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Introduction

Oral squamous cell carcinomas (OSCC) is one of the most common types of cancer, and its incidence is increasing worldwide (1). In spite of considerable advances being made in diagnostics and treatment, the 5-year survival rate for patients with OSCC has not improved during the past few decades and remains <50% (2). Although much scientific research has indicated that local recurrence and nodal metastasis are the major causes of mortality in patients with OSCC, the precise molecular mechanisms remain unclear (3–5). Therefore, in order to improve the diagnosis and management of patients with OSCC, it is important to identify effective diagnostic biomarkers and therapeutic targets.

The mammalian genome encodes large numbers of non-coding transcripts that have structural, regulatory or unknown functions (6,7). Long non-coding RNAs (lncRNAs), a class of transcripts >200 nucleotides in length, have been shown to play significant regulatory roles in epigenetic modulation, as well as transcriptional and post-transcriptional in recent years (8,9). Increasing evidence has indicated that lncRNAs participate in the biological processes of cell proliferation, differentiation, apoptosis and cancer metastasis during cancer development and progression (10–12). However, whether such distinct functions of lncRNAs are involved in the development of OSCC remains unknown. H19 a 2.3 kb of lncRNA molecule which is one of the earliest identified imprinted genes (13). Previous studies have reported that H19 is aberrantly expressed in human cancers, including hepatocellular carcinoma, as well as bladder and breast cancers, and usually correlates with cancer progression, metastasis and a poor prognosis, suggesting that H19 may be used as a biomarker for the diagnosis of these types of cancer (14,15). Moreover, H19 has been found to promote epithelial‑mesenchymal transition (EMT) by antagonizing the activity of microRNAs (miRNAs or miRs) as a competing endogenous RNA (ceRNA), and regulating expression of their downstream genes in several type of cancer, such as pancreatic and colorectal cancer (16,17). Recently, H19 was found to be associated with the risk of OSCC in a Chinese population (18). Zhang et al also demonstrated that H19 played a crucial role in the progression of tongue squamous cell carcinoma (TSCC) by regulating the expression of β-catenin and glycogen synthase kinase (GSK)-3β via enhancer of zeste homolog 2 (EZH2), indicating that the inhibition of H19 may be a potential target for the treatment of TSCC (19). However, the roles and mechanism of H19 in the development and progression of OSCC remain to be elucidated.

miRNAs are a class of endogenous small non-coding RNAs, 20–25 nucleotides in length, which can suppress gene expression by directly binding to the 3′-untranslated region (3′-UTR) of target messenger RNAs (mRNAs) to induce mRNA decay or translational repression (20). Several deregulated miRNAs have also been reported in OSCC, and are involved in OSCC cell growth, apoptosis, migration and invasion (21,22). Xu et al found that miR‑138 was significantly downregulated in OSCC and the overexpression of miR-138 inhibited cell proliferation OSCC in vitro and in vivo (23). However, to date, at least to the best of our knowledge, there are limited studies available on the association between lncRNAs and miRNAs and their role in the development of OSCC; thus, studies are required to shed further insight into this matter.

In the present study, we first analyzed GEO datasets to investigate aberrantly expressed lncRNAs, and found that H19 was significantly upregulated both in OSCC tissues and cell lines. Moreover, the knockdown of H19 inhibited OSCC proliferation and invasion in vitro, and suppressed tumor growth in vivo. Finally, H19 was found to play an oncogenic role in OSCC cells by regulating EZH2 and by targeting miR‑138. These findings provide a novel mechanism of the H19/miR‑138/EZH2 axis in OSCC, and suggest that this axis may be a promising molecular therapeutic target for OSCC.

Materials and methods

Cell lines and tissue samples

Six OSCC cell lines SCC-4 (Cat. no. CRL-1624), SCC-15 (Cat. no. CRL-1623), CAL-27 (Cat. no. CRL-2095) [all from American Type Culture Collection (ATCC), Manassas, VA, USA], HSC-3 (RCB1975), HSC-2 (RCB1945) (both from Riken Cell Bank, Tsukuba, Japan) and Ca9-22 (JCRB0625) [from the Japanese Collection of Research Bioresources (JCRB), Osaka, Japan] were used in this study. All cells were cultured in Dulbecco's modified Eagle's medium (DMEM). The medium was supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a 5% CO2 atmosphere. A non-tumorigenic immortalized oral keratinocyte line (HOK‑16B, generous gift from Dr No-Hee Park, University of California, Los Angeles, CA, USA) was maintained in oral keratinocyte medium, supplemented with 1% keratinocyte growth factor plus epithelial growth factor mixture (Invitrogen, Carlsbad, CA, USA). A total of 42 freshly frozen OSCC tissues, as well as 42 matched controls were obtained from the Department of Oral and Maxillofacial Surgery, Shenzhen Hospital of Southern Medical University, Shenzhen, China. None of the patients with OSCC had received radiotherapy or chemotherapy prior to surgery. This study protocol conformed to the Ethics Committee of Shenzhen Hospital of Southern Medical University (Shenzhen, China). All human materials were obtained with informed consent and approved by the Ethics Committee of Shenzhen Hospital of Southern Medical University.

lncRNA expression profile data from GEO

The micro-array data was downloaded from the open GEO database (https://www.ncbi.nlm.nih.gov/geo/) and the GEO accession number is GSE3524 (24). These microarray expression data were analyzed by GEO2R bioinformatics software (http://www.ncbi.nlm.nih.gov/geo/geo2r/), which can analyze any GEO series. The adjusted P-values (adj. P-value) using the Benjamini and Hochberg (BH) false discovery rate (FDR) method by default were applied to correct for the occurrence of false‑positive results. An adj. P<0.05 and a |logFC|≥1 were set as the cut-off criteria. A heatmap of the 49 lncRNAs which the most significant differences in expression was generated using the online tool Morpheus (https://software.broadinsti-tute.org/morpheus/).

RNA extraction, reverse transcription and quantitative RT-PCR

For lncRNA analysis, total RNA was isolated from the cells and tissues using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and reverse transcribed using the Superscript III first strand synthesis system (Life Technologies, Carlsbad, CA, USA). For miRNA analysis, RNA was extracted from the liver tissues using the miRNeasy mini kit (Qiagen, West Sussex, UK) according to the manufacturer's instructions. The RNA was then reverse transcribed into cDNA. Amplifications were carried out on an ABI 7500 Real-Time PCR system (Life Technologies) using SYBR-Green according to the manufacturer's instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or U6 snRNA were used as endogenous controls. Data were analyzed using 7500 software v.2.0.1 (Applied Biosystems, Foster City, CA, USA), and calculated using the 2−∆∆Cq method (25). All experiments were performed in triplicate.

Cell transfection

The miR-138 mimic, miR-138 inhibitor, mimics negative control (mimics NC) and inhibitor NC were purchased from RiboBio Co., Ltd. (Guangzhou, China). A small interfering RNA against H19 (si-H19) and si-Scramble were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Plasmid cDNA-H19 was constructed by introducing a BamHI-EcoRI fragment containing the H19 cDNA into the same site in the pcDNA3.1 plasmid (Invitrogen). Cell transfections with si-H19, pcDNA-H19 and miR-138 mimic and inhibitor were performed as previously described (26). The HSC-2 and Ca9-22 cells were transfected with H19 siRNA, miR-138 mimic or miR-138 inhibitor using the Lipofectamine® RNAiMAX kit (Thermo Fisher Scientific, Waltham, MA, USA). The CAL-27 cells were transfected with pcDNA-H19 or pcDNA-H19 plus miR-138 mimic using Lipofectamine® 2000 (Life Technologies) according to the manufacturer's instructions. The cells were harvested after 48 h and used for further analysis.

Lentivirus production and infection

Lentiviral constructs carrying shRNA targeting H19 (Lv-shRNA), and an empty negative control vector (LV-GFP) were obtained from GenePharma (Shanghai, China). The 293 cells (CRL-1573, ATCC) were co-transfected with Lenti-Pac HIV Expression Packaging Mix and the lentiviral vectors (or the control lenti-virus vectors) using Lipofectamine 2000 (Life Technologies). After 48 h, lentiviral particles in the supernatant were harvested and ultra-centrifuged to concentrate the lentiviral particles. Subsequently, the HSC-2 and Ca9-22 cells grown on 6-well plates were transduced with the lentiviruses of 10 transduction units (TU) per cell. The expression of H19 in the transduced cells was examined by real-time PCR analysis.

Analysis of cell proliferation, apoptosis and cell cycle progression

Cell proliferation was examined by MTT assay according to the manufacturer's instructions (Roche Applied Science, Basel, Switzerland). Briefly, approximately 1×103 cells were seeded in a 96-well culture plate for 24 h. The cells were then transfected with si-H19, si-Scramble, miR-138 inhibitor or miR-control. At various time points, 0.5 mg/ml MTT solution was added to each well. The absorbance was then recorded at 490 nm on a Bio-Rad model 680 microplate reader, (Bio-Rad Laboratories, Hercules, CA, USA). For the analysis of apoptosis, the cells were stained with Annexin V-FITC and propidium iodide (PI) (TACS Annexin V-FITC, Trevigen Inc., Gaithersburg, MD, USA) and then analyzed with double‑label flow cytometry on a flow cytometer (FACSCanto II; BD Biosciences, San Jose, CA, USA). For cell cycle analysis, the cells were resuspended in PBS, stained with PI containing RNase A for 30 min at 37°C, and analyzed by flow cytometry. All the assays were conducted in triplicate.

In vivo tumor growth assay

All animal procedures were performed according to national guidelines and approved by the Animal Care Ethics Committee of Shenzhen Hospital of Southern Medical University. Twenty female BALB/c nude mice (weighing 20±2 g; 4 weeks old, Laboratory Animal Center of Shanghai, Academy of Science) were used in this study. The HSC-2 and Ca9-22 cells transfected with Lv-shRNA or Lv‑Control were injected into the left flanks of the nude mice (2×106 cells/mouse). After 5 weeks, the mice were sacrificed and tumor tissues were dissected. The tumor weight was measured and the tumor volume was calculated according to the following formula: (length x width2)/2. The largest tumor size and volume in the mice of different groups was 1.30 g and 1,500 mm3 (Lv-Control-transfected HSC-2 cells), 1.25 g and 1,700 mm3 (Lv-Control transfected Ca9-22 cells), 0.68 g and 1,150 mm3 (Lv-siRNA transfected HSC-2 cells), 0.73 g and 1,300 mm3 (Lv-siRNA transfected Ca9-22 cells).

Bioinformatics

In silico prediction of miRNA binding sites within the H19 3′UTR was performed using TargetScan (www.targetscan.org) and PicTar (http://www.pictar.org).

Luciferase assays

The 3′-UTR of H19, with wild-type or mutant (Mut) binding sites for miR-138, was amplified and cloned into the pGL3 vector (Promega, Madison, WI, USA) to generate the plasmid pGL3-WT-H19-1 (wt-H9-1) or pGL3-WT-H19-2 (wt-H9-2). The putative binding site of miR-138 in H19 was mutated by using a QuikChange Site Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA) to synthetize mutant type pGL3‑mut‑H19‑1 (Mut‑H19‑1) or pGL3-mut-H19-2 vector (Mut-H19-2). For the luciferase reporter assay, the 293 cells were co-transfected with the luciferase reporter vectors and miR-138 mimics or corresponding negative control (GenePharma) using Lipofectamine 2000 reagent (Life Technologies). The pRL-TK plasmid (Promega) was used as a normalizing control. After 48 h of incubation, luciferase activity was analyzed using the Dual‑Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.

Wound healing assay

The cells were plated in 6-well plates and transfected when cultured to 95% confluence. The cell layers were then scratched using a 10 μl plastic pipette tip to produce wounds. The wounds were photographed at 0 and 48 h under an inverted phase contrast microscope (IX71; Olympus Corp., Tokyo, Japan). Three random fields were marked and measured. All the assays were carried out in triplicate.

Cell invasion assays

For invasion assays, a total of 3×104 HSC-2 and Ca9-22 cells in 150 μl serum-free medium at post-transfection were seeded into the upper chamber (24‑well insert, pore size 8 μm; Corning, NY, USA) pre‑coated with 30 μg/well Matrigel solution (BD Biosciences), and the lower chambers were filled with 500 μl of 10% FBS medium. Following incubation at 37°C for 48 h, the membranes were fixed with 4% polyoxymethylene and stained with 0.1% crystal violet (Sigma-Aldrich). Five pre-determined fields were counted under a microscope (Olympus Corp., original magnification, ×200). All assays were performed in triplicate.

Western blot analysis

Total proteins were extracted from cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma‑Aldrich) and quantified with a Bicin Choninic Acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). A total of 40 μg of protein were subjected to 10% SDS-PAGE, and subsequently transferred onto a polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The blots were incubated with the primary antibodies specific for cleaved caspase-3 (1:500; Cat. no. 9661), cleaved poly(ADP-ribose) polymerase (PARP; 1:1,000; Cat. no. 5625), Bax (1:1,000; Cat. no. 5023), EZH2 (1:1,000; Cat. no. 5246), zinc finger E‑box‑binding homeobox 1 (ZEB1; 1:1,000; Cat. no. 3396), E-cadherin (1:1,000; Cat. no. 14472), vimentin (1:1,000; Cat. no. 5741), N-cadherin (1:1,000; Cat. no. 13116) and β-actin (1:2,000; Cat. no. 4970). All antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Following 3 washes in TBST, the membranes were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature, and washed with TBST 3 times. The protein bands were visualized by ECL detection reagent (GE Healthcare Life Sciences, Piscataway, NJ, USA). The intensity of the protein fragments was quantified with the Quantity One software (4.5.0 basic; Bio-Rad).

Statistical analysis

Statistical analyses were performed with GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). All data are presented as the means ± SD. Differences were analyzed by a Student's t‑test between two groups or one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison tests between multiple groups. Spearman's analysis was used in correlation analysis. Survival analysis under the circumstance of Kaplan-Meier method. A P-value <0.05 was considered to indicate a statistically significant difference.

Results

lncRNA H1 is overexpressed in OSCC

To identify the lncRNAs involved in the development and progression of OSCC, a GSE dataset was obtained from the GEO database under the accession number GSE3524 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE3524). Analysis of these data revealed that 24 lncRNAs were downregulated and 25 miRNAs were upregulated in the tumor group, compared with the normal group. Among the aberrantly expressed lncRNAs, lncRNA H1 was the most significantly upregulated lncRNA in GSE dataset (Fig. 1A). To validate the microarray analysis finding, we detected H19 expression in a cohort of 42 paired tumor tissues and normal tissues. The detailed patient clinical data are presented in Table I. As shown in Fig. 1B, H19 expression was increased in tumor tissues compared with normal tissues. These data suggested that H19 may be involved in the process of carcinogenesis.

Figure 1 Long non-coding RNA (lncRNA) H19 is highly expressed in OSCC. (A) Hierarchical clustering analysis of 49 lncRNAs that were differentially expressed between OSCC tissues and non-tumor samples (>2.0-fold, P<0.05). Expression values are represented in shades of red and green indicating expression above and below the median expression value across all samples. (B) RT-qPCR was used to validate the expression of H19 in 42 paired OSCC and non-tumor samples. P<0.01 vs. normal group. (C) Expression level of H19 positively correlated with the tumor grade in OSCC. P<0.01 vs. I + II grades. (D) Expression level of H19 positively correlated with nodal invasion in OSCC (P<0.01). The x-axis shows the nodal status (NO or YES), and the y-axis represents H19 levels normalized against U6. (E) H19 expression was classified into 2 groups: H19 low expression group (n=17) and H19 high expression group (n=25). Kaplan‑Meier analyses of the associations between the H19 expression level and overall survival of patients with OSCC (the log-rank test was used to calculate P-values).

Table I Clinicopathogical characteristics of the study subjects.

Table I

Clinicopathogical characteristics of the study subjects.

Characteristic	n (%)
Sex
Male	33 (78.57)
Female	9 (21.43)
Age (years)
≥50	31 (73.81)
<50	11 (26.19)
Tobacco use
Yes	32 (76.19)
No	10 (23.81)
Alcohol use
Yes	30 (71.43)
No	12 (28.57)
Tumor site
Tongue	23 (54.76)
Floor of mouth	8 (19.05)
Alveolar	6 (14.29)
Buccal mucosa	4 (9.52)
Retromolar	1 (2.38)
Tumor stage
I–II	16 (38.10)
III–IV	26 (61.90)
Differentiation
Well and moderate	34 (80.95)
Poor	8 (19.05)
Nodal invasion
Negative	28 (66.67)
Positive	14 (33.33)

To determine whether H19 expression was associated with the grade of malignancy and nodal invasion in OSCC, we examined the expression level of H19. We demonstrated that H19 expression was positively associated with the pathological grades of OSCC and nodal invasion (Fig. 1C and D). Based on the relative expression ratios of <0.5, the 42 clinical cases were divided into 2 groups as follows: the H19 low expression group (n=17) and the H19 high expression group (n=25). We then assessed whether the expression of H19 correlated with the post-operative survival time of patients with OSCC. Kaplan-Meier survival analysis revealed that patients with a high H19 expression had a poorer overall survival than those with a low H19 expression (Fig. 1E). Taken together, these findings indicate that H19 may be used as a biomarker for the diagnosis and prognosis of OSCC.

Knockdown of H19 inhibits the proliferation, increases the G0/G1 phase population and induces the apoptosis of OSCC cells

To examine the effect of H19 on cell growth, we first measured the expression levels of H19 in 6 OSCC cell lines (HSC-2, HSC-3, SCC-4, SCC-15, CAL-27 and Ca9-22) and a normal oral mucosa cell line (HOK-16B). As shown in Fig. 2A, a higher expression of H19 was observed in the OSCC cell lines compared with the normal oral mucosa cell line, particularly in the HSC-2 and Ca9-22 cells. Subsequently, the HSC-2 and Ca9-22 cells were transiently transfected with si-H19 and the endogenous level of H19 was effectively decreased (Fig. 2B). As demonstrated by MTT assays, we found that H19 silencing significantly decreased the viability of the HSC‑2 and Ca9‑22 cells (Fig. 2C and D). Furthermore, the proportion of the cell population undergoing apoptosis was increased after knockdown of H19 in the HSC-2 and Ca9-22 cells (Fig. 2E–G). Flow cytometry also revealed a significant promotion of cells in the G0/G1 phase of the cell cycle in the HSC-2 and Ca9-22 cells transfected with si-H19 (Fig. 2H and I). In addition, western blot analysis revealed that the expression levels of apoptosis-related proteins, including cleaved caspase-3 and cleaved PARP and Bax were markedly increased after H19 knockdown (Fig. 2J–L). Our results thus revealed that H19 knockdown inhibited several malignancy-related parameters of OSCC in vitro.

Figure 2 Effects of H19 on OSCC cell growth in vitro. (A) RT-qPCR analysis of H19 expression in OSCC cell lines. **P<0.01 vs. HOK-16B cells. (B) Relative expression levels of H19 in OSCC cells transfected with si-H19 or si-Scramble. **P<0.01 vs. si-Scramble group. (C and D) MTT assay was performed to determine the effects of transfection with si-H19 or si-Scramble on HSC-2 and Ca9-22 cell proliferation. *P<0.05 and **P<0.01 vs. si-Scramble group. (E–G) Annexin V/propidium iodide-double staining assay was performed to determine the apoptosis of HSC-2 and Ca9-22 cells transfected with si-H19 or si-Scramble. **P<0.01 vs. si-Scramble group. (H and I) The percentage of cells in the G0/G1 phase was increased in cells in which H19 was knocked down (HSC-2 and Ca9-22 cells). **P<0.01 vs. si-Scramble group. (J–L) Relative expression of apoptosis-related protein levels, including caspase-3, PARP and Bax in the HSC-2 and Ca9-22 cells following transfection with si-H19 or si-Scramble. **P<0.01 vs. si-Scramble group.

Knockdown of H19 inhibits tumor growth in vivo

To evaluate the functional roles of H19 in vivo, we established a xenograft mouse mode in which Lv-shRNA or Lv-Control-transfected HSC-2 and Ca9-22 cells were transplanted into the flanks of BALB/c nude mice. Consistent with the results obtained in vitro, after 5 weeks, tumor volumes in the mice injected with cells from the si-H19 group were markedly smaller compared with those in the mice injected with cells from the Lv-Control (Fig. 3A and B). Similarly, tumor weights in the mice injected with cells from the Lv‑shRNA group were significantly lower compared with those in the mice injected with cells from the Lv-Control group (Fig. 3C and D). These results indicate that the knockdown of H19 expression inhibits tumor growth in vivo.

Figure 3 Knockdown of H19 inhibits tumor growth in vivo. Lv-Control or Lv-shRNA was infected into the HSC-2 and Ca9-22 cells, which were injected into nude mice. (A and B) Tumor sizes were measured each week following the Lv‑shRNA injection (5 mice/group). *P<0.05 and **P<0.01 vs. Lv-Control group. (C and D) Tumor weights were measured after the Lv-shRNA injection (5 mice/group). P<0.01 vs. Lv-Control group.

Knockdown of H19 inhibits the migration and invasion of OSCC cells

Based on the above-mentioned results in that the expression of H19 was higher in metastatic tissues, we hypothesized that H19 may be associated with the metastasis of OSCC. Thus, wound healing and Transwell assays were performed to examine the effects of H19 on OSCC cell metastasis. As shown in Fig. 4A, H19 silencing markedly inhibited the migration of monolayer-cultured HSC-2 and Ca9-22 cells. In addition, the numbers of invaded cells were markedly attenuated in the cells in which H19 was knocked down compared with the control cells (Fig. 4B). It is well known that EMT plays a critical role in the invasion and metastasis of OSCC cells (27). In this study, we thus assessed the effect of H19 on the expression of EMT related-genes. Our results demonstrated that H19 silencing significantly decreased the expression levels of vimentin and N-cadherin (mesenchymal markers), but increased the expression levels of ZEB1 and E-cadherin (epithelial markers) (Fig. 4C). These data suggest that the knockdown of H19 suppresses OSCC cell metastasis by inhibiting EMT.

Figure 4 Knockdown of H19 inhibits the migration and invasion of OSCC cells. (A) Wound healing assay was carried out to evaluate the effect of H19 on the migration of HSC-2 and Ca9-22 cells. **P<0.01 vs. si-Scramble group. (B) Invasion assay (with Matrigel Transwell chambers) for investigating cell invasiveness. **P<0.01 vs. si-Scramble group. (C) Relative expression of EMT-related protein levels in HSC-2 and Ca9-22 cells following transfection with si-H19 and si-Scramble. **P<0.01 vs. si-Scramble group. Data are presented as the means ± SD from 3 independent experiments.

H19 directly targets miR-138 and inhibits its level in OSCC cells

Recently, studies have confirmed that lncRNAs can function as ceRNAs or molecular sponges that modulate miRNAs in cancers (28,29). In this study, we thus performed bioinformatics analysis using TargetScan and PicTar, and found that miR-138 contains a binding site for H19. The predicted binding sites for H19 in the miR-138 sequence are illustrated in Fig. 5A. To examine whether H19 directly targets miR-138, we conducted a luciferase assay. As shown in Fig. 5B, luciferase activity was significantly inhibited when wt‑H19‑1 or wt-H19-2 was co-transfected with miR-138 mimics compared with that after mimic NC co-transfection, whereas the inhibitory effect was abolished when the H19 3′-UTR was mutated. This indicated that miR-138 probably interacted with H19.

Figure 5 H19 directly inhibits miR-138 expression in OSCC. (A) The predicted miR-138 binding sites on H19. (B) Luciferase activity in 293 cells co-transfected with miR-138 mimics and luciferase reporters containing wt-H19 or mut-H19 transcript. **P<0.01 vs. miR-138 mimic group. (C) miR-138 expression was validated in OSCC cell lines by RT-qPCR. **P<0.01 vs. HOK-16B group. (D) RT-qPCR analysis of miR-138 expression in HSC-2 cells transfected with si-H19 or si-Scramble. **P<0.01 vs. si-Scramble group. (E) RT-qPCR analysis of miR-138 expression in CAL-27 cells transfected with pcDNA-H19 or pcDNA3.1 plasmids. **P<0.01 vs. pcDNA3.1 group. (F) miR-138 expression was validated in OSCC tissues and corresponding adjacent normal tissues by RT-qPCR (P<0.01 vs. normal group). (G) Pearson's correlation analysis was performed to analyze the correlations between H19 and miR‑138 expression in OSCC tissues (r=‑0.7816; P<0.001).

It has been found that miR-138 functions as a tumor suppressor in human OSCC (23). Thus, we first measured miR-138 expression in 6 OSCC cell lines (HSC-2, HSC-3, SCC-4, SCC-15, CAL-27 and Ca9-22) and a normal oral mucosa cell line (HOK-16B). Consistent with the findings previous of that study, the expression of miR-138 was also expressed at low levels in the OSCC cell lines, particularly in the HSC-2 and Ca9-22 cells (Fig. 5C). To further determine whether H19 affects OSCC cell proliferation and metastasis by regulating miR-138 expression, we measured miR-138 expression after the silencing or overexpression of H19 in HSC-2 and CAL-27 cells, and the results of RT-qPCR indicated that miR-138 expression was upregulated after the knockdown of H19 in the HSC-2 cells (Fig. 5D), whereas the miR-138 level was downregulated after H19 was overexpressed in the CAL-27 cells (Fig. 5E). Furthermore, we detected miR-138 expression in 42 pairs of OSCC tissues and normal tissues. Consistent with the results obtained with the OSCC cell lines, the expression of miR-138 was downregulated in the OSCC tissues (Fig. 5F), and an inverse correlation was observed between the expression of miR-138 and the expression of H19 (Fig. 5G). All these data suggest that H19 negatively regulates the expression of miR-138 in OSCC.

Suppression of miR-138 attenuates the si-H19-induced inhibitory effects on OSCC cells by targeting EZH2

Recent findings have demonstrated that EZH2 acts as an oncogene and correlates with the malignant potential and a poor prognosis in a wide range of cancer types, including OSCC (30–32). Importantly, EZH2 has been identified as a target of miR‑138 in several cancer cells (33–35). Therefore, we sought to determine whether H19 functions as a ceRNA for miR-138 to regulate the expression of EZH2 in OSCC. Western blot analysis revealed that the knockdown of H19 in the HSC‑2 cells significantly decreased the expression levels of EZH2, while transfection with miR‑138 inhibitor restored EZH2 expression in the OSCC cells in which H19 was knocked down (Fig. 6A). By contrast, the overexpression of H19 in the CAL-27 cells increased the expression levels of EZH2, while transfection with miR‑138 mimic inhibited the promotion of EZH2 expression in the cells overexpressing H19 (Fig. 6B). These results suggest that H19 regulates the expression of the oncogene EZH2 by competing with miR-138 in OSCC cells.

Figure 6 H19 regulates EZH2 by sponging miR‑138 in OSCC cells. si‑H19 and miR‑138 inhibitor were transfected into HSC‑2 cells, pcDNA‑H19 and miR‑138 mimic were transfected into CAL‑27 cells. (A and B) Western blot analysis was performed to determine the expression of EZH2 in HSC‑2 and CAL‑27 cells. (C and D) MTT assay was performed to examine the proliferation of HSC-2 and CAL-27 cells. (E and F) Flow cytometric assay was performed to examine the apoptosis of HSC-2 and CAL-27 cells. (G and H) Wound healing assay was performed to determine the percentage of wound closure in the HSC-2 and CAL-27 cells. (I and J) Transwell invasion assay was performed to determine the numbers of invaded HSC-2 and CAL-27 cells. *P<0.05 and **P<0.01 vs. blank group; ##P<0.01 vs. si-H19 group or pcDNA H1 group.

In order to analyze the importance of miR‑138 in H19-mediated OSCC proliferation, apoptosis and invasion, we knocked down the expression of miR-138 in the HSC-2 cells transfected with si-H19 and overexpressed its expression in the CAL-27 cells transfected with pcDNA-H19, separately. The results revealed that miR-138 knockdown blocked the inhibitory effects of H19 on cell proliferation, apoptosis and invasion (Fig. 6C–J), suggesting that the effects of H19 on OSCC growth and invasion are partially mediated by miR-138. Taken together, these data indicated that the effects of H19 on the growth and metastasis of OSCC are partially mediated by regulating the expression of miR-138.

Discussion

In the present study, we found that lncRNA H1 was upregulated in OSCC tissues and cell lines and that a high H19 expression was associated with a poor clinical outcome. Moreover, the knockdown of H19 inhibited OSCC cell proliferation, migration and invasion, induced cell apoptosis and decreased the tumor growth in vivo. Mechanistically, we demonstrated that H19 affects the the biological characteristics of OSCC cells by positively modulating EZH2 expression through competition for miR-138. Collectively, our results demonstrated the roles and functional mechanisms of H19 in OSCC and provide novel insight into potential therapeutic targets for OSCC.

Recent experimental studies have demonstrated that lncRNAs play various roles in tumorigenesis, including OSCC (36–40). For example, HOX transcript antisense RNA (HOTAIR) has been reported to be upregulated in OSCC and its expression has been shown to be associated with the metastasis and poor prognosis of OSCC (41). Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is another reported lncRNA, which contributes to EMT-mediated metastasis in OSCC by modulating the activation of β-catenin and NF-κB pathways (42). However, the roles of lncRNAs in OSCC remain largely unknown. In this study, we analyzed and validated a list of significantly dysregulated lncRNAs in OSCC tissues by retrieving the microarray data in the GEO dataset (accession no. GSE3524). In this study, we found that lncRNA H1 was one of the most significantly differentially expressed lncRNA. Moreover, a high level of H19 positively correlated with clinical stages and was identified as a prognostic parameter for patient survival. These data indicate that H19 may serve as a biomarker for the diagnosis and prognosis of OSCC.

A large body of evidence has indicated that H19 is involved in cancer invasion and metastasis. In esophageal squamous cell carcinoma (ESCC), H19 has been shown to be upregu-lated and to promote cell proliferation and metastasis (43). Xu et al found that H19 functioned as a marker of poor prognosis in cholangiocarcinoma (CCA) and H19 enhanced cell migration and invasion by affecting EMT (44). H19 can also activate Wnt/β-catenin signaling to affect cell proliferation and metastasis in bladder cancer (45). In this study, we proved that the knockdown of H19 inhibited OSCC cell proliferation, migration and invasion, induced cell apoptosis, arrested the cells in the G0/G1 phase and decreased the tumor volume in vivo. Accordingly, apoptosis was increased, conferred by the upregulation of cleaved caspase-3, cleaved PARP and Bax. Moreover, we found that the ectopic expression of H19 decreased the expression of E‑cadherin and ZEB1, and increased the expression of vimentin and N-cadherin in OSCC cells, which suggested that H19 may promote OSCC cell invasion by inducing EMT. Therefore, these data suggest that H19 may serve as an oncogene that promotes OSCC malignant progression.

Recently, increasing evidence has indicated that lncRNAs function as ceRNAs to silence target mRNAs by sponging target miRNAs (46,47). Sui et al found that lncRNA GIHCG promoted hepatocellular carcinoma progression by epige-netically regulating miR-200b/a/429 (48). Another study demonstrated that lncRNA PVT1 promoted cervical cancer progression through the silencing of miR-200b (49). Similarly, lncRNA H1 competitively binds miR-17-5p to regulate YES1 expression in thyroid cancer (50). In this study, we demonstrated that H19 directly targeted miR-138 by bioinformatics analysis and luciferase reporter assays. We also confirmed that miR‑138 was significantly decreased in OSCC tissues and inversely correlated with the expression level of H19. However, the ceRNA mechanisms for H19 deregulation in OSCC have not been thoroughly elucidated.

Previous studies have indicated that miR-138 plays critical roles in various types of cancer by targeting EZH2. For example, miR-138 acts as a tumor suppressor miRNA in human clear cell renal cell carcinoma (ccRCC), induces SN-12 cell senescence by downregulating EZH2 expression (34). The study by Zhang et al demonstrated that miR-138 inhibited tumor growth through the repression of EZH2 in non‑small cell lung cancer (51). A recent study identified EZH2 as a target of miR-138 in osteosarcoma cells (33). In addition, Li et al found that lncRNA H1 regulated EZH2 expression by interacting with miR-630 and promoted cell invasion in nasopharyngeal carcinoma (52). Therefore, it was hypothesized that H19 may also serve as a ceRNA to regulate EZH2 expression by sponging miR‑138. Consistent with the findings of previous studies, we confirmed that H19 regulated the expression of EZH2, and that miR‑138 attenuated the effects of H19 on the expression of EZH2 in OSCC cells. Notably, all the effects of H19 on the biological characteristics of the OSCC cells were blocked by miR-138. Taken together, these data strongly suggest that lncRNA H1 functions as a ceRNA for miR-138 in OSCC.

In this study, the detection of H19 level in OSCC cell lines indicated that H19 had the highest level in HSC-2 cells and the lowest level in CAL-27 cells. Therefore, the HSC-2 cells were selected for the loss-of-function experiments and the CAL-27 cells for gain-of-function experiments. The results revealed that the knockdown of H19 inhibited OSCC cell proliferation and invasion, and induced cell apoptosis, whereas the overexpression of H19 had an opposite result. In addition, the expression of H19 in 6 OSCC cell lines was markedly upregulated compared with that in the HOK-16B cells. However, we did not find OSCC tumor cells in which the expression of H19 was similar to that in the HOK-16B cells. In the future, we aim to find a cell line with which to explore whether this approach would yield the same results in normal H-19-expressing cells in which H19 is knocked down or overexpressed.

In conclusion, in this study, demonstrate that H19 promotes EZH2 expression by competitively binding miR‑138, contributing to the induction of the EMT process in OSCC. We also identified the H19/miR‑138/EZH2 axis as a novel signaling network in OSCC, which may provide a novel therapeutic strategy for the targeted treatment of OSCC.

Notes

[1] Competing interests

The authors declare that they have no competing interests.

References