Sage Journals: Discover world-class research

Abstract

Oral squamous cell carcinoma is a common and lethal malignancy affecting the head and neck region. CCAT2 (colon cancer–associated transcript 2) gene is affiliated with long non-coding RNAs, which are often found to have important regulatory roles in cancers. This study aims to assess the expression and clinical significance of CCAT2 gene, identify its malignant biological behaviors, and explore the possible mechanisms in oral squamous cell carcinoma. CCAT2 expression was detected by quantitative real-time polymerase chain reaction, and its relationship with clinical factors was assayed using the Kaplan–Meier survival curve. The biological behaviors of CCAT2 and its potential mechanisms in oral squamous cell carcinoma were explored by the combined use of CCAT2 knockdown technology and the Wnt/β-catenin pathway agonist lithium chloride (LiCl). Our results showed that CCAT2 functioning as a potential oncogene was upregulated in oral squamous cell carcinoma. CCAT2 with high expression level was correlated with poor differentiation, higher T stage, and clinical stage, which made CCAT2 to be a prognostic biomarker in oral squamous cell carcinoma. LiCl-activated Wnt/β-catenin signaling pathway could partly restore the CCAT2-mediated malignant biological behaviors of oral squamous cell carcinoma cells by suppressing β-catenin, CCND1, and MYC and activating glycogen synthase kinase 3 beta expression. These findings might assist in the discovery of novel potential diagnostic and therapeutic target for oral squamous cell carcinoma, thereby improve the effects of clinical treatment in patients.

Keywords

Long non-coding RNA colon cancer–associated transcript 2 oral squamous cell carcinoma malignant behaviors lithium chloride Wnt/β-catenin signaling pathway

Introduction

Oral cancer is the sixth most common and lethal malignancy of the head and neck. Each year, approximately 11 in 100,000 adults are diagnosed with this type of cancer. Oral squamous cell carcinoma (OSCC), as a representative histological classification of oral cancer, is characterized by invasive growth and regional metastases at diagnosis, which usually caused the poor prognosis.^1,2 Although tremendous advances in the diagnoses and therapeutics including surgical treatment and chemotherapy had been modified recent years, the OSCC morbidity is continuously increasing worldwide, and the patients’ 5-year survival rate is still dismal about 50%.³ Thus, it is urgent and important to find novel and suitable markers for early detection or therapy.

Long non-coding RNAs (lncRNAs) have recently attracted more attention about its role in many biological processes in a variety of diseases, including cancer.^4–6 With the development of bioinformatics and functional genomics studies, accumulating lncRNAs were discovered in abnormal expression of some malignant tumors, and their anomalous functions were related to the tumorigenesis and progression by functioning as oncogenes and tumor suppressors.^7–9 Colon cancer–associated transcript 2 (CCAT2), a novel non-coding RNA, mapped onto 8q24, was originally identified in colon cancer by Ling et al.¹⁰ Recently, CCAT2 gene had been found to be highly expressed in some tumors, including non–small cell lung cancer, esophageal carcinoma, cervical cancer, and bladder cancer, which revealed that CCAT2 functions as an oncogene in these tumors.^11–15 Our microarray assays showed that CCAT2 had over eight-fold upregulation in OSCC tissues than that in adjacent normal tissues (NTs). The result suggested that CCAT2 might be involved in OSCC carcinogenesis. However, the functional roles and regulatory mechanisms of CCAT2 in OSCC are still ambiguous.

The Wnt/β-catenin signaling pathway plays an important role in human diseases, especially in cancer by affecting growth, development, and metabolism, of which β-catenin is one of the key downstream effectors.^16–20 In the canonical Wnt/β-catenin pathway, Wnt first inactivates the β-catenin destruction complex of adenomatous polyposis coli (APC), axin, and glycogen synthase kinase 3 beta (GSK-3β). Then, β-catenin is released from the complex and translocated into nucleus. The nuclear β-catenin interacts with lymphoid-enhancing factor/T-cell factor (LEF/TCF) family that activates target gene transcription. Accumulating evidences have shown that the Wnt/β-catenin signaling pathway is frequently activated during carcinogenesis, including OSCC.²¹ However, the interaction between CCAT2 and Wnt/β-catenin signaling in OSCC has not been explored. Further delineation of the mechanisms underlying the activation of Wnt/β-catenin signaling in OSCC is of great interest.

Lithium chloride (LiCl) is a well-known Food and Drug Administration–approved drug with antimanic properties for 60 years. It also has carcinogenic and anti-carcinogenic properties by inhibiting GSK-3β, an agonist to activate the Wnt/β-catenin signaling.²² In this study, we emphasized the pivotal role of CCAT2 in OSCC carcinogenesis and prognosis, explored the CCAT2 gene participation in Wnt/β-catenin signaling pathway activation by combined use of RNA knockdown technology and GSK-3β agonist LiCl, which might be helpful to improve the effects of clinical treatment in OSCC.

Material and methods

Clinical specimens

Total paired OSCC tissues and adjacent NTs (n = 62) were gathered from Affiliated Stomatological Hospital of China Medical University between February 2011 and December 2011. Collection criteria were as follows: (1) all cases were at an initial diagnosis of OSCC and had undergone no previous treatment; (2) NTs defined as normal tissues from the tumor boundary more than 3 cm (as far as possible) were collected first; and then (3) OSCC tissues with a size of soybean seeds (5 mm × 5 mm) were collected. The clinical pathological data were affirmed by two professional pathologists after surgery. The Ethics Committees of China Medical University approved this study, and permissions of surgical patients were achieved before operation. Tissue samples were immediately frozen in liquid nitrogen and stored at −80°C until use.

Cell culture

The cell lines used in this study (human OSCC Tca8113, Cal27, and normal oral keratinocyte hNOK) were purchased from the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 (Gibco, NY) medium supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 mg/mL of streptomycin (Gibco) in humidified air at 37°C with 5% CO₂. All cells in exponentially growing phase were used in the following experiments.

Quantitative real-time polymerase chain reaction

Total RNA was extracted from the cells or tissues using TRIzol^® reagent (Life Technologies, CA), and the first strand complementary DNA (cDNA) was synthesized with the Reverse Transcription Kit (Applied Biosystems, CA) in accordance with the manufacturer’s protocol. One Step SYBR RT-PCR Kit (TaKaRa, Otsu, Japan) was used to measure the original amount of CCAT2 through the 7500 Real-Time PCR System (Applied Biosystems), and the comparative Ct method was performed to calculate the relative abundance of messenger RNA (mRNA) compared with that of the endogenous reference control glyceraldehyde 3-phosphate dehydrogenase (GAPDH). CCAT2 primers were 5′-AGACAGTGCCAGCCAACC-3′ (sense) and 5′-TGCCAAACCCTTCCCTTA-3′ (antisense). GAPDH primers were 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ (sense) and 5′-ACCCTTCTCCATGGTGGTGAAGAC-3′ (antisense).

Vector construction and cell transfection

The silencer vector pSilencer 4.1-CMV puro-CCAT2 (pS-CCAT2) was synthesized by GenScript (Nanjing, China). To brief, the whole cDNA of CCAT2 gene labeled with BamHI and HindIII enzyme cutting sites was chemically synthesized, followed by cloned into pSilencer 4.1-CMV puro plasmid. The pSilencer 4.1-CMV puro-NC (pS-NC) containing a negative control small interfering RNA (siRNA) template (55 base pairs) between the BamHI and HindIII was provided with the pSilencer 4.1-CMV puro kit (Invitrogen, MA). After sequencing with the primer 5′-AGGCGATTAAGTTGGGTA-3′, the plasmids were purified and transfected into cells with Lipofectamine^TM 3000 (Invitrogen) according to the manufacturer’s instructions. G418 (Invitrogen) was used to establish stable cell lines, and quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the transfection effects.

Experimental design

Human OSCCs Tca8113 and Cal27 were incubated with 20 mmol/L of LiCl for 1 h at 37°C in a humidified atmosphere of 5% CO₂, according to the already published reference.²¹ The four treatment groups (pS-NC, pS-CCAT2, LiCl (20 mmol/L), pS-CCAT2 + LiCl (20 mmol/L)) were evaluated for cell proliferation, apoptosis, invasion ability, and Wnt/β-catenin signaling pathway transcriptional activity (TOP/FOP Flash luciferase reporter assay for TOP/FOP ratio and western blot assay for β-catenin, GSK-3β, CCND1, and MYC).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) can specifically detect living cells and form formazan deposits in the cell. The cell proliferation ability was assayed by the MTT method. In detail, 2 × 10⁴ cells/well were seeded on 96-well plate, and then, 20 μL of 0.5 mg/mL of MTT solution was added. After incubating at 37°C for 4 h, the supernatant was removed carefully, and the wells were added with 0.2 mL of dimethyl sulfoxide (DMSO) for 30 min to dissolve the formazan crystals. Absorbance at 490 nm was recorded using the Infinite F200 microplate reader (Tecan, Switzerland).

Flow cytometry detection

The apoptosis detection was carried out with Annexin V–FITC apoptosis detection kit (Biosea, Beijing, China) through the flow cytometry. In detail, 1 × 10⁶ cells/mL were digested with trypsin as usual. After washing with cold phosphate-buffered saline (PBS), the cells were resuspended in 1× binding buffer provided with the kit for 10 min. Then, 100 μL of cell suspension was incubated with 2.5 μL of fluorescein isothiocyanate (FITC)–Annexin V and 2.5 μL of propidium iodide (PI) for 10 min in the dark at room temperature. The reaction was terminated with the addition of 400 μL of 1× binding buffer and analyzed with FACSCanto II using the Diva 8.0 software (BD Biosciences, USA) according to the manufacturer’s protocol. Cells in the right lower quadrant (FITC–Annexin V positive and PI negative) were regarded as apoptotic, and the experiments were carried out in triplicates.

Transwell invasion assay

Cell invasion ability was assayed using Transwell chamber (Costar, USA) according to the manufacturer’s protocol. In detail, cells were trypsinized and 2 × 10⁵ cells/well were seeded onto the upper chambers in a 25 μL of serum-free medium. The lower chambers were filled with 0.5 mL supernatant of human NIH3T3. After incubating for the appropriate time at 37°C and with 5% CO₂, the cells in the upper chambers were removed by cotton tip. Invading cells at the bottom of the filter were fixed, stained with hematoxylin & eosin, and photographed under a light microscope. The number of invading cells was quantified by counting five randomly fields in each chamber.

Luciferase assay

The TOP Flash (wild type (wt) TCF binding sites, Catalog # 21-170) luciferase reporter plasmid along with FOP Flash (mutant TCF binding sites, Catalog # 21-169) purchased from Biovector NTCC Ltd (Beijing, China) enables quantitation of Wnts signaling in cells transfected with these constructs. For reporter assays, cells were transiently co-transfected with luciferase plasmid and CCAT2 silencer vector using Lipofectamine^TM 3000 (Invitrogen), followed by treated with 20 mmol/L LiCl for 1 h. Reporter assays were performed 48 h post-transfection using Dual-Lucy Assay Kit from Vigorous Biotech (Beijing, China), with firefly luciferase used as a base line and renilla luciferase used as the internal control.

Western blotting

Proteins were extracted by radioimmunoprecipitation assay (RIPA; Beyotime, Shanghai, China) lysis buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl (pH 7.4)), and the concentration was determined using the bicinchoninic acid (BCA) assay (Beyotime) according to manufacturer’s instructions. Totally, 30 μg of protein was subjected to 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a 0.22 μm polyvinylidene difluoride (PVDF) membrane (Millipore, MA). The membrane was blocked in 5% powdered non-fat milk in Tris-buffered saline (TBS) containing 0.05% Tween 20 for 1 h. After incubating with β-catenin, GSK-3β, CCND1, and MYC antibodies (Cell Signaling Technology, MA) on a shaker for overnight at 4°C, signals were visualized using the infrared-labeled secondary antibody and scanned with the Dual Color Infrared Laser Imaging System (Gene, Hong Kong, China) by normalizing to the inference gene of GAPDH (Cell Signaling Technology). The intensity of the indicated bands was quantified using ImageJ (National Institutes of Health, MD).

Statistical analysis

All data were showed as mean ± standard deviation (SD) of three independent experiments and analyzed with SPSS 21.0 software (IBM, NY). Data from surgical resection to overall survival time of the patients were analyzed using the Kaplan–Meier survival curve. The difference in comparisons between them was analyzed using one-way analysis of variance (ANOVA) and paired Student’s t test. p < 0.05 means statistically significant difference.

Results

CCAT2 expression is upregulated in OSCC

To explore the role of CCAT2 in OSCC, we analyzed the CCAT2 expression by lncRNAs array. The results from assay showed that in four pairs of OSCC tissues (T) and their corresponding adjacent NTs (P), CCAT2 showed an over eight-fold upregulation in OSCC tissues than that in the P tissues (Figure 1(a), p < 0.05). Furthermore, we expanded our investigation to clinical samples and cell lines in order to assess the expression level of CCAT2 in 62 human OSCC and in OSCC cell lines Tca8113 and Cal27 by qRT-PCR. As shown in Figure 1(b), CCAT2 expression level was found to be markedly increased both in OSCC tissues and cell lines as compared with the adjacent NTs and hNOK cells. These results suggest that CCAT2 expression is upregulated in human OSCC and may play a role in OSCC carcinogenesis.

Figure 1.

CCAT2 was upregulated in OSCC. (a) Representative microarray analysis of CCAT2 in four OSCC tissues and adjacent NTs (1T–4T: OSCC tissues, 1P–4P: NTs). (b) qRT-PCR assay of the CCAT2 expression levels in OSCC tissues (n = 62) and OSCC cells. (c) Kaplan–Meier overall survival curves by CCAT2 expression level (*p < 0.05).

CCAT2 is an independent prognostic biomarker for OSCC patients

To further explore the prognostic value of CCAT2, we divided the patients with OSCC into two groups according to CCAT2 expression levels (high expression and low expression) and assessed the association between the CCAT2 expression level and overall survival through Kaplan–Meier analysis and log-rank test. Clinical characteristic analysis showed that increased CCAT2 expression was positively correlated with poor differentiation grade (p < 0.001), higher T stage (p = 0.0354), and clinical stage (p = 0.0168). However, CCAT2 expression was not associated with other parameters such as gender (p = 0.5057), age (p = 0.8907), smoking (p = 0.2778), drinking (p = 0.5626), position (p = 0.1337), and N stage (p = 0.1138) in OSCC (Table 1). These findings provided initial evidence that CCAT2 may play an important role in the OSCC progression. Furthermore, Kaplan–Meier survival analysis revealed that patients with high CCAT2 expression level had a significantly poorer overall survival than those with low expression level (p = 0.0287, Figure 1(c)). These results further verified that CCAT2 was an independent prognostic biomarker for OSCC patients.

Table 1.

Correlation between CCAT2 expression and clinical parameters in OSCC patients.

Factors	n	Mean ± SD	p value
Gender
Male	40	3.31 ± 0.52	0.5057
Female	22	3.22 ± 0.48	0.5057
Age (years)
⩽50	15	3.26 ± 0.45	0.8907
>50	47	3.28 ± 0.50	0.8907
Smoking
Yes	23	3.34 ± 0.39	0.2778
No	39	3.24 ± 0.32	0.2778
Drinking
Yes	15	3.33 ± 0.39	0.5626
No	47	3.26 ± 0.41	0.5626
Position
Tongue	31	3.29 ± 0.41	0.1337
Gingiva	15	3.41 ± 0.36
Buccal mucosa	8	3.24 ± 0.44
Lip	3	3.34 ± 0.42
Palate	5	2.86 ± 0.33
Differentiation
Well/moderately	44	2.73 ± 0.53	<0.001*
Poorly	18	4.62 ± 0.62	<0.001*
T stage
T1 and T2	41	3.17 ± 0.49	0.0354*
T3 and T4	21	3.48 ± 0.62	0.0354*
N stage
N0	38	3.19 ± 0.53	0.1138
N1–N3	24	3.42 ± 0.58	0.1138
Clinical stage
I and II	32	3.11 ± 0.46	0.0168*
III and IV	30	3.46 ± 0.65	0.0168*

CCAT2: colon cancer–associated transcript 2; OSCC: oral squamous cell carcinoma; SD: standard deviation.

Significant difference.

CCAT2 silencing inhibits the malignant biological behaviors of OSCC cells

As for the important role of CCAT2 in OSCC, we further investigated the biological role of CCAT2 in OSCC cells by loss of function. Tca8113 and Cal27 cells were stably transfected with pS-CCAT2/pS-NC/Lipofectamine^TM 3000 control, and qRT-PCR was performed to confirm the downregulation of CCAT2. The results showed that cells transfected with pS-CCAT2 presented a significantly decreased CCAT2 expression level compared with the pS-NC group in both cells, while there was no statistical difference between Lipofectamine^TM 3000 control group and pS-NC group (Figure 2(a), p < 0.05). The MTT assay showed that CCAT2 silencing in Tca8113 and Cal27 cells significantly inhibited the MTT response as compared with that of pS-NC control cells (Figure 2(b) and (c), p < 0.05). This result demonstrates that CCAT2 silencing in OSCC cells is correlated with impaired cell growth. Flow cytometry assay showed that apoptotic rate of cells transfected with pS-CCAT2 was notably elevated compared with the pS-NC control group (Figure 2(d) and (e)). Transwell assays showed that the numbers of CCAT2 silenced cells in the bottom well were significantly reduced compared with the pS-NC control cells, which indicated that CCAT2 might suppress the cell invasion (Figure 2(f) and (g), p < 0.05). Taken together, these results suggest that CCAT2 silence could inhibit the malignant biological behaviors of human OSCC cells.

Figure 2.

CCAT2 and Wnt signaling agonist LiCl affect the malignant potential in OSCC cells. (a) qRT-PCR assay of CCAT2 expression levels after pS-CCAT2 or pS-NC transfection. (b and c) MTT assay analyzing the cell proliferation ability in Tca8113 and Cal27 cells. (d and e) Flow cytometry analysis of apoptosis in Tca8113 and Cal27 cells. (f and g) Transwell chamber assay analyzing the invasion ability of Tca8113 and Cal27 cells (magnification: 200×; *p < 0.05 vs pS-NC; ^#p < 0.05 vs pS-CCAT2).

CCAT2 silencing inhibits Wnt/β-catenin signaling pathway

Next, we sought to explore the driving mechanisms behind the effects of CCAT2 on OSCC cell proliferation, apoptosis, as well as invasion. We first predicted the probability of adjacent mRNA by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and found that there was a positive relationship between CCAT2 and Wnt/β-catenin activation. Together with the published references,²³ we have chosen Wnt/β-catenin signaling pathway as a target to highlight the CCAT2-associated malignant behavior in OSCC.

To further confirm whether CCAT2 regulates the Wnt/β-catenin signaling pathway, luciferase assay of TCF reporter (TOP/FOP) was conducted. As shown in Figure 3(a), pS-CCAT2 transfection could significantly suppress the TOP/FOP ratio, which indicated that CCAT2 silencing inhibited the Wnt/β-catenin signaling pathway transcriptional activity in Tca8113 and Cal27 cells. As expected and shown in Figure 3(b)–(e), CCAT2 silencing could decrease the expression of β-catenin in both cytoplasm and nucleus in comparison with the control cells. Next, we explored the mechanisms of CCAT2 regulating the β-catenin signaling. We found that CCAT2 silencing increased the expression of GSK-3β (Figure 3(b)–(e)), suggesting that GSK-3β mediates the modulatory role of CCAT2 in Wnt/β-catenin signaling in OSCC. Moreover, CCAT2 silencing reduced the expression of CCND1 and MYC (classic downstream genes of the Wnt/β-catenin signaling pathway) in both Tca8113 and Cal27 cells (Figure 3(b)–(e)). Collectively, these data indicate that Wnt/β-catenin signaling is a target downstream of CCAT2 in OSCC.

Figure 3.

CCAT2 and LiCI regulate Wnt/β-catenin signaling in OSCC cells. (a) TOP/FOP Flash luciferase reporter assay of Wnt/β-catenin signaling activity in OSCC cells. (b–e) Western blot assay was used to detect the level of GSK-3β, β-catenin, CCND1, and MYC in OSCC cells (*p < 0.05 vs pS-NC; ^#p < 0.05 vs pS-CCAT2).

Activition of Wnt/β-catenin signaling partly restores CCAT2 effects on OSCC cells

To better understand the CCAT2-regulated Wnt/β-catenin signaling in OSCC cells, 20 mmol/L LiCl was used to examine whether activation of Wnt/β-catenin signaling could restore CCAT2 effects on OSCC cells. Using the pS-CCAT2 group and the Wnt/β-catenin signaling agonist LiCl group of the Tca8113 and Cal27 cells, as well as pS-CCAT2 combined with LiCl group, we found that LiCl could not only affect the malignant biological behaviors of human OSCC cells by promoting cell proliferation and invasion and inhibiting cell apoptosis but also could activate the Wnt/β-catenin signaling pathway transcriptional activity, increase the β-catenin recruitment, promote the classic downstream genes of the Wnt/β-catenin signaling pathway CCND1 and MYC expression, and reduce the expression of GSK-3β (Figures 2(b)–(g) and 3(a)–(e)).

While combining the use of pS-CCAT2 and LiCl, both Tca8113 and Cal27 cells showed significantly increased cell proliferation and invasion abilities and reduced cell apoptosis ability compared with those of pS-CCAT2 group. However, pS-CCAT2 and LiCl could not restore the biological abilities to the original level of LiCl group (Figure 2(b)–(g)). These results indicated that LiCl could partly reverse the CCAT2-mediated malignant biological behaviors of OSCC cells.

In addition, the TOP/FOP Flash luciferase reporter assay and western blot assay were employed in Tca8113 and Cal27 cells. The results showed that combination of pS-CCAT2 and LiCl could partly increase the Wnt/β-catenin signaling pathway transcriptional activity, elevate the β-catenin recruitment, increase the downstream genes CCND1 and MYC expression, and reduce the expression of GSK-3β compared with the pS-CCAT2 group, although the levels were not back to the LiCl group (Figure 3(a)–(e)). Thus, these results suggest that activation of Wnt/β-catenin signaling pathway could partly restore the CCAT2-mediated malignant biological behaviors of OSCC cells by suppressing β-catenin and activating GSK-3β expression.

Discussion

LncRNAs were novel class molecules with more than 200 nucleotides, which generally lack an open reading frame and are involved, in numerous ways, in many cancers. Recently, lncRNAs are emerging as convenient and minimally invasive diagnostic/prognostic/therapeutic markers. Some lncRNAs such as PCA3 is now routinely used in the clinical diagnosis of prostate cancer.²⁴ CCAT2 was also discovered to be a prognostic biomarker but showed poor prognosis in prostate cancer, ovarian cancer, esophageal squamous cell carcinoma, and lung cancer.^14,25–27 In addition, Wang J reported that CCAT2 detected in serum of esophageal squamous cell carcinoma patients could be a potential serum prognostic biomarker of esophageal squamous cell carcinoma.²⁸ In our research, we presented that the expression of CCAT2 was significantly upregulated in OSCC tissues and cell lines, and the high expression of CCAT2 correlated with poor differentiation grade, higher T clinical stage, and lower overall survival time. These results also verified that CCAT2 could be an independent prognostic biomarker for OSCC patients.

As for the important clinical significance of CCAT2, we next explored the potential function of CCAT2 in OSCC by using MTT method, flow cytometry detection, and transwell chamber assay. At present, the oncogenic role of CCAT2 has been increasingly demonstrated in human cancers.²⁹ CCAT2 upregulation is frequently reported, and high expression of CCAT2 is often associated with tumor progression and poor clinical outcomes. Functionally, knockdown of CCAT2 could induce cancer cell apoptosis and suppress cell proliferation and invasiveness.^14,23,30 Our research showed that suppression of CCAT2 expression could inhibit the malignant biological behaviors of OSCC cells by inducing OSCC cell apoptosis and inhibiting OSCC cell proliferation and invasion, which revealed that CCAT2 also functions as an oncogene in the process of OSCC.

Recent literatures reported that lncRNAs could affect biological behaviors of malignant cancers by regulating some target gene expression, such as TUSC7 and miR-23b, and MEG3 and miR-21.^7,8 CCAT2 promoted proliferation and invasion by regulating the E-cadherin and LATS2 expression in gastric cancer and activating the Wnt/β-catenin signaling pathway in breast cancer.^12,23 Our GO and KEGG analysis showed that there was a positive relationship between CCAT2 and Wnt/β-catenin activation. The canonical Wnt signaling pathway, also known as the Wnt/β-catenin signaling pathway, likewise regulates a wide array of biological processes by inhibiting GSK-3β activity, thus allowing β-catenin to accumulate and enter the nucleus, where it associates with TCF/LEF, leading to the transcription of Wnt signaling genes.^16–20,31 Therefore, we got the hypothesis that CCAT2 might affect malignant biological behaviors of OSCC cells through Wnt/β-catenin pathway by regulating β-catenin and GSK-3β expression levels in OSCC, which are two key downstream effectors in the Wnt/β-catenin signaling pathway.

To further verify this, we used TOP/FOP Flash luciferase reporter assay and western blot assay to confirm that CCAT2 silencing inhibited the Wnt/β-catenin signaling pathway activity by suppressing β-catenin and promoting GSK-3β expression, followed by inhibiting the classic downstream genes of Wnt/β-catenin signaling pathway CCND1 and MYC expression. By combining used CCAT2 silencer pS-CCAT2 and the Wnt/β-catenin pathway agonist LiCl in both Tca8113 and Cal27 cells, we found that LiCl could partly restore the CCAT2 effects on OSCC cells. There were other mechanisms like epithelial–mesenchymal transition and allele-specific reprogramming that might regulate the CCAT2-associated biological behaviors in OSCC, which needs to be further explored.

In conclusion, our results clarify that CCAT2 is upregulated in OSCC tissues and cell lines and correlated with poor differentiation in OSCC patients, which made CCAT2 to be a prognostic biomarker. CCAT2 silencing induced OSCC cell apoptosis and suppressed cell proliferation and invasion abilities. Furthermore, CCAT2-mediated oncogenic effects are partially through Wnt/β-catenin signaling pathway. LiCl-activated Wnt/β-catenin signaling pathway could partly restore the effects by suppressing β-catenin, CCND1, and MYC and activating GSK-3β expression. Our findings elucidate a potential mechanism underlying the tumor-oncogenic role of CCAT2 in OSCC and aid in the discovery and design of more effective and evidence-based Wnt/β-catenin signaling personalized therapy in OSCC.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (No. 81301834).

References

de Morais

Mafra

Gonzaga

. Prognostic factors of oral squamous cell carcinoma in young patients: a systematic review. J Oral Maxillofac Surg. Epub ahead of print 21 December 2016. DOI: 10.1016/j.joms.2016.12.017.

Krishna Rao

Mejia

Roberts-Thomson

. Epidemiology of oral cancer in Asia in the past decade—an update (2000–2012). Asian Pac J Cancer Prev 2013; 14: 5567–5577.

Siegel

Naishadham

Jemal

Cancer statistics, 2013. CA Cancer J Clin 2013; 63(1): 11–30.

Viereck

Thum

Circulating noncoding RNAs as biomarkers of cardiovascular disease and injury. Circ Res 2017; 120(2): 381–399.

Chen

. Association of nsv823469 copy number loss with decreased risk of chronic obstructive pulmonary disease and pulmonary function in Chinese. Sci Rep 2017; 7: 40060.

Zheng

Song

Xiao

. LncRNA GAS5 contributes to lymphatic metastasis in colorectal cancer. Oncotarget 2016; 7(50): 83727–83734.

Shang

Guo

Hong

. Long non-coding RNA TUSC7, a target of miR-23b, plays tumor-suppressing roles in human gliomas. Front Cell Neurosci 2016; 10: 235.

Zhang

Yao

Wang

. Long noncoding RNA MEG3 is downregulated in cervical cancer and affects cell proliferation and apoptosis by regulating miR-21. Cancer Biol Ther 2016; 17(1): 104–113.

Chen

. Detection and analysis of Wnt pathway related lncRNAs expression profile in lung adenocarcinoma. Pathol Oncol Res 2016; 22(3): 609–615.

10.

Ling

Spizzo

Atlasi

. CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer. Genome Res 2013; 23(9): 1446–1461.

11.

Zhao

Wang

. LncRNA CCAT2 promotes tumorigenesis by over-expressed Pokemon in non-small cell lung cancer. Biomed Pharmacother 2017; 87: 692–697.

12.

Wang

Liu

. Long non-coding RNA CCAT2 promotes gastric cancer proliferation and invasion by regulating the E-cadherin and LATS2. Am J Cancer Res 2016; 6(11): 2651–2660.

13.

Zhang

. Elevated expression of CCAT2 is associated with poor prognosis in esophageal squamous cell carcinoma. J Surg Oncol 2015; 111(7): 834–839.

14.

Jin

Zhang

. Roles of long non-coding RNA CCAT2 in cervical cancer cell growth and apoptosis. Med Sci Monit 2016; 22: 875–879.

15.

Zhuang

Liu

. shRNA targeting long non-coding RNA CCAT2 controlled by tetracycline-inducible system inhibits progression of bladder cancer cells. Oncotarget 2016; 7(20): 28989–28997.

16.

Lin

Angeli

Chung

. sFRP2 activates Wnt/β-catenin signaling in cardiac fibroblasts: differential roles in cell growth, energy metabolism, and extracellular matrix remodeling. Am J Physiol Cell Physiol 2016; 311(5): C710–C719.

17.

Rothe

Kanwal

Dietmann

. An Epha4/Sipa1l3/Wnt pathway regulates eye development and lens maturation. Development 2017; 144(2): 321–333.

18.

Chen

Luo

Xiang

. EZH2 promotes colorectal cancer stem-like cell expansion by activating p21cip1-Wnt/β-catenin signaling. Oncotarget 2016; 7(27): 41540–41558.

19.

Chopra

Al-Sammarraie

Lai

. Increased canonical WNT/β-catenin signalling and myxomatous valve disease. Cardiovasc Res 2017; 113(1): 6–9.

20.

Serafino

Sferrazza

Colini Baldeschi

. Developing drugs that target the Wnt pathway: recent approaches in cancer and neurodegenerative diseases. Expert Opin Drug Discov 2017; 12(2): 169–186.

21.

Liang

Zhang

Wang

. LncRNA, TUG1 regulates the oral squamous cell carcinoma progression possibly via interacting with Wnt/β-catenin signaling. Gene 2017; 608: 49–57.

22.

Kahn

Can we safely target the WNT pathway?

Nat Rev Drug Discov 2014; 13(7): 513–532.

23.

Cai

Zhang

Long noncoding RNA CCAT2 promotes breast tumor growth by regulating the Wnt signaling pathway. Onco Targets Ther 2015; 8: 2657–2664.

24.

Chandra Gupta

Nandan Tripathi

. Potential of long non-coding RNAs in cancer patients: from biomarkers to therapeutic targets. Int J Cancer. Epub ahead of print 7 December 2016. DOI: 10.1002/ijc.30546.

25.

Zheng

Zhao

. The up-regulation of long non-coding RNA CCAT2 indicates a poor prognosis for prostate cancer and promotes metastasis by affecting epithelial-mesenchymal transition. Biochem Biophys Res Commun 2016; 480(4): 508–514.

26.

Huang

Qing

Huang

. The long non-coding RNA CCAT2 is up-regulated in ovarian cancer and associated with poor prognosis. Diagn Pathol 2016; 11(1): 49.

27.

Chen

. LncRNA CCAT2 predicts poor prognosis and regulates growth and metastasis in small cell lung cancer. Biomed Pharmacother 2016; 82: 583–588.

28.

Wang

Qiu

. Long noncoding RNA CCAT2 correlates with smoking in esophageal squamous cell carcinoma. Tumour Biol 2015; 36(7): 5523–5528.

29.

Xin

Zheng

. CCAT2: a novel oncogenic long non-coding RNA in human cancers. Cell Prolif. Epub ahead of print 28 February 2017. DOI: 10.1111/cpr.12342.

30.

Hao

Qui

. High expression of long noncoding RNA CCAT2 indicates poor prognosis of gastric cancer and promotes cell proliferation and invasion. Minerva Med. Epub ahead of print 1 March 2017. DOI: 10.23736/S0026-4806.17.04703-6.

31.

Gordon

Nusse

Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem 2006; 281(32): 22429–22433.

Silencing of long non-coding RNA CCAT2 depressed malignancy of oral squamous cell carcinoma via Wnt/β-catenin pathway

Abstract

Keywords

Introduction

Material and methods

Clinical specimens

Cell culture

Quantitative real-time polymerase chain reaction

Vector construction and cell transfection

Experimental design

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay

Flow cytometry detection

Transwell invasion assay

Luciferase assay

Western blotting

Statistical analysis

Results

CCAT2 expression is upregulated in OSCC

CCAT2 is an independent prognostic biomarker for OSCC patients

CCAT2 silencing inhibits the malignant biological behaviors of OSCC cells

CCAT2 silencing inhibits Wnt/β-catenin signaling pathway

Activition of Wnt/β-catenin signaling partly restores CCAT2 effects on OSCC cells

Discussion

Footnotes

Declaration of conflicting interests

Funding

References