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Introduction

Oral squamous cell carcinoma (OSCC) is the most frequent malignant tumor occurring in the oral-maxillofacial region; OSCC ranks sixth in incidence among all types of tumors worldwide and represents nearly 3% of all newly diagnosed cancer cases (1). Although the diagnostic and therapeutic methods have been improved over the past two decades, the survival rate of patients with OSCC has not significantly increased, and the 5-year survival rate remains at <50% (2). As reported previously, OSCC is highly malignant, with strong local infiltration and metastasis, and its inevitable relapse and metastasis constitute the principal cause of mortality for patients with OSCC (3,4). Therefore, investigating the molecular mechanism of OSCC metastasis contributes to identifying an optimal therapeutic target to improve the prognosis of patients with OSCC.

Long non-coding RNAs (lncRNAs) are a class of functional RNA molecules with transcripts >200 nucleotides, which cannot be translated into proteins (5). In recent years, lncRNAs have been identified to be closely associated with tumor progression by affecting the growth, apoptosis, infiltration and metastasis of tumor cells (6). Meanwhile, epithelial-mesenchymal transition (EMT), one of the effective processes that endow tumor cells with the ability for invasion and metastasis, serves an important role in the invasion and metastasis of epithelium-originated tumors (7). It has been further confirmed that the presence of EMT markers in OSCC cells predicts high malignancy and a poor prognosis in patients with OSCC (8). Accumulating evidence has suggested that numerous lncRNAs in OSCC, including urothelial cancer-associated 1 (9), gastric carcinoma proliferation-enhancing transcript 1 (10) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) (11), may regulate the invasion and migration of tumor cells and affect the disease progression through mediating EMT.

Colorectal neoplasia differentially expressed (CRNDE), which is located at 16q12.2 on the long arm of human chromosome sixteen, was previously known as LOC388279 or LOC643911 (12). As demonstrated by the published literature, CRNDE exerts significant effects on the proliferation, invasion and migration of various types of malignant tumors, including colorectal cancer (13), ovarian cancer (14) and glioma (15). For instance, the study by Wang et al (16) detected the overexpression of CRNDE in glioma, which could facilitate the growth and migration of glioma cells in vivo and in vitro. Additionally, Chen et al (17) identified that CRNDE was also increased, upregulating the expression of nuclear factor-κB and p-protein kinase B (AKT) via the negative modulation of microRNA (miR)-384, thereby promoting hepatic carcinoma cell proliferation, migration and invasion (17). However, there was no evidence clearly demonstrating whether CRNDE influences the invasion and migration of OSCC cells through the regulation of the EMT process. Therefore, the present study was conducted to provide a novel perspective regarding the targeted treatment of OSCC in the hope of preventing recurrence and metastasis, and improving the prognosis of patients with OSCC.

Materials and methods

Ethics statement

The present study was conducted in accordance with the protocols in the Helsinki Declaration (18), and was approved by Clinical Trial Ethics Committee of Jingzhou Central Hospital (Jingzhou, China). All patients involved in the present study were informed of the experiments and provided written informed consent. The animal experiments were approved by the Ethics Committee of Jingzhou Central Hospital, The Second Clinical Medical College, Yangtze University (Jingzhou, China).

OSCC patients and experimental cell lines

Between April 2012 and October 2013, OSCC specimens were collected from 52 patients (including 35 males and 17 females, aged between 32 and 65 years with a mean age of 58.6±9.1 years) who received surgical excision in the Department of Stomatology at Jingzhou Central Hospital. Normal oral mucosa tissue specimens from 25 healthy individuals (including 16 males and 9 females, aged between 28 and 62 years with a mean age of 57.8±8.9 years) were obtained as the control group. None of the patients had received chemotherapy or radiation therapy prior to surgery. The tumor and normal tissues were examined and confirmed by three pathologists, and all the specimens were then preserved at −80°C until subsequent experimentation. The immortalized human oral keratinocyte (HOK) cell line (catalog no. BNCC340217) and the OSCC cell lines, Tca8113, SCC-9, TSCCA, CAL-27 and SCC-15, used in the present study were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in minimal essential medium (MEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 50 µg/ml streptomycin, and were incubated with 5% CO2 at 37°C. When cell confluence reached 80–90%, the cells were digested with 0.25% trypsin prior to passaging.

In situ hybridization (ISH)

ISH was performed on the basis of the manufacturer's protocol of a commercial ISH Detection kit (catalogue no. AR0149; Wuhan Boster Bio Technology, Ltd., Wuhan, China, http://www.boster.com.cn/product/ish_ar0149.html). In brief, the 4-µm thick paraffin-embedded sections were deparaffinized with xylene and rehydrated with 100, 90, 70 and 50% ethanol (5 min each) at room temperature. The samples were digested with proteinase K and fixed in 4% paraformaldehyde for 10 min at room temperature, followed by hybridization with a 5′-digoxin-labeled CRNDE probe (Wuhan Boster Bio Technology, Ltd.), which had the sequence 5′-CCTCAGTTGTCACGCAG-AAG-3′, at 55°C overnight and subsequent incubation with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (1:5,000; part of the ISH Detection kit) for 30 min at 4°C. Diaminobenzidine was used to develop the stain with a colorimetric reaction for 30 min at room temperature. The ISH-stained tissue sections were independently reviewed and scored by two pathologists in a blinded manner. Disagreements were resolved by a third pathologist. A simple and reproducible scoring system based on the intensity and proportion of CRNDE-positive cells was used according to a previous study (19).

Cell grouping and transfection

CAL-27 and SCC-15 cells were seeded at a density of 105 cells per well on a 6-well plate 24 h prior to transfection. Cells were assigned into three groups: The control group (non-transfected cells), the si-NC group (cells transfected with control siRNA) and the si-CRNDE group (cells transfected with CRNDE siRNA). The CRNDE siRNA and control siRNA used in the present study were provided by Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences were as follows: CRNDE siRNA forward, 5′-GUGCUCGAGUGGUUUAAAUTT-3′ and reverse, 5′-AUUUAAACCACUCGAGCACTT-3′; and control siRNA forward, 5′-GCGACGAUCUGCCUAAGAUTT-3′ and reverse, 5′-AUCUUAGGCAGAUCGUCGCTT-3′. The transfection of cells was performed with Lipofectamine™ 3000 (Thermo Fisher Scientific, Inc.) for a final concentration of 50 nM siRNA/well, according to the manufacturer's protocol. Cells were harvested 48 h post-transfection for further analyses.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from OSCC tumor and normal tissues, and CAL-27 and SCC-15 cells with a TRIzol reagent kit (Invitrogen; Thermo Fisher Scientific, Inc.), the optical density (OD)260/280 ratio was determined with an ultraviolet spectrophotometer, and the RNA concentration was then calculated. The RNA specimens were preserved at −80°C for later experiments. The primer design software Primer 5.0 (Premier Biocoft International, Palo Alto, CA, USA) was used to design the following primers: LncRNA CRNDE forward, 5′-CGCGCCCGCGCGGCGGAGGA-3′ and reverse, 5′-TATGAATTGCAGACTTTGCAGA-3′; and GAPDH forward, 5′-GTCAGCCGCATCTTCTTTTG-3′ and reverse, 5′ GCGCCCAATACGACCAAATC-3′. RT-PCR was performed on total RNA according to the protocols of the cDNA transcription kit (Thermo Fisher Scientific, Inc.), and RT-qPCR was performed following the steps for the SYBR Green PCR Master Mix kit (Takara Bio, Inc., Otsu, Japan). PCR conditions included an initial step of 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 55°C for 30 sec and 72°C for 30 sec. The PCR results were obtained and the relative expression of the target genes was calculated by the 2−ΔΔCq method (20), with GAPDH as the internal reference.

Western blotting

The concentrations of proteins extracted from the CAL-27 and SCC-15 cells were determined with a BCA kit (Wuhan Boster Biological Technology, Ltd.). Subsequent to the addition of SDS gel loading buffer (Wuhan Boster Biological Technology, Ltd.), the proteins were heated for 10 min at 95°C, and 30 µg protein was loaded into each well of a 10% polyacrylamide gel (Wuhan Boster Biological Technology, Ltd.). Next, electrophoresis was used for the separation of proteins, which were transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was later placed in 5% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 1 h at room temperature, followed by the addition of primary antibodies overnight at 4°C. The primary antibodies included GAPDH (1:500 dilution; catalog no. 5174; Cell Signaling Technology, Inc., Danvers, MA, USA), E-cadherin (1:1,000 dilution; catalog no. 3195; Cell Signaling Technology, Inc.), glycogen synthase kinase 3β (GSK-3β; 1:1,000 dilution; catalog no. ab93926; Abcam, Cambridge, UK), phosphorylated (p-)GSK-3β (1:1,000 dilution; catalog no. ab131097; Abcam), β-catenin (1:1,000 dilution; catalog no. ab16051; Abcam), vimentin (1:1,000 dilution; catalog no. SC-6260; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), N-cadherin (1:1,000 dilution; catalog no. SC-393933; Santa Cruz Biotechnology, Inc.) and Snail (1:1,000 dilution; catalog no. SC-393172; Santa Cruz Biotechnology, Inc.). The next day, the membrane was rinsed with Tris-buffered saline with Tween-20 (TBST) three times for 5 min each prior to the addition of the HRP-conjugated anti-mouse IgG secondary antibody (1:2,000; catalog no. HS201-01; Beijing Transgen Biotech Co., Ltd., Beijing, China) for 1 h of incubation at room temperature. Following incubation with the secondary antibody, the membrane was washed again with TBST three times for 5 min each. Finally, a chemiluminescence reagent was used for detection and visualization, and the gray values of the target bands were analyzed with ImageJ software version 1.43 (National Institutes of Health, Bethesda, MD, USA), with GAPDH as the loading control.

Immunofluorescence staining

Briefly, CAL-27 and SCC-15 cells cultured on cover slips were washed twice with cold PBS and fixed with cold methanol/acetone (1:1) for 10 min at −20°C. Following washing with PBS three times, the cells were blocked for 40 min at room temperature with 0.1% Triton X-100 and 2% normal donkey serum (Chemicon International; Thermo Fisher Scientific, Inc.) in PBS buffer, and incubated with the aforementioned specific primary antibodies, and were then stained at 37°C for 10 min with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody (1:500; catalog no. FI-4000; Vector Laboratories, Inc., Burlingame, CA, USA) secondary antibody. To visualize the nuclei, the cells were double-stained with 4,6-diamidino-2-phenylindole for 10 min at 37°C and viewed with a Nikon Eclipse 80i epifluorescence microscope, which was equipped with a digital camera (DS-Ri1, Nikon Corporation, Toyko, Japan). All immunofluorescence images were obtained with identical exposure settings.

Cell proliferation measurement by MTT assay

An MTT assay was used to detect cell proliferation. First, CAL-27 and SCC-15 cells in the logarithmic growth phase were obtained and seeded into 96-well plates at a density of 3,000 cells/well. Four replicate wells were established for each group. Subsequently, adherent cells were spread on the bottom and walls of the plate, MTT was detected at 0, 24, 48 and 72 h post-inoculation by adding 20 µl of 5 mg/ml MTT solution to each well and incubating the plate for 4 h at 37°C. The supernatant was discarded, 200 µl dimethyl sulfoxide (DMSO) was added, and, after 10 min of oscillation, the absorbance value (OD value) at a wavelength of 570 nm was detected by a microplate reader.

Cell apoptosis determination by flow cytometry

The rate of cell apoptosis was detected by an Annexin V kit (BD Biosciences, Franklin Lakes, NJ, USA). CAL-27 and SCC-15 cells were digested with an appropriate amount of trypsin to create a single cell suspension, which was centrifuged at 500 × g for 5 min at 37°C. Next, cells were washed with PBS three times for 5 min each. The supernatant was removed, 100 µl of buffer (provided in the kit) was added to each Eppendorf tube and 5 µl of propidium iodide (PI) and FITC was added to the tubes, which were then held at room temperature for 5 min, followed by the addition of 400 µl buffer in an ice bath for 30 min and the determination of cell apoptosis by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA). The data were analyzed by CELL Quest 3.0 software (BD Biosciences).

Cell migration examination by wound-healing assay

CAL-27 and SCC-15 cells (1×106 cells/well) were seeded in 6-well plates, and a vertical line was drawn across the middle of the plate with the head of a 200-µl transfer liquid gun. Cells were subsequently washed with PBS buffer three times, followed by the addition of 200 µl serum-free MEM. At 0 and 48 h post-incubation at 37°C, the plate was placed under an inverted microscope for observation and imaging. Cells were counted in five random fields (magnification, ×200). The distances between the edges of the scratches were measured by Image-Pro Plus 6.0 software (National Institutes of Health).

Cell invasion detection by Transwell assay

CAL-27 and SCC-15 cells obtained in the logarithmic growth phase were subjected to routine digestion and were adjusted to a cell density of 1×105 cells/ml with serum-free MEM. Next, the cells were inoculated into the upper chamber of a Transwell chamber with 100 µl/well Matrigel. After 24 h, the remaining cells and Matrigel on the upper chamber were removed with a cotton swab. Next, the cells that had migrated to the lower chamber, which contained 600 µl Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS, were fixed for 20 min with 4% paraformaldehyde at 37°C, washed with DPBS 3 times for 2 min each, stained with 0.1% crystal violet for 3–5 min at 37°C, and rinsed with DPBS 3 times for 2 min each. The polycarbonate membrane was sliced into pieces and fixed to a slide with neutral resin, which was inverted under a microscope (×200 magnification) for the observation of 3 randomly selected fields of view. The number of cells penetrating the membrane was counted prior to and following transfection in each group.

In vitro generation of xenograft tumors in nude mice

For this experiment, 15 BALB/c-nu (specific pathogen-free) male mice, aged 4–6 weeks and weighing 15–18 g, were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China). CAL-27 cells were transfected with short hairpin CRNDE (sh-CRNDE) plasmid or with empty vector according to the previously outlined transfection protocol and were harvested from 6-well plates, washed with PBS and resuspended at 2×107 cells/ml. The nude mice were divided into three groups (n=5 per group), namely the control group (implanted with CAL-27 cells only), the empty vector group (implanted with CAL-27 cells transfected with empty vector) and the sh-CRNDE group (implanted with CAL-27 cells transfected with sh-CRNDE plasmid). Subsequently, 100 µl of suspended cells were injected into the right flank of each mouse. Every 3 days, the length (a) and width (b) of the tumors were measured by Vernier calipers, and the tumors were observed for 6 weeks continuously to allow a tumor growth curve to be drawn. The volume of the tumors was calculated according to the following formula: V=(ab2)/2. Following the experiment, the animals were sacrificed and the tumor-bearing specimens were removed for weighing, formalin fixation, paraffin embedding and sectioning.

Detection of EMT-related proteins in tumor tissues of nude mice by immunohistochemistry

The avidin-biotin-peroxidase complex immunohistochemical method was utilized to evaluate the expression of EMT-related proteins in tumor tissues of nude mice. First, paraffin-embedded sections were dewaxed with 100, 90, 70, and 50% alcohol solutions (5 min each at 37°C), followed by heat-induced repair in 0.01 mol/l citrate buffer (pH 6.0), 20 min of endogenous peroxidase inhibition with 0.3% hydrogen peroxide, 30 min of incubation at room temperature in 20% normal goat serum (Vector Laboratories, Inc.) and overnight incubation at 4°C with the aforementioned primary antibodies, including E-cadherin, vimentin, N-cadherin and Snail. The sections were then incubated for an additional 1 h at 37°C, washed with 0.01 mol/l PBS and incubated for 20 min at 37°C with anti-mouse HRP-conjugated IgG secondary antibody (1:2,000; catalog no. HS201-01; Transgen Biotech Co., Ltd.). After development with 3,3′-diaminobenzidine reagent for 5 min at room temperature, sections were observed for staining under a light microscope. Finally, hematoxylin was used for 30 sec of counterstaining; sections were then rinsed with running water for 5 min, hyalinized and mounted with neutral resin prior to observation under a light microscope (magnification, ×400).

Statistical method

All data were analyzed with the statistical software package SPSS 21.0 (IBM Corp., Armonk, NY, USA). The results are presented as the mean ± standard deviation. Comparisons between two groups of data conforming to a normal distribution were conducted using unpaired Student's t-test, while the difference among multiple groups was analyzed by one-way analysis of variance followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Expression of CRNDE in human OSCC tissues and cell lines

The expression of CRNDE in human OSCC tissues and cell lines was detected by RT-qPCR. As illustrated in Fig. 1A, compared with normal oral mucosa tissues, human OSCC tissues exhibited a significant increase in CRNDE expression (P<0.05). Compared with immortalized HOK cells, the human OSCC cell lines, Tca8113, SCC-9, TSCCA, CAL-27 and SCC-15, displayed a statistically significant increase in the expression of CRNDE (all P<0.05; Fig. 1B). Among the OSCC cell lines, CAL-27 and SCC-15 presented with the most significant upregulation of CRNDE (P<0.05), therefore, these two cell lines were selected for subsequent experiments. In addition, the present study confirmed that CRNDE expression was markedly upregulated in OSCC tissues by in situ hybridization (Fig. 1C); CRNDE was identified to be primarily expressed in the cytoplasm, with expression in 80.77% (42/52) of the OSCC samples, but in only 16.00% (4/25) of the normal oral mucosa tissues.

Figure 1. Expression of lncRNA CRNDE in human OSCC tissues and cell lines. (A) The relative expression of lncRNA CRNDE in human OSCC and normal oral mucosa tissues, as determined by RT-qPCR. (B) The relative expression of lncRNA CRNDE in human HOK cells and the OSCC cell lines, Tca8113, SCC-9, TSCCA, CAL-27 and SCC-15, as determined by RT-qPCR. *P<0.05 vs. HOK cells. (C) Expression analysis of CRNDE in human OSCC tissues and normal oral mucosa tissues by in situ hybridization. Magnification, ×200. lncRNA, long non-coding RNA; CRNDE, colorectal neoplasia differentially expressed; OSCC, oral squamous cell carcinoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; HOK, human oral keratinocyte.

Expression of CRNDE and the effects of si-CRNDE on proliferation and apoptosis in each group of CAL-27 and SCC-15 cells

Following the silencing of CRNDE, RT-qPCR was performed to detect the transfection efficiency in each group of CAL-27 and SCC-15 cells. As illustrated in Fig. 2A and B the expression of CRNDE was significantly downregulated in the si-CRNDE group compared with that in the control and si-NC groups (P<0.05); however, no observable difference was identified between the control and the si-NC groups (P>0.05). In addition, cell proliferation and apoptosis were evaluated by an MTT assay and flow cytometry, respectively. Compared with the control group, the si-CRNDE group exhibited a marked decrease in cell proliferation and an appreciable increase in cell apoptosis (P<0.05; Fig. 2C-F); however, there was no significant difference between the control group and the si-NC group with respect to the proliferation and apoptosis rates at each time point (all P>0.05).

Figure 2. Expression of CRNDE and the effects of si-CRNDE on proliferation and apoptosis in each group of CAL-27 cells. Comparison of the relative expression of lncRNA CRNDE in (A) CAL-27 and (B) SCC-15 cells in each transfection group as determined by reverse transcription-quantitative polymerase chain reaction. The proliferation of (C) CAL-27 and (D) SCC-15 cells in each group following CRNDE silencing, as examined by an MMT assay. The apoptosis of (E) CAL-27 and (F) SCC-15 cells in each group following CRNDE silencing, as evaluated by flow cytometry. Comparison of the apoptosis rate in (D) CAL-27 and (H) SCC-15 cells in each transfection group. *P<0.05 vs. control group. si-NC, small interfering-negative control; CRNDE, colorectal neoplasia differentially expressed; lncRNA, long non-coding RNA; PI, propidium iodide; FITC, fluorescein isothiocyanate; OD optical density.

Effects of si-CRNDE on migration and invasion in each group of CAL-27 and SCC-15 cells

A wound-healing assay was used to detect the migration ability of each group of CAL-27 and SCC-15 cells. When the scratch lines at the 0 and 48 h time points were compared, it was evident that the migration ability of the CAL-27 and SCC-15 cells in the si-CRNDE group was significantly lower than that in the control and si-NC groups (P<0.05), and that the latter two groups exhibited no observable difference in cell migration (P>0.05) (Fig. 3A and B). In addition, the Transwell assay demonstrated that the number of invasive CAL-27 and SCC-15 cells in the si-CRNDE group was decreased significantly compared with that in the control and si-NC groups (P<0.05); however, there was no significant difference between the control group and the si-NC group with respect to the number of invasive cells (P>0.05) (Fig. 3C and D).

Figure 3. Effects of si-CRNDE on migration and invasion in each group of CAL-27 cells. Magnification, ×200. The migration ability of (A) CAL-27 and (B) SCC-15 cells in each group, as detected by a wound-healing assay. The invasion ability of (C) CAL-27 and (D) SCC-15 cells in each group, as detected by a Transwell assay. *P<0.05 vs. the control group. si-NC, small interfering-negative control; CRNDE, colorectal neoplasia differentially expressed.

Expression of proteins associated with the Wnt/β-catenin pathway or with EMT in each group of CAL-27 and SCC-15 cells

Western blotting was performed to detect the expression of proteins associated with the Wnt/β-catenin pathway or with EMT in each group of CAL-27 and SCC-15 cells following treatment with si-CRNDE (Fig. 4). Compared with the cells in the control and si-NC groups, those in the si-CRNDE group demonstrated significantly enhanced expression of E-cadherin protein (P<0.05), but a significant reduction in the expression of p-GSK-3β, β-catenin, N-cadherin, vimentin and Snail protein (all P<0.05). The expression of total GSK-3β protein did not exhibit a statistically significant difference among the three groups in the CAL-27 and SCC-15 cells. Furthermore, the control group was not significantly different from the si-NC group in terms of the expression of Wnt/β-catenin pathway-related proteins and EMT-associated proteins.

Figure 4. Expression of proteins associated with the Wnt/β-catenin pathway and EMT in each group of CAL-27 and SCC-15 cells following lncRNA CRNDE silencing, as detected by western blotting. (A) Wnt/β-catenin pathway and EMT-associated proteins were detected by western blotting. Comparison of the expression of proteins associated with the Wnt/β-catenin pathway and EMT in (B) CAL-27 and (C) SCC-15 cells. *P<0.05 vs. control group. EMT, epithelial-mesenchymal transition; lncRNA, long non-coding RNA; CRNDE, colorectal neoplasia differentially expressed; si, small interfering; NC, negative control; p-GSK-3β, phosphorylated glycogen synthase kinase 3β.

Effects of si-CRNDE on EMT in each group of CAL-27 and SCC-15 cells

To further investigate the effect of CRNDE targeting on EMT, immunofluorescence staining was performed to analyze the expression of epithelial and mesenchymal markers in CAL-27 and SCC-15 cells. As illustrated in Fig. 5, CAL-27 and SCC-15 cells in the control group displayed a phenotypic conversion, as demonstrated by the loss of E-cadherin and the induction of N-cadherin, vimentin and Snail. However, treatment with CRNDE siRNA resulted in an increase in E-cadherin, yet a decrease of N-cadherin, vimentin and Snail compared with the expression of these proteins in the control cells. These results suggest that downregulation of CRNDE led to a robust blockade of EMT-like transformation in CAL-27 and SCC-15 cells.

Figure 5. Inhibition of CRNDE suppresses EMT in CAL-27 and SCC-15 cells. CRNDE siRNA upregulated E-cadherin and downregulated N-cadherin, vimentin and Snail in CAL-27 and SCC-15 cells, as determined by immunofluorescence staining (×1,000 magnification). CRNDE, colorectal neoplasia differentially expressed; EMT, epithelial-mesenchymal transition; siRNA, small interfering RNA.

Effect of CRNDE on the growth of subcutaneous tumors and on EMT in nude mice

Tumors formed ~1 week after tumor cell inoculation in nude mice, with a tumor formation rate of 100%. Compared with the control group, the sh-CRNDE group exhibited a noticeably lower tumor growth rate and a significantly lighter tumor weight (P<0.05), while the empty vector group did not exhibit an apparent difference in the two indices (P>0.05) (Fig. 6A-C). Following the detection of EMT-associated proteins in tumor tissues of nude mice with immunohistochemical staining, the present study identified that, compared with the control group and the empty vector group, the sh-CRNDE group exhibited greatly increased expression of E-cadherin protein, but appreciably decreased expression of N-cadherin, vimentin and Snail protein (P<0.05) (Fig. 6D). However, the control group and the empty vector group were not noticeably different in terms of the expression of these EMT-related proteins (Fig. 6D).

Figure 6. Effect of lncRNA CRNDE on the growth of subcutaneous tumors and on EMT in nude mice. (A) Representative images of xenograft tumors from the nude mice. (B) The growth curves for subcutaneous tumors in each group of nude mice. (C) Comparison of tumor weights from each group of nude mice. *P<0.05 vs. the control group. (D) The expression of EMT-associated proteins in tumor tissues of each group, as detected by immunochemistry (×200 magnification). lncRNA, long non-coding RNA; CRNDE, colorectal neoplasia differentially expressed; EMT, epithelial-mesenchymal transition; sh, short hairpin.

Discussion

LncRNA CRNDE, was initially identified to be highly expressed in colorectal cancer, and has become an area of increasing focus for research; CRNDE, overexpressed in other types of tumor cells, may function as a novel oncogene through its effect on cellular proliferation, migration, invasion and apoptosis (12). For instance, in the study by Jing et al (15), CRNDE was apparently increased in glioma tissue samples, as determined by RT-qPCR, which could be considered an independent prognostic factor associated with the progression of glioma (15). In chronic lymphocytic leukemia, CRNDE was also detected to be upregulated, and this upregulation was negatively correlated with the CRNDE methylation level, and patients with hypermethylation of CRNDE had a significantly worse prognosis and survival rate than those with hypomethylation of CRNDE (21). In the present study, the expression of CRNDE was determined to be markedly increased in OSCC tissues and cell lines, which was consistent with the results of the aforementioned studies. In addition, Ellis et al (22) revealed that CRNDE was implicated in the epigenetic regulation of chromatin and that its transcriptional expression was regulated by insulin and insulin-like growth factors mediating two signaling pathways, namely, the phosphoinositide 3-kinase/AKT/mechanistic target of rapamycin (mTOR) and RAF proto-oncogene serine/threonine-protein kinase/mitogen-activated protein kinase pathways (22). Similarly, as indicated by Wang et al (16), CRNDE was the most upregulated lncRNA in gliomas owing to histone acetylation in the promoter region, which is also influenced by mTOR signaling (16); these findings further highlight the oncogenic role of CRNDE in cancer types such as OSCC.

To better understand the underlying mechanism of CRNDE in OSCC, CAL-27 cells were selected for transfection with CRNDE siRNA in vitro in the present study. As a result of CRNDE silencing, the proliferation, invasion and migration of the OSCC cells were noticeably reduced, while cell apoptosis was clearly elevated. Similar to the findings of the present study, the findings of Gao et al (23) demonstrated that CRNDE acted as a competing endogenous RNA to regulate the expression of miR-136 and that knockdown of CRNDE significantly inhibited the invasion and migration of colorectal cancer cells (23). Additionally, downregulation of CRNDE led to a reduction in piwi-like RNA-mediated gene silencing 4 protein and an elevation in miR-384 levels, consequently resulting in tumor regression (24). In addition, another important finding from the present study was that the silencing of CRNDE could reduce the expression of p-GSK-3β and β-catenin, downregulate the protein expression of N-cadherin, vimentin and Snail, and enhance the protein expression of E-cadherin, suggesting that the silencing of CRNDE may slow the EMT process in OSCC through the inhibition of the Wnt/β-catenin pathway and thereby inhibit the growth, invasion and migration of OSCC cells. Consistent with this possibility, as reported by Zhang et al (25), downregulation of lncRNA H19 in tongue squamous cell carcinoma (TSCC) cells was able to inhibit the activation of the β-catenin/GSK3β signaling pathway, thus increasing the expression of E-cadherin, decreasing the expression of N-cadherin, vimentin, Snail1, Twist1, and ZEB1, and eventually inhibiting tumor growth. Moreover, MALAT1 is also able to promote EMT in TSCC cells through the activation of the Wnt/β-catenin pathway (11).

EMT has been recognized as an inevitable stage of early tumor metastases and is characterized by common cytological changes, including downregulation of epithelial cell markers, including E-cadherin, upregulation of mesenchymal phenotype markers, including vimentin and N-cadherin, and the increased expression of EMT-inducing cytokines and transcription factors, including Snail, Slug and Twist, which results in reduced intercellular adhesion ability (26,27). During this process, the tumor cells gradually lose certain characteristics of epithelial cells and acquire the characteristics of interstitial cells, which have stronger migration and movement abilities, eventually giving rise to the proliferation and metastasis of tumor cells (28,29). Thus far, it has been demonstrated that the abnormal activation of the Wnt/β-catenin signaling pathway is involved in the occurrence of EMT (30). When the pathway is abnormally activated, Wnt binds to its receptor, frizzled protein (Frz), and acts on disheveled protein (Dsh) in the cytoplasm in order to block GSK-3β-mediated phosphorylation/degradation of β-catenin. The accumulated β-catenin in the cytoplasm then enters the nucleus to interact with T cell factor/lymphoid enhancer factor (TCF/LEF), thereby activating the expression of Snail (31,32). In addition, as an important transcription factor in the EMT process, Snail can bind to mSin3A and histone deacetylase to form a complex, which can further reduce the expression of E-cadherin and destroy the E-cadherin/β-catenin complex on the cell membrane, eventually enhancing the expression of β-catenin in the cytoplasm and promoting the transcription of TCF/LEF (33,34). Therefore, Snail can induce the occurrence of EMT, mediate the degradation of the extracellular matrix by matrix metalloproteinase-2/9 and mediate the transcription of numerous target genes, including c-myc and cyclin D1, thus regulating the proliferation, apoptosis, invasion and migration of cells (35). Notably, the Wnt/β-catenin pathway, which can induce the EMT process and promote the invasion and migration of tumor cells, has been confirmed to be activated in OSCC (36). In breast cancer cells, Huan et al (37) identified the critical role of CRNDE with regard to activation of the Wnt/β-catenin pathway via direct repression of miR-136. Meanwhile, CRNDE also promoted the development and chemoresistance of colorectal cancer through the regulation of Wnt/β-catenin signaling, as reported by Han et al (38). Considering the aforementioned findings, silencing CRNDE may regulate the downstream mRNAs and inhibit the activation of the Wnt/β-catenin pathway, thus suppressing the EMT process and regulating the proliferation, migration and invasion of OSCC cells. Nevertheless, the specific mechanism remains to be further studied in the future. Finally, through the tumor xenograft experiment, the present study found that the silencing of CRNDE could slow down the tumor growth rate and inhibit EMT in tumor-bearing nude mice, which further supported the hypothesis that CRNDE can affect tumor growth through the regulation of the EMT process.

In conclusion, the silencing of CRNDE may ameliorate the EMT process via inactivation of the Wnt/β-catenin signaling pathway, and may thus inhibit the growth but promote the apoptosis of human OSCC cells, which provides a novel theoretical basis for the targeted treatment of OSCC.

Acknowledgements

Not applicable.

Funding

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Author's contributions

JD and JWM contributed equally to this study. JD and JWM designed the study and analyzed the data. HM performed the experiments and generated the figures. JD and JWM drafted and revised the manuscript. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

The present study was approved by Clinical Trial Ethics Committee of Jingzhou Central Hospital (Jingzhou, China). All patients involved in the present study provided written informed consent. The animal experiments were approved by the Ethics Committee of Jingzhou Central Hospital, The Second Clinical Medical College, Yangtze University (Jingzhou, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References