Background
Head and neck cancer is the sixth most common malignant cancer worldwide [
1‐
3], and epithelial tumors arising in the oral cavity are the most frequent tumors in the head and neck region. Tongue squamous cell carcinoma (TSCC) is the most common epithelial cancer identified in the oral cavity and accounts for approximately 25 to 40% of cases [
1,
3]. TSCC is characterized by its high metastatic and proliferative ability and is a considerable threat to human health worldwide [
3]. Over the past decades, although recent developments have been achieved in the therapeutic management of TSCC, such as surgery, chemotherapy and radiotherapy, the overall survival among TSCC patients with locally advanced disease and cervical lymph node metastasis remains dismal [
4]. The five-year survival rate of TSCC remains lower than 50%, and the patient quality of life is poor [
3,
4]. Therefore, it is necessary to clarify the underlying molecular mechanisms involved in TSCC invasion and metastasis and to develop novel therapeutic strategies for TSCC.
Long non-coding RNAs (lncRNAs) are a type of endogenous RNA over 200 nucleotides in length that completely lack or possess limited protein-coding capacity [
5‐
8]. Several studies have demonstrated that lncRNAs played a vital regulatory role in cellular and developmental processes [
6,
7]. Aberrant expression of lncRNAs is involved in cancer biology through a variety of mechanisms ranging from transcriptional levels to post-transcriptional levels [
5,
6,
9‐
12]. Recently, emerging evidence has indicated that lncRNAs are involved in the tumor initiation and progression of various malignant tumors, including TSCC [
6,
13‐
21]. For example, overexpression of lncRNAs
LINC00152 and
LINC00673 promoted TSCC cell invasion and metastasis and was associated with the poor prognosis of TSCC [
20,
21]. Huang et al. demonstrated that lncRNA
NKILA inhibits the migration and invasion of TSCC cells via suppressing epithelial-mesenchymal transition (EMT) [
22]. LncRNA
MALAT1 modulated metastatic potential, inhibited apoptosis and induced EMT in TSCC cells through the regulation of small proline rich proteins and the Wnt/β-catenin signaling pathway [
23,
24]. Moreover, overexpression of lncRNA
HOTTIP is an independent poor prognostic factor and might serve as a predictor of poor prognosis for TSCC patients [
25].
UCA1 is highly expressed in TSCC and might be correlated with cancer metastasis [
26].
LncRNA actin filament associated protein 1 antisense RNA1 (
AFAP1-AS1) is upregulated and acts as an oncogene in a variety of cancers, such as hepatocellular carcinoma, esophageal carcinoma, pancreatic ductal adenocarcinoma, colorectal cancer, cholangiocarcinoma, gallbladder cancer, and nasopharyngeal carcinoma [
27‐
36]. However, the expression and detailed function of
AFAP1-AS1 in TSCC remains largely unknown and must be investigated. In this study, we sought to determine the expression of
AFAP1-AS1 in TSCC tissues and paired noncancerous tissues and the relationship between the expression of
AFAP1-AS1 and clinical characteristics. Further functional studies revealed that knockdown of
AFAP1-AS1 could result in the inhibition of cell proliferation and invasion in vitro and tumor growth in vivo.
Methods
Human tissue samples
Patients with TSCC who were diagnosed, treated, and followed up at the Department of Oral and Maxillofacial Surgery, The Second Xiangya Hospital, Central South University, Hunan, China, were included in the study. This study was approved by the hospital institutional review board and written informed consent was obtained from all the patients. All the protocols were reviewed by the Joint Ethics Committee of the Central South University Health Authority and performed following national guidelines. Tissue samples were collected at surgery, immediately frozen in liquid nitrogen and stored until total RNA or proteins were extracted.
Quantitative real-time-PCR analysis
The tissue sample was grinded in pre-chilled mortars with liquid nitrogen. TRIzol reagent (1 ml per 50-100 mg) was added when homogenizing. Then, the powders were transferred to 2-ml or 1.5-ml microcentrifuge tubes. The cultured cells were lysed directly in the dish with 0.3-0.4 ml of TRIzol reagent per 1 × 10
5-10
7 cells. Then, RNA was isolated from harvested cells, xenograft tumors, or human tissues with TRIzol reagent according to the manufacturer’s instructions (Invitrogen, CA, USA). Real-time PCR reactions were performed using SYBR Premix DimerEraser (Takara, Dalian, China), and human GAPDH was used as an endogenous control for mRNA detection. The expression of each gene was quantified by measuring Ct values and normalized using the 2
-ΔΔct method relative to GAPDH. The gene-specific primers are shown in Table
1.
Table 1
The primers of the genes
AFAP1-AS1
| F: 5′- AATGGTGGTAGGAGGGAGGA −3’ R: 5’- CACACAGGGGAATGAAGAGG −3’ |
NANOG
| F: 5’- GAACTCTCCAACATCCTGAA −3′ R: 5′- TATTCTTCGGCCAGTTGTTT −3’ |
CADN
| F: 5’- ATGGCTACTCAA GCTGATT −3′ R: 5′- TCGAGTCATTGCATACTGT −3’ |
NESTIN
| F: 5’- CGGGCTACTGAAAAGTTCC -3′ R: 5′- CTGAAAGCTGAGGGAAGTC -3’ |
SMAD22
| F: 5’- GAGGTTCGATACAAGAGGC -3′ R: 5′- CAGCAGTCTCTTCACAACT −3’ |
SLUG
| F: 5’- AGATCTGCCAGACGCGAACT −3′ R: 5′- GCATGCGCCAGGAATGTTCA −3’ |
SNAIL1
| F: 5’- TCAAGATGCACATCCGAAGCC -3′ R: 5′- TTGTGGAGCAGGGACATTCG −3’ |
SOX2
| F: 5’- TGGAAACTTTTGTCGGAGAC -3′ R: 5′- CAGCGTGTACTTATCCTTCT −3’ |
TWIST1
| F: 5’- CAGCGCACCCAGTCGCTGAA −3′ R: 5′- CCAGGCCCCCTCCATCCTCC -3’ |
ZEB1
| F: 5’- GCACAACCAAGTGCAGAAGA −3′ R: 5′- GCCTGGTTCAGGAGAAGATG −3’ |
ZEB2
| F: 5′- GATGAAATAAGGGAGGGTGG −3′ R: 5′- CCTCAAAATCTGATGTGCAA −3’ |
GAPDH
| F: 5’- ATCAAGATCATTGCTCCTCCTGAG-3′ R: 5′- CTGCTTGCTGATCCACATCTG −3’ |
Cells culture
Human TSCC cell lines SCC-15, Tca8113, SCC-4, SCC-9 and CAL-27 cells were maintained in RPMI 1640 medium (Gibco, Waltham, MA, USA), supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air.
Vector construction and cell transfection
The pLKO.1-puro vector used for the stable expression of shRNA against AFAP1-AS1 contained a puromycin resistance gene. The scrambled control shRNA sequence had no homology to any human genomic sequences. The cultured cells (5 × 105cells/well) were seeded in 6-well culture plates and were maintained in RPMI 1640 medium with 10% FBS for 24 h before transfection. Cell transfection was performed using Lipofectamine 2000 (Invitrogen–Life Technologies, Carlsbad, CA, USA) as per the manufacturer’s instructions. For screening, puromycin (10 μg/mL) was added to the medium 72 h after transfection. The medium was replaced every 2 days for 2-3 weeks. CAL-27 and SCC-9 cells with high endogenous AFAP1-AS1 were selected for silencing. The expression of AFAP1-AS1 was confirmed by qRT-PCR. The sequence of AFAP1-AS1 shRNA and scrambled control shRNA were as follow: forward, 5’-CCGGAGCGGT CTCAGCCGAATGACTCTCGAGAGTCATTCGGCTGAGACCGCTTTTTTG-3′ and reverse, 5′ -AATTCAAAAAAGCGGTCTCAGCCGAATGACTCTCGAGAGTCATTCGGCTGAGACCGC T-3′; scrambled control shRNA, forward 5’-CCGGTTTCTCCGAACGTGTCACGTCTCGAGA CGTGACACGTTCGGAGAATTTTTG-3′ and reverse, 5′ - AATTCAAAGTTCTCGAACGTGT CACGTCTCGAGACGTGACACGTTCGGAGAA- 3′.
CCK-8 assay
Cell viability was determined using the CCK-8 assay. Briefly, 2000 cells/well were seeded into 96-well plates, and the absorptions of the cells were measured using a CCK-8 kit (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer’s instructions at different indicated time points. Data were from three separate experiments with four replications each time.
From each group, nearly 1 × 104 cells were plated in each well of a 6-well culture plate. Each cell group consisted of three wells. The cells were incubated at 37 °C for 14 days with growth media being replaced every third day. Then, the cells were washed twice with PBS and stained with 0.5% crystal violet. The number of colonies containing ≥ 50 cells was counted under a microscope [plate clone formation efficiency = (number of colonies/number of cells inoculated) × 100%]. These experiments were performed in triplicate.
Cell cycle analyses by flow cytometry
Cell cycle analyses were performed using the Cell Cycle and Apoptosis Analysis Kit (Beyotime Institute of Biotechnology, Jiangsu, China) as per the manufacturer’s instructions. Cells were harvested and fixed in 70% ethanol overnight at 4 °C. Then, the cells were stained with 25 μg/ml propidium iodide containing 1 μg/ml RNase at 37 °C for 30 min. The cells were analyzed for their distribution in different phases of the cell cycle on FACScalibur flow cytometer using CellQuestPro software (Becton Dickinson, USA).
Cell migration/invasion assay
Cell migration was evaluated using a transwell assay. A total of 2 × 104 cells in 200 μl of serum-free medium were added to the top chamber of the transwell (8 μm pore size, BD Biosciences, New Jersey, USA) at 24 h after siRNA transfection. The bottom well contained growth medium with 20% FBS. Cells were incubated at 37 °C for 24 h. After 24 h, the cells that had migrated to the lower face of the filters were fixed with 100% methanol and stained with 0.5% crystal violet and counted. Matrigel invasion assays were performed as follows. Filters coated with Matrigel in the upper compartment were loaded with 200 μl serum-free medium containing 5 × 104 transfected cells, and the lower compartment was filled with 20% FBS. After 24 h, the migrated cells on the bottom surface were fixed with 100% methanol and counted after staining with 0.5% crystal violet. Numbers of invaded cells were counted in six randomly selected fields under a microscope, and the average value was calculated. Each experiment was conducted in triplicate.
Wound-healing assay
Cells were cultured until they reached 90% confluence in 6-well plates. Cell layers were scratched using a 10-μL tip to form wounded gaps, washed with PBS twice and cultured. The wounded gaps were photographed at different time points and analyzed by measuring the distance of migrating cells from five different areas for each wound.
Western blotting
For total cell lysates, cells were lysed in lysis buffer that contained 25 mM Tris (pH 7.4), 2 mM NaVO4, 10 mM NaF, 10 mM Na4P2O7, 1 mM EGTA, 1 mM EDTA, and 1% NP-40. Protease inhibitor cocktail and PhosSTOP were added fresh to the lysis buffer before each experiment. The proteins were separated on SDS-PAGE and then transferred to PVDF membrane (Merck Millipore). The membrane was blocked in Tris-buffered saline (TBS; pH 7.4) with 5% skim milk for 2 h, and then, the membranes were incubated overnight at 4 °C with diluted primary antibodies overnight. Antibodies against β-catenin (#8480), AKT (#9272), phospho-AKT (#9271), GSK3β (#12456) and phospho-GSK3β (#5558) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against AFAP1 (sc-374,655) and GAPDH (sc-32,233) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). After incubation, the membranes were washed three times in TBST, followed by incubation with secondary antibody (1:5000, Santa Cruz Biotechnology, CA, USA) in TBST 10% blocking reagent for 1 h, and washed again in TBST (3 times for 20 min). Immunoblots were developed using ChemicalDocTM XRS+ (Bio-Rad, Berkeley, CA, USA). The intensity of the protein fragments was quantified using QuantityOne software (Bio-Rad, Berkeley, CA, USA).
Tumorigenicity assays in nude mice
All the animal procedures were performed in accordance with local institutional ethical guidelines. Ethical approval was obtained from the Experimental Animal Ethics Committee of the Central South University Health Authority. CAL-27 cells (1 × 107) with stably transfected shR-AFAP1-AS1 or shR-NC were suspended in 0.1 ml PBS and injected into the left flank of 4-week-old female BALB/c athymic nude mice. Tumor volumes were calculated using hand calipers every week after the injection using the following formula: tumor volume (mm3) = (length × width2)/2. At 35 days, mice were sacrificed and tumor volumes and weights were recorded.
Statistical analysis
All experiments were performed three times, and the data were analyzed with GraphPad Prism 5 (La Jolla, CA, USA). Results are presented as mean ± SD. The differences between TSCC tissues group and the adjacent normal tissues groups group were was tested using paired Student’s t-test. Differences between groups were tested using paired and unpaired Student’s t-test, a one-way ANOVA and χ2 tests where appropriate with the SPSS 17.0 program. A p-value of <0.05 was considered statistically significant.
Discussion
In recent years, aberrant expression or dysfunctional activities of lncRNAs has been discovered in multiple tumor types, and substantial evidence indicates that lncRNAs participate in all steps of tumor initiation and development [
6,
13,
17,
37]. Many studies have shown that lncRNAs are of great importance in the diagnosis and treatment of tumors, and that lncRNAs are useful as novel prognostic tumor biomarkers [
22,
38‐
42]. LncRNA
AFAP1-AS1 is oriented in an antisense direction to the protein-coding gene
AFAP1 in the opposite strand [
36,
43,
44]. High expression of
AFAP1-AS1 is associated with malignancy, metastasis and poor prognosis in various cancers, such as hepatocellular carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, colorectal cancer, cholangiocarcinoma, and gallbladder cancer [
27‐
35].
AFAP1-AS1 is upregulated in hepatocellular carcinoma, and its higher expression is associated with tumor size, TNM stage, vascular invasion, and poor prognosis [
45]. Furthermore, silencing
AFAP1-AS1 significantly reduce cell proliferation, clonal growth, cell migration, and invasion and increase apoptosis through RhoA/Rac2 signaling [
31,
45]. LncRNA
AFAP1-AS1 also promote tumor growth and invasion in cholangiocarcinoma [
29,
32], esophageal squamous cell carcinoma, nasopharyngeal carcinoma and gallbladder cancer [
27,
28,
34,
35]. Consistent with these studies, in the present study, lncRNA
AFAP1-AS1 was also upregulated in TSCC tissues when compared with pair matched normal tissues, and the upregulation of
AFAP1-AS1 was associated with clinical characteristics and poor overall survival. We also determined the effects of
AFAP1-AS1 on TSCC cells. Inhibition of
AFAP1-AS1 inhibited the proliferation, migration and invasion of TSCC cells in vitro and in vivo. Collectively, our results demonstrated that
AFAP1-AS1, acting as an oncogene, may be a potential diagnostic and prognostic biomarker as well as a therapeutic target in TSCC.
Cell migration and invasion are significant aspects of cancer progression, and the EMT is a critical biological process in tumor cell migration and invasion [
5‐
7,
46,
47]. The Wnt/β-catenin pathway plays a critical role in cellular proliferation, survival, differentiation and EMT in cancer cells [
48‐
50]. When Wnt ligands bind to transmembrane receptors, Wnt signaling can be initiated [
49]. β-catenin accumulates in the nucleus and forms the β-catenin/TCF/LEF transcriptional complex [
48]. As a result, Wnt target genes are activated and promote EMT [
49]. Accumulating evidence supports that many important oncogenes or tumor suppressor genes regulate the Wnt/β-catenin signaling pathway, and thus modulate EMT, migration and invasion in tumor cells [
51‐
54]. In addition, lncRNAs also participate in this regulatory process [
24,
55‐
57]. LncRNA
UCA1 enhances the Wnt/β-catenin signaling pathway to promote the EMT of breast cancer cells [
55]. LncRNA
PTCSC3 inhibites the proliferation and invasion of glioma cells by suppressing the Wnt/β-catenin signaling pathway [
56]. In tongue cancer,
MALAT1 induces EMT and inhibits apoptosis through the Wnt/β-catenin signaling pathway [
24]. Moreover, overexpression of lncRNA
CTD903 inhibits colorectal cancer invasion and migration by repressing Wnt/β-catenin signaling and predicts a favorable prognosis [
57]. In the present study, we tested the effect of
AFAP1-AS1 knockdown on the Wnt/β-catenin signaling pathway. Compared with the control group, silencing
AFAP1-AS1 decreased the phosphorylation of AKT and GSK3β and the total level of β-catenin in both SCC-9 and CAL-27. Total levels of AKT and GSK3β did not show obvious differences. Additionally, the expression of EMT-related genes was determined by qRT-PCR. Downregulation of
AFAP1-AS1 significantly downregulated EMT-related genes
SLUG, SNAIL1, VIM, CADN, ZEB1, ZEB2, and
TWIST1 in SCC-9 cells. The expression levels of
SLUG, VIM, CADN, ZEB1, SMAD2, and
TWIST1 were downregulated in CAL-27 cells after inhibiting the expression of
AFAP1-AS1. Additionally, we obtained similar results in the xenograft tumor model.
SLUG, SNAIL1, VIM, ZEB1, NANOG, SMAD2, NESTIN and
SOX2 were down-regulated when the expression of
AFAP1-AS1 was silenced in xenograft tumors. The above data indicated that
AFAP1-AS1 could regulate the activity of the Wnt/β-catenin signaling pathway and affect the expression of several EMT-related genes in TSCC cells.
Previous studies have demonstrated that lncRNA activity was partly dependent on its genomic location. Antisense lncRNAs such as
AFAP1-AS1 are oriented in an antisense direction with respect to a protein-coding gene in the opposite strand and usually act as a regulator of this gene [
58‐
60].
AFAP1-AS1 is localized in the antisense DNA strand of the
AFAP1 gene [
61]. We demonstrated that
AFAP1-AS1 expression increased AFAP1 protein levels. However, it remains unknown whether the effects of
AFAP1-AS1 on the regulation of tumor cell metastasis potential are mediated by the changed
AFAP1 protein levels.