modified Eagle’s medium for 24 h; the medium was then replaced with fresh Dulbecco’s modified Eagle’s medium and the cells were cultured for 48 h.
Cell proliferation assay
The proliferation of Hep-2 cells was evaluated using cell count kit 8 (CCK-8) cell proliferation kit according to the manufacturer’s instructions. Briefly, the cells were seeded into 96-well plates at 5 × 103 cells/well and cultured for 48 h. Then 10 μl CCK-8 solution were added to each well and incubated for 1 h. The absorbance (A) at 450 nm was measured using a microplate reader. Results were representative of three individual experiments in triplicate.
Wound-healing migration assay
Cells were seeded in 6-well plates and grown to about 90 % confluence before being wounded using a 200-mL plastic tip across the monolayer cells. Debris was removed by washing three times with phosphate-buffered saline (PBS), and then cells were cultured with fresh DMEM containing 5 % FBS. Wound healing was observed at different time points within the scrape line, and representative scrape lines for each cell line were photographed. The migration of the cells was evaluated by measuring the width of the wounds (at × 400 magnification). Images were captured immediately after 24 h post-wounding. Each assay was performed in triplicate in at least two independent experiments.
Transwell filters (pore size, 8 μm; Falcon; BD Biosciences) were coated on the lower side with 8 μg/μL Matrigel and placed on a 24-well plate containing DMEM. Hep-2 cells (1 × 105) were added to the upper compartment of a transwell chamber and allowed to migrate for 24 h at 37 °C. The cells were then harvested and suspended in DMEM with 1 % bovine serum albumin. After 24 h, Matrigel and cells remaining on the upper side of the membrane were wiped off with cotton swabs, and the cells that had migrated to the bottom surface of the membrane were fixed in 3.7 % paraformaldehyde in PBS. Once fixed, the cells were stained with crystal violet for 10 min at room temperature. Cell invasion was quantified by counting the number of cells in three inserts. Data were expressed as the average number of cells per insert.
Cell cycle analysis
Cells were collected and fixed with cold ethanol at 4 °C for 1 h before being stored at −20 °C. Fixed cells were washed and resuspended in 1 mL PBS containing 50 μg/mL RNase A and 50 μg/mL ethidium bromide. After incubating for 20 min at 37 °C, cells were analyzed for DNA content by flow cytometry (FACS Calibur; Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). For each sample, 20,000 events were acquired, and cell cycle distribution was determined using cell cycle analysis software (Modfit; LT for mac V 3.0).
Apoptosis assay by flow cytometry
The cells were washed twice with cold 10 mM PBS and resuspended in 1 × binding buffer (BD Biosciences, San José, CA, USA) at a concentration of 1 × 106 cells/ml. Cells were stained with Annexin V/ APC and propidium iodide (PI), using the Annexin V apoptosis detection kit (KeyGen Biotech, Nanjing, China). Hep-2 cells without any treatment were used as control, and the experiments were repeated at least three times.
Sixteen BALB/c mice (5 to 6 weeks old) were provided by Vital River Laboratories (Beijing, China) and kept in aseptic conditions with a constant humidity and temperature according to standard guidelines under a protocol approved by Harbin Medical University. All mice were injected subcutaneously in the dorsal scapula region with 100 μL suspension (1 × 106) of Hep-2 cells. The size of the tumor was measured twice a week with calipers, and the volume of tumor was determined using the simplified formula of a rotational ellipsoid (length × width2 × 0.5). Once tumors reached approximately 0.5 to 0.6 cm3, mice received an injection into the tumor once a week for 3 weeks. Mice in the experimental group (n = 8) were treated with 100 μL NEAT1 siRNA lentivirus; mice in the control group (n = 8) received an injection of 100 μL GFP lentivirus. Tumors were harvested 1 week after the end of treatment.
Transmission electron microscopy
Apoptotic morphological changes of cells were detected by transmission electron microscopy. Tumor tissues were fixed for 1 h with 2.5 % glutaraldehyde using 0.1 mol/L Na cacodylate buffer (pH 7.3), and then postfixed with 1 % osmium tetroxide for 1 h. Fixed samples were rinsed and dehydrated in a graded ethanol series. Ultrathin sections were cut, mounted on copper grids, and stained with uranyl acetate and lead citrate by standard methods. Stained grids were examined and photographed in a JEM 1200 EX transmission electron microscope (Tokyo, Japan).
Apoptosis in vivo was detected using the terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL) in situ apoptosis detection kit (Roche) according to the manufacturer’s instructions. After deparaffinization and dehydration and inactivation of peroxidase activity, 20 paraffin sections of each tumor specimen were incubated with 2 μg/mL proteinase K at 37 °C for 15 min. Then, the sections were treated with terminal deoxynucleotidyl transferase (TdT) and biotinylated dUTP, and observed under microscopy. Controls were treated in the same manner except that TdT was replaced with dH2O.
Western blot analysis
Hep-2 cells were collected and CDK6 expression was detected as described previously using CDK6 antibody (1:400 dilution, Boster, Wuhan, China) . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.
Luciferase report assay
3′UTR of CDK6 gene harboring miR-107 binding site was inserted downstream of firefly luciferase (f-luc) in pGL3 plasmid and named as 3′UTR. 3′UTR of CDK6 gene with the deletion of miR-107 binding site was inserted downstream of f-luc in pGL3 plasmid and named as 3′UTR-NC. 3′UTR of CDK6 gene with three mismatch mutations in miR-107 seed complementary site was inserted downstream of f-luc in pGL3 plasmid and named as 3′UTR-MU. HEK293T cells were cultured in 96-well plates and co-transfected with 400 ng of either 3′UTR, 3′UTR-NC or 3′UTR-MU, 50 ng of pRL-TK (Promega, USA) and 50 nmol/L of miR-107 or scramble miRNA negative control (miR-NC). The pRL-TK Renilla luciferase plasmid was used as internal control. 48 h after transfection, firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay (Promega, USA). The results were expressed as relative luciferase activity (firefly luciferase/Renilla luciferase).
All quantitative data were presented as the mean ± SD of at least three independent experiments. The differences between groups were analyzed by Student’s t test. P < 0.05 was considered as statistically significant.
NEAT1 is overexpressed in LSCC
NEAT1 knockdown inhibits the proliferation and invasion of LSCC cells
NEAT1 knockdown induces G1 phase arrest and apoptosis of Hep-2 cells
NEAT1 knockdown inhibits the growth of LSCC xenografts
NEAT1 knockdown induces the apoptosis of LSCC cells in vivo
NEAT1 regulates CDK6 expression through modulating miR-107
To further confirm NEAT1 can regulate CDK6 expression which is mediated by miR-107, we performed rescue experiments. In Hep-2 cells with high expression of NEAT1, overexpression of miR-107 led to significant reduction of CDK6 expression (Compared lane 2 to lane 5, Fig. 8f). In contrast, while NEAT1 knockdown decreased CDK6 expression (compared lane 7 to lane 2, Fig. 8f), downregulation of miR-107 (by lentivirus transduction of Anti-miR-107) could restore CDK6 expression inhibited by NEAT1 knockdown (compared lane 7 to lane 1, Fig. 8f). Taken together, these results suggest that NEAT1 regulates CDK6 expression through modulating miR-107.
lncRNAs are a class of noncoding RNA transcripts with no or little protein-coding capacity. Recent studies have suggested that more genomic sequences were transcribed into lncRNAs than protein-coding RNAs . lncRNAs play important role in carcinogenesis . Through regulating gene expression by a variety of mechanisms, such as transcription, post-transcriptional processing, chromatin modification and the regulation of protein function, lncRNAs are involved in multiple processes of tumorigenesis . However, the function of lncRNAs in LSCC is not well characterized. Our previous studies showed that lncRNA HOTAIR played oncologic role in LSCC [18, 19]. In this study, by real-time PCR we found that lncRNA NEAT1 is overexpressed in LSCC. NEAT1 is transcribed from the familial tumor syndrome multiple endocrine neoplasia (MEN) type 1 locus on chromosome 11 . Paraspeckles are a new class of subnuclear bodies and contain RNA-binding proteins in the interchromatin space of mammalian cells. Paraspeckle can regulate gene expression by retaining mRNAs for editing in the nucleus . NEAT1 localizes to paraspeckles and serves as an architectural component of these nuclear bodies . Recent studies showed that NEAT1 is upregulated and possesses oncogenic properties in various cancers such as lung, breast and prostate cancer [13, 20, 23]. In the present study, our data indicate that NEAT1 RNA is involved in LSCC aggressiveness. NEAT1 was upregulated in LSCC specimens compared with the control tissues. In particular, patients with grade T3 to T4, lymph node metastasis, poor differentiation, or advanced clinical stages expressed higher levels of NEAT1.
To further understand the biological function of NEAT1 in LSCC progression, both in vitro and in vivo assays were performed. Our data showed that siRNA mediated knockdown of NEAT1 led to significant inhibition of cell proliferation and invasion, while induced cell cycle arrest at G1 phase and apoptosis in LSCC cells. Furthermore, our results demonstrate that NEAT1 knockdown suppressed tumor growth in BALB/c mice xenografts and induced tumor cell apoptosis in vivo. These results indicate that high expression of NEAT1 promotes the progression of LSCC and NEAT1 plays oncogenic role in LSCC.
The molecular mechanism of NEAT1 is complex. Hirose et al. suggested that paraspeckles/NEAT1 attenuated the cell death pathway . Choudhry et al. showed that NEAT1 expression correlated with poor survival of breast cancer and was induced by HIF-2 in hypoxia . Chakravarty et al. identified NEAT1 as a potential target of ERα in prostate cancer . Moreover, NEAT1 was reported to be regulated by miR-449a in lung cancer . miR-107 belongs to the miR-103/107 family and functions as a tumor suppressor in multiple cancers including head and neck squamous cell carcinoma . CDK6, a member of the CDK family, is a key regulator during the G1/S cell cycle transition. Cdk6 is highly expressed in head and neck squamous cell carcinoma and significantly correlates with tumor progression . Targetscan predicted that CDK6 may be a target of miR-107 and Starbase database suggested the target sites between NEAT1 and miR-107. Thus, we speculated that NEAT1 can regulate CDK6 thorough miR-107 network in LSCC. Through luciferase reporter assay, we found that CDK6 is a direct target gene of miR-107. Furthermore, we showed that NEAT1 siRNA increased miR-107 level and decreased CDK6 protein level. These results are consistent with our predication and suggest that NEAT1 can regulate CDK6 through miR-107. These data provide new insights into noncoding RNA regulation network, suggesting that lncRNA not only regulate proteins but also modulate the target gene of miRNA indirectly through affecting miRNA expression.
This is the first study to demonstrate oncogenic role of NEAT1 in LSCC. The molecular mechanism of NEAT1 function in LSCC is associated with the regulation of miR-107/CDK6 pathway. NEAT1 may serve as a marker for LSCC progression and a potential target for therapeutic intervention of LSCC.
This study was supported by grants from the National science Foundation of china (No. 81272965, 81241085, 81402257) and Foundation of Health Department of Heilongjiang Province (No. 2013115).
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