Minghe Li1, Dongsheng Yu2, Zhihong Li3, Cong Zhao1, Chang Su1, Jun Ning4. 1. Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Jilin University, Changchun, Jilin 130021, P.R. China. 2. Department of Orthodontics, Hospital of Stomatology, Jilin University, Changchun, Jilin 130021, P.R. China. 3. Department of Thoracic Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China. 4. Department of Gynaecology II, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China.
Abstract
Long non‑coding RNA (lncRNA) actin filament‑associated protein 1 antisense RNA 1 (AFAP1‑AS1) has been reported to serve important roles in multiple types of cancer. However, the biological function and underlying mechanism of AFAP1‑AS1 in oral squamous cell carcinoma (OSCC) remain largely unknown. The present study aimed to investigate the biological roles and clarify the potential mechanism of AFAP1‑AS1 in OSCC. The expression levels of AFAP1‑AS1 in OSCC tissues and cells were determined using reverse transcription‑quantitative PCR. Cell proliferation, colony formation, migration and invasion were analyzed using Cell Counting Kit‑8, colony formation, wound healing and Transwell invasion assays, respectively. The potential binding between AFAP1‑AS1 and microRNA (miR)‑145 was validated using dual luciferase reporter and RNA pull‑down assays. A xenograft tumor model was established to evaluate the effect of AFAP1‑AS1 in vivo. The results revealed that AFAP1‑AS1 expression levels were markedly upregulated in OSCC tissues and cells. In addition, patients with OSCC with high expression levels of AFAP1‑AS1 had a poor prognosis. Functionally, the knockdown of AFAP1‑AS1 in OSCC cells significantly inhibited cell proliferation, migration and invasion in vitro. Similarly, in vivo AFAP1‑AS1 knockdown prevented tumor growth and reduced tumor size and weight. Mechanistically, AFAP1‑AS1 was discovered to regulate the expression levels of Homeobox A1 (HOXA1) by competing with miR‑145. The inhibition of miR‑145 partially attenuated the inhibitory effects of AFAP1‑AS1 knockdown on OSCC cells. In conclusion, the findings of the present study suggested that AFAP1‑AS1 may promote the progression of OSCC by regulating the miR‑145/HOXA1 axis.
Long non‑coding RNA (lncRNA) actin filament‑associated protein 1 antisense RNA 1 (AFAP1‑AS1) has been reported to serve important roles in multiple types of cancer. However, the biological function and underlying mechanism of AFAP1‑AS1 in oral squamous cell carcinoma (OSCC) remain largely unknown. The present study aimed to investigate the biological roles and clarify the potential mechanism of AFAP1‑AS1 in OSCC. The expression levels of AFAP1‑AS1 in OSCC tissues and cells were determined using reverse transcription‑quantitative PCR. Cell proliferation, colony formation, migration and invasion were analyzed using Cell Counting Kit‑8, colony formation, wound healing and Transwell invasion assays, respectively. The potential binding between AFAP1‑AS1 and microRNA (miR)‑145 was validated using dual luciferase reporter and RNA pull‑down assays. A xenograft tumor model was established to evaluate the effect of AFAP1‑AS1 in vivo. The results revealed that AFAP1‑AS1 expression levels were markedly upregulated in OSCC tissues and cells. In addition, patients with OSCC with high expression levels of AFAP1‑AS1 had a poor prognosis. Functionally, the knockdown of AFAP1‑AS1 in OSCC cells significantly inhibited cell proliferation, migration and invasion in vitro. Similarly, in vivo AFAP1‑AS1 knockdown prevented tumor growth and reduced tumor size and weight. Mechanistically, AFAP1‑AS1 was discovered to regulate the expression levels of Homeobox A1 (HOXA1) by competing with miR‑145. The inhibition of miR‑145 partially attenuated the inhibitory effects of AFAP1‑AS1 knockdown on OSCC cells. In conclusion, the findings of the present study suggested that AFAP1‑AS1 may promote the progression of OSCC by regulating the miR‑145/HOXA1 axis.
Oral squamous cell carcinoma (OSCC) is one of the most common types of head and neck cancer, and its incidence is increasing worldwide (1). Despite significant improvements being made in the treatment and diagnosis of OSCC, the 5-year survival rate for patients with OSCC has remained unsatisfactory during the past few decades (2,3). Therefore, understanding the underlying mechanisms associated with OSCC progression remains a priority to determine novel diagnostic biomarkers and therapeutic strategies for improving the prognosis of this disease.Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs of >200 nucleotides in length that lack protein-coding abilities (4). Accumulating evidence has revealed that lncRNAs are involved in various physiological and pathological processes, such as cell proliferation, apoptosis, differentiation and carcinogenesis (5,6). In addition, previous studies have identified that the dysregulation of lncRNAs may be associated with the occurrence and development of various types of disease, including multiple types of cancer (7,8). Notably, in OSCC, a number of lncRNAs have been identified to serve crucial roles in OSCC progression, where they function as either oncogenes or tumor suppressors (9,10).Actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1), a 6.8-kb lncRNA located on chromosome 4p16.1, has been reported to be associated with carcinogenesis in a number of types of cancer (11), including breast cancer (12), non-small lung cancer (13), nasopharyngeal carcinoma (14), colorectal cancer (15), hepatocellular carcinoma (16) and gastric cancer (17). AFAP1-AS1 expression levels were also previously reported to be upregulated in OSCC (18); however, to the best of our knowledge, the molecular mechanisms and the role of AFAP1-AS1 in the migration and invasion of OSCC cells have not been investigated.In the present study, the expression levels and clinical significance of AFAP1-AS1 in OSCC were investigated. The biological function and regulatory mechanisms of AFAP1-AS1 in OSCC were also determined.
Materials and methods
Ethical statement
The patient studies were approved by the Ethics Committee of Jilin University (approval no. JLU20160328) and were performed according to the principles of the Declaration of Helsinki. Written informed consent was obtained from all participating patients prior to surgery. All animal experiments were approved by the Ethics Committee of Jilin University (approval no. JLUA2016084) and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health).
Patient samples
Between March 2014 and December 2015, OSCC and adjacent normal mucosal tissues (≥1.5 cm away from the unaffected margins of the tumor) were obtained from 48 patients at the Department of Oral and Maxillofacial Surgery. All tissues were stored in liquid nitrogen immediately after resection until use. All of the patients involved in the present study were diagnosed by pathology, and had not received treatment, such as radiotherapy or chemotherapy, prior to surgery. The clinicopathological data was collected and is presented in Table I.
Table I.
Associations between clinicopathological features in 48 patients with OSCC and AFAP1-AS1 expression.
Three OSCC cell lines (SCC9, SCC15 and SCC25) were purchased from the American Type Culture Collection. Human oral keratinocytes (HOKs) were obtained from ScienCell Research Laboratories, Inc. All cells were cultured in DMEM supplemented with heat-inactivated 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.), and maintained in an incubator with 5% CO2 at 37°C.
Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was extracted from cultured cells and tissues using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) or a miRNeasy mini kit (Qiagen, Inc.), according to the manufacturer's protocols. Total RNA was reverse transcribed into cDNA using a PrimeScript RT reagent kit with gDNAEraser (Perfect Real Time) (cat no. RR047A; Takara Bio, Inc.). qPCR was subsequently performed on an ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR-Green (Takara Biotechnology Co., Ltd.), according to the manufacturer's instructions. The thermocycling conditions used were as follows: Initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 58°C for 40 sec. The primer sequences used for the qPCR were described previously (15,19) and were as follows: For AFAP1-AS1 5′-CGTTCACTTCAATAGCCGC-3′ (forward) and 5′-GGAGAAGGGATCGTCCCA-3′ (reverse). The mRNA expression levels were calculated using the 2−∆∆Cq method (20) and 7500 Real-time PCR software v.2.0.1 (Applied Biosystems; Thermo Fisher Scientific, Inc.). GAPDH or U6 snRNA were used as endogenous loading controls for AFAP1-AS1/HOXA1 and miR-145, respectively.
Cell transfection
The miR-145 mimics (5′-GUCCAGUUUUCCCAGGAAUCCCU-3′), miR-145 inhibitor (anti-miR-145; 5′-AGGGAUUCCUCCCAAAACUGGAC-3′), mimics negative control (miR-NC; 5′-GUAGGAGUAGUGAAAGGCC-3′) and inhibitor NC (anti-miR-NC; 5′-GGCCUUUCACUACUCCUAC-3′) were purchased from Guangzhou RiboBio Co., Ltd. Two small interfering RNAs (siRNA/si) targeting AFAP1-AS1 (si-AFAP1-AS1#1; 5′-GGACCATTTGGTGTATCTTT-3′ and si-AFAP1-AS1#2; 5′-GGTGGAGAATGAACATTCUTT-3′) and the corresponding control scramble NC (si-NC; 5′-TTCTCCGAACGTGTCACGTTT-3′) were purchased from Santa Cruz Biotechnology, Inc. Short hairpin RNA (shRNA/sh) targeting AFAP1-AS1 (sh-AFAP1-AS1; 5′-TTATTTTGCTAATTCAAC-3′) and the corresponding control (sh-NC; 5′-CCTAACCACAAACTCTACGGC-3) were synthesized and packaged in lentiviral vectors (Lv) by Shanghai GenePharma Co., Ltd. SCC9 cells were transfected with each transfectant (600 ng) using 3 µl Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following incubation at 37°C for 6 h, the serum-free DMEM medium was replaced with fresh RPMI-1640 medium containing 10% FBS, and then cells were cultured for 24–72 h for further assays.
Cell Counting Kit-8 (CCK-8) assay
Cell proliferation was analyzed using a CCK-8 assay (Roche Diagnostics), according to the manufacturer's protocol. Briefly, ~5×103 transfected cells/well were seeded into a 96-well culture plate and incubated for 24, 48 or 72 h. Following the incubation, 10 µl CCK-8 solution was added to each well and incubated for an additional 4 h in DMEM medium supplemented with 10% FBS at 37°C. The absorbance at 450 nm was then measured on a Model 680 microplate reader (Bio-Rad Laboratories, Inc.).
Colony formation assay
For the colony formation assay, ~1×103 transfected cells/well were seeded into a 6-well culture plate and cultured for 14 days. The cells were subsequently washed twice with PBS, fixed in 70% methanol for 30 min at 37°C and then stained with 0.1% crystal violet for 5 min at 37°C. The number of visible colonies (>50 clones) was counted using ColCount colony counter (Oxford Optronix Ltd.).
Wound healing assay
Cell migration was analyzed using a wound healing assay. Briefly, 2×105 transfected cells/well were plated into 6-well plates and cultured to 100% confluence. A scratch was then made in the cell monolayer using a 200-µl plastic pipette tip. Cells were subsequently cultured in serum-free medium for 24 h and the wound healing distance was measured at 0 and 24 h to assess cell migration under a GX53 light microscope (Olympus Corporation) at a magnification of ×100.
Cell invasion assay
Cell invasion was determined using 24-well BioCoat cell culture inserts (Corning, Inc.) containing a polyethylene terephthalate membrane (8-µm pores) precoated with Matrigel (BD Biosciences). Briefly, 5×104 transfected cells suspended in 100 µl serum-free medium were plated into the upper chambers. The lower chambers were filled with 600 µl medium supplemented with 20% FBS. Following 48 h of incubation, the membranes were fixed with 4% methanol at 37°C for 30 min and stained with 0.1% crystal violet at 37°C for 10 min. The number of invasive cells was determined by counting five pre-determined fields of view under a GX53 light microscope (magnification, ×200).
Subcellular fraction
The separation of SCC9 cells into nuclear and cytoplasmic fractions was conducted using a PARIS Kit (Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The expression levels of AFAP1-AS1 in the nuclear and cytoplasmic fractions of SCC9 cells were analyzed using RT-qPCR.
Dual luciferase reporter assay
Bioin-formatics softwar LncBase V3 (https://diana.e-ce.uth.gr/lncbasev3/interactions) was used to predict AFAP1-AS1 interaction with miR-145. The 3′-untranslated region (3′-UTR) of AFAP1-AS1 containing wild-type (wt) or mutant (mut) binding sites for miR-145 were amplified and cloned into the PGL-3 luciferase reporter vector (Promega Corporation) to generate pGL3-wt-AFAP1-AS1 and pGL3-mut-AFAP1-AS1 plasmids. The luciferase reporter plasmids (pGL3-wt-AFAP1-AS1 or pGL3-mut-AFAP1-AS1) and miR-145 mimics or miR-NC were co-transfected into OSCC cells using Lipofectamine 2000 reagent. Following 48 h of transfection, the cells were collected, and the relative luciferase activity was measured using a Dual Luciferase Reporter assay kit (Promega Corporation). The Renilla luciferase activity was normalized to firefly luciferase activity.
RNA pull-down assay
SCC9 cells were transfected with 50 nM biotin-labeled wt-bio-miR-145, mut-bio-miR-145 or bio-miR-NC (Wuhan GeneCreate Biological Engineering Co., Ltd.). Subsequently, the cells were collected and incubated in specific lysate buffer (Ambion; Thermo Fisher Scientific, Inc.) and M-280 streptavidin beads (cat. no. S3762; Sigma-Aldrich; Merck KGaA). The bound RNAs were isolated and subjected to RT-qPCR to analyze the expression levels of AFAP1-AS1 as aforementioned.
Western blotting
Total protein of tissues and cultured cells were extracted using RIPA buffer (Beyotime Institute of Biotechnology), and was quantified by a BCA Protein Quantification Kit. Total protein (20 µg) was separated using 10% sodium dodecyl sulfonate-polyacrylamide gel (SDS-PAGE; Beyotime Institute of Biotechnology). Then the proteins (20 µg/lane) were transferred onto polyvinylidene difluoride membranes (PVDF; EMD Millipore). Membranes were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) in TBS with 0.1% Tween-20 (Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Next, the membranes were incubated with primary antibodies against HOXA1 (1:500 dilution; product code ab230513) and GAPDH (1:3,000 dilution; product code ab181602; both from Abcam) overnight at 4°C and then HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5,000 dilution; product code ab205718; Abcam) was added for 2 h at room temperature. Finally, the protein bands were observed using an enhanced chemiluminescence reagent (EMD Millipore) and analyzed by ImageJ software (v1.8.0; National Institutes of Health).
In vivo tumorigenicity assay
A total of 10 male BALB/c nude mice (age, 5 weeks old; weight, 18–22 g) were purchased from the Laboratory Animal Research Center of Jilin University (Changchun, China) and bred under specific pathogen-free conditions at 25°C and 45% humidity with a 12-h light/dark cycle, with free access to food and water. A total of 2×106 SCC-9 cells (100 µl) stably infected with Lv-sh-AFAP1-AS1 or Lv-sh-NC were subcutaneously injected into the left-hand side of the armpit region of each mouse (5 mice per group). The tumor volume was measured every 5 days for 30 days by measuring the maximum long diameter (L) and minimum short diameter (w) of the tumor with a Vernier caliper. The tumor volume (V) was calculated using the following formula: V=0.5×LxW2. Subsequently, 30 days after inoculation, the mice were sacrificed by cervical dislocation. The tumors were excised, weighed and photographed. The xenograft tissues were stored in liquid nitrogen and then used to analyze the expression levels of AFAP1-AS1, miR-145 and HOXA1.
Immunohistochemistry (IHC)
IHC was performed to analyze HOXA1 expression levels in xenograft tumors were assessed according to a previous study (21). Briefly, the tumor tissues were dissected and fixed in 4% paraformaldehyde at 25°C overnight, embedded in paraffin and then dissected into sections (5 µm), deparaffinized in xylene, rehydrated with graded alcohols (100, 90, 70 and 50%). Subsequently, the sections were autoclaved for 15 min at 121°C using sodium citrate buffer (0.01 M, pH 7.0) for antigen retrieval. After being blocked with 10% FBS in PBS at 37°C for 30 min, the sections were stained with an anti-HOXA1 primary antibody (1:400 dilution; product code ab230513; Abcam) at 4°C overnight. Subsequently, the sections were incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (1:1,000 dilution; product code ab205718; Abcam) for 30 min at 37°C. Then the sections were incubated with DAB for 2 min at 25°C, counterstained with hematoxylin, dehydrated and stabilized with mounting medium (product code ab64230; Abcam). Images were acquired using an IX73 inverted microscope (magnification, ×40; Olympus Corporation). The expression levels of HOXA1 were semi-quantified using Image-Pro Plus software (Media Cybernetics, Inc.).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 5.01 software (GraphPad Software, Inc.) and SPSS 19.0 software (IBM Corp.). A χ2 test was used to analyze the relationship between AFAP1-AS1 expression levels and the clinicopathological characteristics of patients with OSCC. Survival curves were plotted using the Kaplan-Meier method, and the P-values were analyzed using a log-rank test. A two-tailed Student's t-test and a one-way ANOVA followed by Tukey's test were used to determine the statistical differences between two independent or ≥3 groups, respectively. The correlations between the expression of AFAP1-AS1, miR-145 and HOXA1 were analyzed using Pearson's correlation coefficient analysis. All data are presented as the mean ± SD from ≥3 independent experiments. P<0.05 was considered to indicate a statistically significant difference.
Results
AFAP1-AS1 expression levels are upregulated in OSCC and are associated with a poor prognosis in patients with OSCC
The expression levels of AFAP1-AS1 in 48 OSCC and matched adjacent non-tumor tissues were analyzed using RT-qPCR. As revealed in Fig. 1A, AFAP1-AS1 expression levels were significantly upregulated in OSCC tissues compared with adjacent normal tissues. The expression levels of AFAP1-AS1 in OSCC cell lines (SCC9, SCC15 and SCC25) were also analyzed and it was revealed that they were increased compared with HOKs (Fig. 1B). Furthermore, the association between AFAP1-AS1 expression levels and the clinicopathological variables of patients with OSCC was determined. The results revealed that upregulated AFAP1-AS1 expression levels were positively associated with an advanced clinical stage and lymph node metastasis (Table I). Kaplan-Meier survival analysis also demonstrated that patients with high AFAP1-AS1 expression had a poorer overall survival (OS) compared with those with low AFAP1-AS1 expression (Fig. 1C). These results indicated that AFAP1-AS1 may be involved in the tumorigenesis and development of OSCC.
Figure 1.
AFAP1-AS1 expression levels are upregulated and associated with a poor prognosis in patients with OSCC. (A) Expression levels of AFAP1-AS1 were upregulated in OSCC tissues compared with adjacent normal tissues. (B) Expression levels of AFAP1-AS1 were upregulated in three OSCC cell lines (SCC9, SCC15 and SCC25) compared with HOKs. (C) Association between AFAP1-AS1 expression levels and overall survival was analyzed using Kaplan-Meier analysis. All experiments were performed in triplicate and data are expressed as the mean ± SD. **P<0.01. AFAP1-AS1, actin filament-associated protein 1 antisense RNA 1; OSCC, oral squamous cell carcinoma; HOK, human oral keratinocytes.
Knockdown of AFAP1-AS1 inhibits OSCC proliferation, migration and invasion
To determine the possible regulatory effect of AFAP1-AS1 in OSCC cells, two siRNAs targeting AFAP1-AS1 (si-AFAP1-AS1#1 and si-AFAP1-AS1#2) and one scrambled siRNA (si-NC) were transfected into SCC9 cells. AFAP1-AS1 expression levels were revealed to be downregulated by >69% in the si-AFAP1-AS1#1 group and by >86% in the si-AFAP1-AS1#2 group compared with the si-NC group 48 h post-transfection (Fig. 2A). Therefore, si-AFAP1-AS1#2 (referred to as si-AFAP1-AS1 henceforth) was selected for use in subsequent experiments. The results of the CCK-8 assay revealed that cell proliferation was significantly reduced following the knockdown of AFAP1-AS1 (Fig. 2B). In addition, AFAP1-AS1 knockdown significantly inhibited colony formation in SCC9 cells (Fig. 2C). The impact of AFAP1-AS1 on the migration and invasion of OSCC cells was determined using wound healing and Matrigel invasion assays, respectively. The results demonstrated that AFAP1-AS1 knockdown notably decreased the migratory and invasive abilities compared with the si-NC group (Fig. 2D and E). These results indicated that the knockdown of AFAP1-AS1 may inhibit OS progression.
Figure 2.
Knockdown of AFAP1-AS1 inhibits oral squamous cell carcinoma proliferation, migration and invasion. (A) The expression levels of AFAP1-AS1 were downregulated in SCC9 cells transfected with si-AFAP1-AS1#1, si-AFAP1-ASA#2 compared with SCC9 cells transfected with si-NC. (B) Cell proliferation, (C) colony formation, (D) migration and (E) invasion were inhibited in SCC9 cells transfected with si-AFAP1-AS1. All experiments were performed in triplicate and data are expressed as the mean ± SD. *P<0.05, **P<0.01. AFAP1-AS1, actin filament-associated protein 1 antisense RNA 1; si, small interfering RNA; NC, negative control.
AFAP1-AS1 directly targets miR-145 and downregulates its expression levels in OSCC cells
It is well known that cytoplasmic lncRNAs can act as competing endogenous RNAs (ceRNAs) or molecular sponges that regulate miRNAs in various types of cancer (22,23). To detect the potential molecular mechanism of AFAP1-AS1, cytoplasmic and nuclear fractions were isolated from SCC9 cells to identify the distribution of AFAP1-AS1 in SCC9 cells. The results demonstrated that AFAP1-AS1 was mainly located in the cytoplasm of SCC9 cells (Fig. 3A). Bioinformatics analysis revealed that miR-145 contained a binding site for AFAP1-AS1 (Fig. 3B). Thus, to validate whether AFAP1-AS1 directly targeted miR-145, dual luciferase reporter assays were performed. As revealed in Fig. 3C, the overexpression of miR-145 markedly inhibited the relative lucif-erase activity of pLG3-wt-AFAP1-AS1 in SCC9 cells, but not that of pLG3-mut-AFAP1-AS1. To further verify the interaction between AFAP1-AS1 and miR-145, RNA pull-down assays were performed. The results indicated that AFAP1-AS1 expression levels were significantly upregulated in the bio-wt-miR-145 group compared with the bio-mut-miR-145 and bio-miR-NC groups (Fig. 3D). In addition, the knockdown of AFAP1-AS1 significantly upregulated the expression levels of miR-145 in SCC9 cells (Fig. 3E). However, the overexpression or knockdown of miR-145 did not alter AFAP1-AS1 expression levels in SCC9 cells (Fig. 3F). These results indicated that AFAP1-AS1 may function as a ceRNA for miR-145.
Figure 3.
AFAP1-AS1 directly targets miR-145 and downregulates its expression levels in oral squamous cell carcinoma cells. (A) AFAP1-AS1 was observed to be primarily located in the cytoplasm of SCC9 cells. (B) Predicted binding and mutated sites between AFAP1-AS1 and miR-145. (C) Dual luciferase reporter assay revealed that the transfection with miR-145 mimics decreased the relative luciferase activity of wt-AFAP1-AS1, but not that of mut-AFAP1-AS1-AS1. (D) Association between AFAP1-AS1 and miR-145 was determined using an RNA pull-down assay. (E) miR-145 expression levels were upregulated in SCC9 cells transfected with si-AFAP1-AS1 compared with SCC9 cells transfected with si-NC. (F) AFAP1-AS1 expression levels were analyzed in SCC9 cells transfected with miR-145 mimics, miR-NC, miR-145 inhibitors (anti-miR-145) or anti-miR-NC. All experiments were performed in triplicate and data are expressed as the mean ± SD. **P<0.01. AFAP1-AS1, actin filament-associated protein 1 antisense RNA 1; miR, microRNA; NC, negative control; Wt, wild-type; Mut, mutant; si, small interfering RNA.
Knockdown of miR-145 partially reverses the AFAP1-AS1 knockdown-induced inhibitory effects on OSCC cells
To investigate the importance of miR-145 in AFAP1-AS1-mediated OSCC proliferation, migration and invasion, the expression levels of miR-145 were knocked down using a miR-145 inhibitor in SCC9 cells transfected with si-AFAP1-AS1. The results revealed that the miR-145 inhibitor reversed the upregulated miR-145 expression levels induced by AFAP1-AS1 knockdown in SCC9 cells (Fig. 4A). Furthermore, miR-145 inhibition partially blocked the inhibitory effects of AFAP1-AS1 on cell proliferation, colony formation, migration and invasion in SCC9 cells (Fig. 4B-E).
Figure 4.
Knockdown of miR-145 partially reverses the AFAP1-AS1 knockdown-induced inhibitory effects on OSCC. (A) Expression levels of miR-145 were analyzed in SCC9 cells transfected with si-NC, si-AFAP1-AS1 and si-AFAP1-AS1 + miR-145 inhibitor (anti-miR-145). Knockdown of miR-145 partially reversed the AFAP1-AS1 knockdown-induced inhibitory effects on OSCC (B) cell proliferation, (C) colony formation, (D) migration and (E) invasion. All experiments were performed in triplicate and data are expressed as the mean ± SD. *P<0.05, **P<0.01. miR, microRNA; AFAP1-AS1, actin filament-associated protein 1 antisense RNA 1; OSCC, oral squamous cell carcinoma; si, small interfering RNA; NC, negative control; NS, non-significant.
AFAP1-AS1 mediates HOXA1 expression levels through miR-145 in OSCC cells
HOXA1 has been previously identified as a target of miR-145 in OSCC (19). Therefore, the present study sought to determine whether AFAP1-AS1 functioned as a ceRNA for miR-145 to regulate the expression levels of HOXA1 in OSCC. The knockdown of AFAP1-AS1 significantly downregulated HOXA1 expression levels at both the mRNA and protein level (Fig. 5A and B), while the knockdown of miR-145 partially reversed this trend (Fig. 5A and B). HOXA1 expression levels were revealed to be upregulated in OSCC tissues and cell lines (Fig. 5C and D), and its expression was observed to be positively correlated with AFAP1-AS1 expression levels (Fig. 5E) and negatively correlated with miR-145 expression levels (Fig. 5F).
Figure 5.
AFAP1-AS1 mediates HOXA1 expression levels through miR-145 in OSCC cells. (A) mRNA and (B) protein expression levels of HOXA1 were determined in SCC9 cells transfected with si-NC, si-AFAP1-AS1 and si-AFAP1-AS1 + anti-miR-145. (C) Expression levels of HOXA1 were upregulated in OSCC tissues and adjacent normal tissues. (D) Expression levels of HOXA1 were upregulated in three OSCC cell lines (SCC9, SCC15 and SCC25) compared with normal oral mucosal HOK cells. Correlations between (E) AFAP1-AS1 and HOXA1 expression levels and (F) miR-145 and HOXA1 expression levels in OSCC tissues were analyzed using Pearson's correlation analysis. All experiments were performed in triplicate and data are expressed as the mean ± SD. *P<0.05, **P<0.01. AFAP1-AS1, actin filament-associated protein 1 antisense RNA 1; HOXA1, homeobox A1; miR, microRNA; OSCC, oral squamous cell carcinoma; si, small interfering RNA; NC, negative control.
AFAP1-AS1 knockdown suppresses tumor growth in vivo
Finally, the role of AFAP1-AS1 in vivo was determined using tumor xenograft mice. As revealed in Fig. 6A, tumor growth in the sh-AFAP1-AS1 group was decreased compared with the sh-NC group. Following sacrifice, the tumor tissues were obtained and weighed; the weight and volume of the tumors in the sh-AFAP1-AS1 group were significantly decreased compared with those in the sh-NC group (Fig. 6B and C). In addition, RT-qPCR analysis revealed that, compared with the sh-NC group, the expression level of AFAP1-AS1 was downregulated in the sh-AFAP1-AS1 tumor tissues (Fig. 6D). Furthermore, the expression level of miR-145 was increased in the sh-AFAP1-AS1 group compared with the sh-NC group (Fig. 6E). In addition, the expression level of HOXA1 was downregulated in the sh-AFAP1-AS1 group compared with the sh-NC group (Fig. 6F). Finally, HOXA1 expression levels were analyzed in xenograft tumors using immunohistochemistry and it was revealed that the number of HOXA1-positive cells was decreased in the sh-AFAP1-AS1 group compared with the sh-NC group. These results indicated that the knockdown of AFAP1-AS1 may significantly inhibit the growth of tumors in vivo.
Figure 6.
AFAP1-AS1 knockdown suppresses tumor growth in vivo. (A) Tumor growth curve. (B) Images of tumors. (C) Tumor weight was measured at the end of the experiment. mRNA expression levels of (D) AFAP1-AS1, (E) miR-145 and (F) HOXA1 were analyzed in the xenograft tumors using reverse transcription-quantitative PCR. (G) HOXA1 expression levels were analyzed in xenograft tumors using immunohistochemistry. All experiments were performed in triplicate and data are expressed as the mean ± SD. *P<0.05, **P<0.01. AFAP1-AS1, actin filament-associated protein 1 antisense RNA 1; miR, microRNA; HOXA1, homeobox A1.
Discussion
Over the last several decades, accumulating evidence has revealed that lncRNAs are involved in the tumorigenesis of OSCC through the regulation of several biological characteristics of OSCC (9,10). Therefore, an improved understanding of the activities of cancer-related lncRNAs in OSCC may help determine potential novel targets for the diagnosis, prevention and treatment of the disease. In the present study, AFAP1-AS1 expression levels were revealed to be upregulated in OSCC tissues, and that knockdown of AFAP1-AS1 inhibited the progression of OSCC.AFAP1-AS1 has been reported to exert oncogenic actions during the tumorigenesis of multiple types of cancer (11–17). For example, AFAP1-AS1 promoted non-small cell lung cancer cell proliferation and invasion by upregulating interferon regulatory factor 7 expression levels and the RIG-I-like receptor signaling pathway (13). In addition, the upregulation of AFAP1-AS1 expression levels was revealed to promote breast cancer tumorigenesis and cell invasion by regulating the Wnt/β-catenin signaling pathway (12). AFAP1-AS1 also promoted the tumorigenesis and epithelial-mesenchymal transition of osteosarcoma by regulating the RhoC/Rho-associated protein kinase 1/p38MAPK/Twist1 signaling pathway (24). However, although a previous study reported that AFAP1-AS1 expression levels were upregulated in OSCC (18), the involvement of AFAP1-AS1 in OSCC progression remains elusive. The present study analyzed the expression levels of AFAP1-AS1 in OSCC tumor tissue and cell lines, and revealed that its expression levels were upregulated in OSCC tissues and cell lines. The clinical significance of AFAP1-AS1 in patients with OSCC was also investigated in further detail; the upregulated expression levels of AFAP1-AS1 were significantly associated with an advanced clinical stage, lymph node metastasis and a poor prognosis in patients with OSCC. In addition, the specific functions of AFAP1-AS1 with respect to OSCC progression were determined, and it was revealed that AFAP1-AS1 knockdown significantly decreased OSCC cell proliferation, migration and invasion in vitro, and inhibited tumor growth in vivo.Accumulating evidence has suggested that cytoplasmic lncRNAs may exert a biological role by interacting with certain miRNAs, and then regulating the expression of the target genes of those miRNAs (22,23,25). The results of the present study identified that AFAP1-AS1 was mainly located in the cytoplasm of SCC9 cells. To investigate the regulatory mechanisms by which AFAP1-AS1 affected the malignant characteristics of OSCC cells in vitro and in vivo, LncBaseV3 software was used to identify potential miRNAs that could bind with AFAP1-ASI. miR-145 was further selected as the study focus based on its previously reported role in cancer progression (26,27); for example, miR-145 expression levels were revealed to be downregulated in multiple types of cancer, including OSCC, where it functioned as a tumor suppressor (19,28–30). The present study validated that AFAP1-AS1 could bind with miR-145 and regulate its expression levels in OSCC cells. Notably, miR-145 inhibition reversed the inhibitory effect induced by AFAP1-AS1 knockdown in SCC9 cells. These results suggested that AFAP1-AS1 may serve an oncogenic role in OSCC cells by functioning as a ceRNA for miR-145.HOXA1 was previously confirmed as a direct target of AFAP1-AS1 in OSCC cells (19). HOXA1, an important member of the HOXA family, was reported to be upregulated in multiple types of malignant cancer and was closely associated with tumor progression and a poor prognosis (31,32). In particular, HOXA1 expression levels were found to be highly upregulated and associated with a poor prognosis in patients with OSCC (33). In addition, HOXA1 could promote OSCC carcinogenesis by increasing tumor cell proliferation and invasion. The results of the present study demonstrated that the knockdown of AFAP1-AS1 significantly downregulated the expression levels of HOXA1 in OSCC cells, while the knockdown of miR-145 reversed this trend. In addition, HOXA1 expression levels were determined to be upregulated in OSCC tissues, and a positive correlation was identified between HOXA1 and miR-145 expression levels. These results suggested that AFAP1-AS1 may regulate the expression levels of HOXA1 by sponging miR-145.In conclusion, the findings of the present study revealed that AFAP1-AS1 expression levels were upregulated and closely associated with poorer clinical parameters and a poor prognosis in patients with OSCC. The knockdown of AFAP1-AS1 expression was revealed to reduce the aggressive behavior of OSCC cells in vitro and in vivo through the miR-145/HOXA1 axis. However, the study has several limitations and in future studies, gain-of-function experiments should be performed to investigate the biological function of AFAP1-AS1 in OSCC. Secondly, AFAP1-AS1 could target multiple miRNAs to regulate its role in OSCC, therefore other potential target miRNAs should be investigated. Overall, these findings suggested that the AFAP1-AS1/miR-145/HOXA1 axis may serve a crucial role in the progression of OSCC, and that targeting this axis may be an effective strategy for treating patients with OSCC.
Authors: Carolina Cavalcante Bitu; Maria Fernanda de Souza Setúbal Destro; Manoela Carrera; Sabrina Daniela da Silva; Edgard Graner; Luiz Paulo Kowalski; Fernando Augusto Soares; Ricardo D Coletta Journal: BMC Cancer Date: 2012-04-12 Impact factor: 4.430
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