Background
Laryngeal carcinoma is the second most frequently diagnosed head and neck cancer worldwide [
1], with an estimated 17,590 new cases and 3230 deaths projected to occur in the United States in 2018 [
2]. Laryngeal squamous cell carcinoma (LSCC) is the most common type of laryngeal carcinoma and accounts for ~ 95% of laryngeal carcinoma cases. Currently, the main treatment for LSCC is surgery, followed by radiotherapy and chemotherapy [
3,
4]. Disease specific survival rates for limited cancers (stages I, II) typically range from 60 to 90%. However, once recurrence or distant metastasis occours, patients remains worse 5-year survival rate of 64% and almost die of this disease [
5,
6]. Therefore, there is an urgent need to study the underlying molecular mechanisms that contribute to LSCC migration.
Circular RNAs (circRNAs) are a new class of endogenous non-coding RNAs [
7]. Unlike linear RNAs containing a 5′ caps and 3′ tails, circRNAs are characterized by covalently closed-loop structures and therefore do not have 5′ and 3′ ends [
8]. Recently, increasing evidence has confirmed that circRNAs play critical roles in carcinogenesis and cancer progression [
9], including proliferation, apoptosis, migration and invasion. For example, circHIPK3 derived from exon 2 of the HIPK3, regulates hepatocellular carcinoma proliferation and migration [
10]; circITCH acts as a tumor suppressor contributing to bladder cancer proliferation [
11]; and circRNA_104916 regulates colon cancer cell apoptosis, migration, and invasion [
12]. A previous study has found that 302 circRNAs are upregulated, while 396 are downregulated in LSCC tissues compared with that in non-tumor tissues as per a microarray analysis [
13]. However, the biological functions of circRNAs in LSCC metastasis remain unclear.
A previous study demonstrated that the expression of hsa_circ_0092012, termed circFLNA form exon-9 to exon-15 of the filamin A (FLNA) gene, is upregulated in human LSCC via high-throughput circRNA microarray [
13]. Nevertheless, the role of circFLNA in LSCC remains unknown. In the present study, we first confirmed that circFLNA was present and upregulated in LSCC tissues and cell lines. A functional study showed that the overexpression of circFLNA promoted LSCC cell migration. Mechanistically, circFLNA sponged miR-486-3p in LSCC cells, relieving the miR-486-3p effect, leading to elevation of migration-related FLNA expression thus affecting LSCC migration and progression.
Methods
Tissues collection
All 39 pairs of LSCC tissues and adjacent normal epithelial tissues were obtained from patients who had undergone surgery and received primary surgical resection of LSCC between September 2017 and July 2018 in the Department of Otolaryngology, Second Hospital of Hebei Medical University. None of patients with LSCC were treated with radiotherapy or chemotherapy prior to surgery. All patients with LSCC were histopathologically and clinically diagnosed. The patients were further divided into two groups according to lymph node metastasis. The study protocol was approved by the Ethics Committee of Second Hospital of Hebei Medical University and written consent was obtained from each patient (HebMU20080026). All of experiments in this paper obey World Medical Association Declaration of Helsinki.
Cell culture condition and transfection
Three human LSCC cell lines (Tu212, SCC-2 and SCC-40) were purchased from American Type Culture Collection (Manassas, VA, USA). A normal human keratinocyte cell line (HOK), and LSCC cell line Hep2 were available in our own lab. All cells were maintained in RPMI-1640 (Gibco, Beijing, China) with 10% fetal bovine serum (FBS) (Clark Bio, Claymont, DE, USA), 100 U/ml penicillin and 100 μg/ml streptomycin. According to the manufacturer’s protocol, the transfection was carried out using Lipofectamine 2000 (Invitrogen). The miR-486-3p mimic, mimic-negative control (NC), miR-486-3p inhibitor, inhibitor-NC, miR-574-5p mimic, miR-1275 mimic, sh-FLNA, sh-circFLNA and shCtl were purchased from GenePharma Co., Ltd (Shanghai, China). The sequences of sh-RNA were shown in follows: sh-circFLNA-1#: GUGCCAGCUCCCUGAAGGGTT; si-circFLNA-2#: GCCAGCUCCCUGAAGGGGCTT; sh-FLNA-1#: CCGCCAAUAACGACAAGAATT; sh-FLNA-2 #: CAGGCAACAUGGUGAAGAATT. Overexpression vector and luciferase reporter vector were purchased from GENEWIZ Company (Suzhou, China).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNA was extracted using QIAzol Lysis Reagent (Qiagen, 79306) according to the manufacture’s protocol. The concentration and purity of total RNA were measured using a NanoDrop
® spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). For miRNA, the miScripIIRT kit (QIAGEN GmbH, D-40724 Hilden, GERMANY) was used for reverse transcription, and the miScript SYBR
® Green PCR kit was used for qRT-PCR according to the manufacturer’s protocol. For circRNA and mRNA expression, the M-MLV First Strand Kit (Life Technologies) was used to synthesize to cDNA from RNA. The Platinum SYBR Green qPCR Super Mix UDG Kit (Invitrogen) was used for the qRT-PCR. qPCR was carried out using Platinum SYBR Green qPCR Super Mix UDG Kit (Invitrogen). U6 and GAPDH were used as control, respectively. The primers were designed as follows: miR-34a-5p:GGCTGGCAGTGTCTTAGCTGGTTG; miR-92b-5p:AGGGACGGGACGCGGTGC; miR-296-3p:GCGAGGGTTGGGTGGAGGCTC; miR-486-3p:GCCGGGGCAGCTCAGTACAG; miR-661:TGCCTGGGTCTCTGGCCTGC; miR-574-5p:GGCTGAGTGTGTGTGTGTGAGTGTG; miR-760:CGGCTCTGGGTCTGTGGGG; miR-486-3p:GGGGCTGGGGCCGGGGCC; miR-1226-5p:GTGAGGGCATGCAGGCCTG; miR-1271-3p:AGTGCCTGCTATGTGCCAGGC; miR-1275:GGCGTGGGGGAGAGGCTGTC; miR-1287-5p:GGCTGCTGGATCAGTGGTTCGAG; FLNA-F:AATGTGACGACAAGGGCGAC, FLNA-R:AGCACGTGAACGGCATACTC; circFLNA-F:CCAGCTGAGGCTCTACCGTGCC, circFLNA-R:GAGGCGTCAGCATCCCCAACAG, linearFLNA; F:GCTTGGCCAACAGTGACAGTGTAGG, linearFLNA-R:CAGCTACCAGCCCACCATGGAG. All data were analyzed by adopting 2-ΔΔCt method as described previously [
14].
RNA pull-down analysis
RNA pull-down analysis was performed as described previously [
15]. Briefly, the Hep2 cells were incubated with biotin (Bio)-labeled oligonucleotide probes against circFLNA (Bio-5′-CAACAGCCCCTTCAGGGAGCTGGCACGGGC (GenePharma Co., Shanghai, China) at 37 °C for 4 h. M-280 Streptavidin Dynabeads (Life Technologies) were added per 100 pmol of biotin-DNA oligos, and the mixture was then rotated for 30 min at 37 °C. The beads were captured by magnets (Life Technologies) and washed five times. Each experiment was replicated in triplicate.
Western blot analysis
The cultured cells were lysed with lysis buffer. Equal amounts of protein were run on 10% SDS-PAGE, and electro-transferred to a polyvinylidene fluoride (PVDF) membranes (Millipore). Following blocking in 5% nonfat milk, the membranes were incubated with special primary antibodies as follows: anti-MKL1 (1:1000, ab49311), anti-MMP2 (1:1000, ab37150), anti-FLNA (1:1000, ab51217) and anti-β-actin (1:1000, sc-47778). The blots were treated with the Immobilon™ Western (Millipore), and detected by ECL (enhanced chemiluminescence) Fuazon Fx (Vilber Lourmat). Images were captured and processed using FusionCapt Advance Fx5 software (Vilber Lourmat). All experiments were replicated in triplicate.
Luciferase assay
Luciferase assay was performed as previously described [
15]. For circFLNA-binding-miRNA luciferase assays, the Hep2 cells were co-transfected with an miRNA mimic (Gene pharma; Shanghai) or NC mimic (200 pmol) combined with 100 ng of circFLNA-luciferase reporter or an empty vector; For the miR-486-3p-FLNA luciferase assay, the Hep2 cells were co-transfected with a miRNA-486-3p mimic or NC mimic combined with FLNA-3′UTR-luciferase reporter (wt or mut). Dual-Glo Luciferase Assay system (Promega, Madison, WI) was used to detected luciferase activity according to the manufacturer’s protocols. Firefly luciferase activity was measured and normalized against the Renilla luciferase (RLuc) activity.
Transwell migration assay
Cell migration ability was tested by 8-μm pore size transwell filiters (Costar, Cambridge, Massachusetts). In brief, Hep2 cells (1 × 105 cells/well) were transferred onto the upper chambers of a serum-free culture. RPMI-1640 containing 10% FBS was added to the lower chambers. Following incubation at 37 °C and 5% CO2 for 24 h, migratory cells on the upper side of the chamber and medium part of the lower chamber was scraped off with a cotton swab. Then the membranes were stained by crystal violet solution. The migratory cell number was counted in three randomly areas using a microscope.
Statistical analysis
Data were presented as mean ± SEM. Student’s t test was used to analyze differences between two groups. Spearman’s correlation analysis was use to evaluate the correlation analysis. Values of P < 0.05 were considered statistically significant. Graphpad Prism 7.0 software was using to perform the statistical analysis (GraphPad Software, San Diego, CA, USA).
Discussion
In the last few decades, several circRNAs functions have been identified, including as competing endogenous RNAs or miRNA sponges [
17], interaction with RNA binding proteins [
18], modulation of the stability of mRNAs [
19], translation of proteins and regulation of gene transcription [
20]. circRNAs has been confirmed as sponges for miRNAs that influence gene expression by reducing the inhibitory effect of miRNA on its target gene [
21]. Accumulating evidence suggests that circRNAs play specific biological roles in cancer migration and development. Yang has found that circAMOTL1L contributes to prostate cancer migration and EMT by sponging miR-193a-5p and affecting Pcdha level [
15]. Bi reported that circRNA_102171 overexpression promoted papillary thyroid cancer migration via activation of Wnt/β-catenin pathway [
22]. Additionally, circRNA_0023642 [
23] and circRNA_103809 [
24] have been reported to correlate with cancer migration. In the present study, we first confirmed that circFLNA was present in LSCC tissues and cell lines. As expected, circFLNA was upregulated in LSCC tissues and its expression level was correlated with lymph node metastasis. Thereafter, using a series of in vitro assays, we found that circFLNA acted as an important migration promotor in LSCC, suggesting that circFLNA may be a potential biomarker or a therapeutic target in LSCC.
FLNA is important for organogenesis during development owing to its ability to induce cell migration via its actin-binding properties [
25]. It serves as a scaffold for over 90 binding partners and is involved in multiple cell functions, of which cell migration and adhesion are particularly critical [
26]. Mechanistically, FLNA promotes cell adhesion and migration by directing β1 integrin to the site of cell attachment and through its correlation with vimentin [
27,
28]. Recently, FLNA has been considered to be a tumor-promoting protein with an important role in tumor development and metastasis, including bladder cancer [
16], lung cancer [
29], melanoma tumor [
30] and breast cancer [
31]. Moreover, high level of FLNA implicates poor survival and drug resistance [
32]. Therefore, FLNA may be a possible target for future therapies. In the present study, we found that overexpression of circFLNA in Hep2 cells increased the FLNA protein expression but did not affect the FLNA mRNA expression. miR-486-3p, as an important mediate, directly targeted the FLNA 3′UTR inhibiting FLNA expression that promoted LSCC migration. Meanwhile, LSCC tissues had a higher expression of FLNA mRNA than their corresponding non-tumorous tissues, as supported by the data from TCGA. A higher FLNA expression indicated poor prognosis in patients with LSCC. Based on these interesting findings, it was inferred that higher linear FLNA mRNA may directly inregulate the transcription level in LSCC; however, circFLNA indirectly enhances the level of FLNA protein in the post-transcriptional level. As previously mentioned, the expression of circRNA could not always be consistent with the level of which the circRNA-derived linear RNA [
33]. Therefore, the underlying mechanisms of how and why both circFLNA and linear FLNA are upregulated in LSCC requires further investigation.
miRNA, as the most important non-coding RNA, post-transcriptionally regulates target gene protein expression through interaction with the 3′-UTR of their target genes in a sequence-specific base pairing manner [
34]. Various aberrantly expressed miRNAs are associated with the progression and prognosis of LSCC. miR-370 targeted FoxM1 functions as a tumor suppressor in LSCC [
35]; miR-1290 acts as a novel potential oncomiR in LSCC [
36]; miR-1297 mediates PTEN expression and contributes to LSCC cell progression [
37]. circRNAs contain one or more types of miRNA binding sites, and the association between miRNA and disease indicates that circRNAs may play a regulatory role by sponging miRNAs. Increasing evidence suggests that circRNAs mainly function in forming circRNA-miRNA-mRNA axis to play its biological effect in gene regulation. For example, circMTO1 acts as the sponge of miR-9 to suppress hepatocellular carcinoma progression [
38]; circTCF25 serves a the regulatory role on the pathway in bladder carcinoma by binding to miR-103a [
39]; circACTA2/miR-548-5p axis acts as a novel regulatory mechanism in smooth muscle alpha-actin expression [
40]. In the present study, we confirmed that circFLNA could sponge miR-486-3p and prevent miR-486-3p from binding FLNA 3′UTR in LSCC cells. The results also indicated that miR-486-3p reduced LSCC cell migration by negative moderation of FLNA protein level. Interestingly, miR-486-3p is not significant different between LSCC tissues and adjacent normal tissues. It is suggested that circFLNA only contributes to miR-486-3p functions but not its expression. These findings suggest that miR-486-3p plays a critical role in LSCC migration.
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