Aberrant DNA hypermethylation-silenced SOX21-AS1 gene expression and its clinical importance in oral cancer
© The Author(s). 2016
Received: 17 March 2016
Accepted: 15 November 2016
Published: 26 November 2016
Long noncoding RNAs (lncRNAs) are more than 200 nucleotides in length and lack transcriptional ability. The biological function of lncRNAs in oral squamous cell carcinoma (OSCC) remains unclear. The aim of this study was to identify the dysfunction of lncRNA in OSCC.
We analyzed the transcriptome profiles of human OSCC tissues and paired adjacent normal tissues from two patients through a next-generation sequencing approach. A total of 14 lncRNAs were upregulated (fold change ≥3) and 13 were downregulated (fold change ≤−3) in OSCC tissues compared with the adjacent normal tissues. SOX21-AS1 was subjected to further analysis, revealing that the expression levels of SOX21-AS1 significantly decreased in OSCC compared with the adjacent normal tissue. The promoter activity of SOX21-AS1 was obviously suppressed by in vitro methylation. The DNA methylation status of the SOX21-AS1 promoter was analyzed using combined bisulfite restriction analysis, revealing that the aberrant promoter hypermethylation of SOX21-AS1 was observed frequently in OSCC tissues. The effects of SOX21-AS1 on cell proliferation and invasion were examined through transient transfection. Our data showed that SOX21-AS1 could significantly suppress oral cancer cell growth and invasion. Furthermore, the low expression level of SOX21-AS1 was significantly correlated with an advanced stage (P = 0.047), large tumor size (P = 0.033), and poor disease-specific survival in OSCC patients (P = 0.002).
SOX21-AS1 was identified as susceptible dysfunction correlated with promoter hypermethylation in OSCC. Low SOX21-AS1 expression may be an adverse prognostic biomarker for OSCC.
Oral cancer is one of the most common cancers in developing countries and occurs in the anterior tongue, cheek, floor of the mouth, gingiva, and other parts of the oral cavity . Oral squamous cell carcinoma (OSCC) is the most common oral cancer and 300,400 incident cases and 145,400 deaths have been reported worldwide . Betel quid chewing, tobacco smoking, and alcohol drinking contribute to the increasing incidence and mortality rates of oral cancer .
Long noncoding RNAs (lncRNAs) are over 200 nucleotides in length and lack transcriptional ability . Some lncRNAs, such as transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), and spliceosomal RNAs, are crucial for maintaining normal cellular mechanisms . Except for these functional lncRNAs, most lncRNAs have been frequently considered functionless sequences [6–8]. Recently, lncRNAs have been reported as crucial for regulating cell development, growth, cell cycles, and cancer metastasis in human cancer . Several aberrant lncRNAs were identified from high-throughput profiles and were associated with OSCC progression . Fang et al. reported that urothelial cancer associated 1 (UCA1) is an overexpression in tongue squamous cell carcinomas (TSCCs) and may play an oncogenic role in tumorigenesis . Jia et al.  reported that the loss of miR-26a expression resulted in MEG3 downregulation in TSCC tissues compared with adjacent normal tissues. Low MEG3 expression was highly correlated with poor prognosis of patients. Furthermore, MEG3 overexpression in SCC-15 and CAL27 cells inhibited cell proliferation and cell cycle progression and promoted apoptosis . Oncogenic lncRNA, HOX transcript antisense RNA (HOTAIR), was significantly upregulated in OSCC, and the upregulated expression was associated with poor survival and metastasis. HOTAIR knockdown can suppress oral cancer cell proliferation, colony formation, cell migration, and invasion . However, the role of lncRNA remains largely unknown, and the detailed mechanisms and functions of dysfunctional lncRNAs in OSCC have not yet been fully elucidated. On the basis of the analysis of the expression profile of two pairs of OSCC tissue samples, as well as the experimental studies of two OSCC cell lines and 86 pairs of OSCC tissue samples in this study, we determined that SOX21-AS1 expression was silenced with an aberrant methylation promoter; moreover, the low expression was highly correlated with poor prognosis of patients with OSCC.
Long noncoding RNAs identified in OSCC tissues through next-generation sequencing
LncRNA candidates were dysregulation in OSCC
Fold change (T1/N1)
Fold change (T2/N2)
DNA methylation-silenced SOX21-AS1 expression in oral cancer cells
DNA hypermethylation results in SOX21-AS1 silencing in OSCC
SOX21-AS1 suppresses oral cancer cell growth and invasion
We made two additional deletion constructs, pCMV-SOX21-AS1-E1 and pCMV-SOX21-AS1-E2, for determining which region is responsible for cell growth and motility inhibition. As shown in Fig. 6a, b, both SOX21-AS1-E1 and SOX21-AS1-E2 were significantly overexpressed (>80-fold increase) compared with the vector control group. The effects of SOX21-AS1-E1 and SOX21-AS1-E2 on the proliferation, migration, and invasion of SAS cells were then respectively examined after transient transfection. Our data revealed that ectopic overexpression of SOX21-AS1-E1 revealed no effect on proliferation or motility ability in SAS cells. Ectopic SOX21-AS1-E2 expression in SAS cells significantly suppressed the growth and motility ability of SAS cells (Fig. 6c–h). To understand the putative mechanism of SOX21-AS1 suppressed cell growth and invasion ability, we further examined the epithelial-mesenchymal transition (EMT) genes and growth-related genes. As shown in Additional file 4: Figure S3C, the expression level of E-cadherin was increased, and fibronectin and cyclin D1 were decreased in SAS cells with SOX21-AS1 overexpression. However, the details of the mechanisms of SOX21-AS1 suppressed cell growth and invasive ability remain unclear and require further study.
Correlation between SOX21-AS1 expression and clinicopathological characteristics
The relationship of expression levels of SOX21 and SOX21-AS1 with clinicopathologic data of OSCC patients
SOX21 (n = 86)
SOX21-AS1 (n = 86)
Buccal and other oral mucosal sites
AJCC pathological stage
II, III, IV
T2, T3, T4
Univariate and multivariate Cox’s regression analysis of SOX21-AS1 expression for disease-specific survival and disease-free survival of OSCC patients
Number of events
After the entire human genome was completely sequenced in 2003, it was revealed that only approximately 2% of the human genome is used for protein coding . High-throughput sequencing data revealed that 80–90% of the human genomic DNA is actively produced by RNA transcripts [20–22]. Most of these transcripts are nonprotein coding genes, including small RNAs and lncRNAs. In the present study, we comprehensively analyzed OSCC profiles by using an NGS approach, which revealed that the expression of 27 lncRNAs significantly differed between OSCC and the corresponding adjacent normal tissues (Table 1). Among lncRNAs, lnc00152 has been reported to play an oncogenic role in promoting gastric cancer cell growth, migration, and invasion and in suppressing cell apoptosis by sponging miR-18a-5p, miR-195-3p, miR-139-5p, and miR-31-5p expression [23, 24]. In addition, lnc00152 was significantly overexpressed in pancreatic cancer . However, in-depth biological function and the effect of dysfunctional lnc00152 on oral cancer remain unclear and require further investigation.
In this study, our data revealed that SOX21 and SOX21-AS1 share a head-to-head promoter, and DNA methylation of the promoter leads to simultaneous suppression of their expression in OSCC. Our data showed that SOX21-AS1 expression was increased in oral cancer cells treated with 5-Aza-dC. An in vitro methylation assay demonstrated that SOX21-AS1 promoter activity could be suppressed with complete DNA methylation. However, only the methylation status of CpG2 was well correlated with low SOX21-AS1 expression in OSCC, but CpG regions 1 and 3 were not thus correlated (Additional file 3: Figure S2). These conflicting results may be due to two putative reasons: (1) A COBRA assay does not completely represent DNA methylation status; (2) DNA methylation is not the only factor for transcriptional silencing.
A previous study revealed that SOX21 expression was controlled through DNA methylation and was a suitable biomarker for colorectal cancer diagnosis . In human glioma cells, SOX21 overexpression inhibits SOX2 expression and induces apoptosis because of the interaction between SOX21 and SOX2 [26, 27]. Ferletta et al. reported that the knockdown of SOX2 expression can suppress SOX21 expression at the protein and mRNA levels in glioma cells, implying that SOX2 can positively regulate the transcriptional activity of SOX21 . SOX21 in combination with SOX2 exerts a tumor-suppressive effect on carcinogenesis. Although SOX21 and SOX21-AS expression were reduced simultaneously in OSCC, SOX21 expression does not contribute to the clinical manifestations of patients with OSCC (Tables 2 and 3). Conversely, low SOX21-AS1 expression was significantly correlated with poor clinicopathologic features and a short survival time of patients with OSCC (Tables 2 and 3).
Although SOX21-AS1 can suppress oral cancer cell growth and invasion, the details of the underlying mechanism remain unknown. LncRNAs can modulate cellular function through different mechanisms such as RNA degradation, epigenetic modification, transcriptional regulation, and an miRNA decoy . We analyzed the subcellular fractionation localization of SOX21-AS1 in SAS cells (Additional file 4: Figure S3). Furthermore, a considerable increase in SOX21-AS1 expression was observed in the nuclei compared with the cytoplasms. According to its location and tumor-suppressive role, SOX21-AS1 is probably an enhancer that reinforces other tumor suppressor mRNAs or acts as a decoy removing certain transcription factors from binding to the DNA to prevent oncogene transcription. In conclusion, low SOX21-AS1 expression correlated with its sense gene SOX21 and can serve as an independent biomarker for poor prognosis of OSCC. SOX21-AS1 and SOX21 expression in tumors may be downregulated through DNA hypermethylation. However, the details of the biological functions of SOX21-AS1 must be further evaluated in OSCC.
Clinical tissue samples were collected from OSCC patients who provided signed informed consent and accepted a surgical operation at the Department of Dentistry and Department of Otorhinolaryngology, Kaohsiung Veterans General Hospital. This study protocol was approved by the Institutional Review Board of Kaohsiung Veterans General Hospital (Kaohsiung, Taiwan; IRB number VGHKS14-CT6-18). The total RNA and DNA of the tissues were extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the instruction manual.
Next-generation sequencing and analysis
In this study, the transcriptome profiles of paired tumors and adjacent normal tissue samples from two OSCC patients were analyzed using an NGS approach; the detailed clinical characteristics of the samples are shown in Additional file 6: Table S2. High-purity normal and tumor tissues were individually separated through laser capture microdissection and were transferred to an RNA extraction kit (Qiagen, Hilden, Germany) for RNA extraction. All procedures of the RNA transcriptome were performed according to the manufacturer protocol from Illumina. Library construction of all samples was performed using TruSeq RNA Sample Prep Kits v2 for 50 single-end base pair sequencing on the Solexa platform. Finally, raw sequences were obtained from the Illumina GA Pipeline software CASAVA version 1.8 and expected to generate 30 million reads per sample. After low-quality data were filtered, qualified reads were analyzed using TopHat/Cufflinks for estimating gene expression, which was calculated as RPKM. For differential expression analyses, CummeRbund was used for statistically analyzing the gene expression profiles. The reference genome and gene annotations were retrieved from the Ensembl database.
Cell lines and demethylation treatment
Two human OSCC cell lines, SAS (human tongue SCC) and CAL27 (human tongue SCC) cell lines, were provided by Dr. Michael Hsiao (Genomics Research Center, Academia Sinica, Taipei, Taiwan). The oral cancer cell lines, SAS and CAL27, were cultured in Dulbecco's modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA). To examine the role of methylation in the regulation of lncRNA expression, SAS and CAL27 cells were cultured in the presence or absence of 10 μM 5-aza-2′-deoxycytidine (5-Aza-dC) for 4 days and treated with 0.25 μM TSA on the third day.
Reverse transcriptase polymerase chain reaction of lncRNAs
For RT-PCR, 2 μg of total RNA was reverse transcribed using random primers and SuperScript III reverse transcriptase according to manufacturer instructions (Invitrogen, Carlsbad, CA, USA). Gene expression was detected using an SYBR Green I assay (Applied Biosystems, Foster City, CA, USA), and lncRNA expression was normalized to that of GAPDH (△Ct = target lncRNA Ct-GAPDH Ct). The individual primers used in the present study were as follows: SOX21-F: GCACAACTCGGAGATCAGCA and SOX21-R: CCGGGAAGGCGAACTTGTC; SOX21-AS1-exon1-F: CCGATGGGAAACCCCCAATC and SOX21-AS1-exon1-R: AACGCTTGCTCAAGCCTCAT; SOX21-AS1-exon2-F: TCACTTACATGCGCTGCTGA and SOX21-AS1-exon2-R: GCCGCAGCATACCAAAAAGT; GAPDH-F: TGCACCACCAACTGCTTAGC and GAPDH-R: GGCATGGACTGTGGTCATGAG.
SOX21-AS1 promoter and expression construction
The SOX21-AS1 promoter was amplified using the promoter-specific primer pairs (SOX21AS1-promoter-F: ATGAAGCTTCCATGAAGGCGTTCATGGGCCG and SOX21AS1-promoter-R: ATGAAGCTTAGAGGAAGACTCGAGAGGCAGGT). PCR products were digested with the restriction enzyme HindIII and cloned into the pGL4.21 luciferase expression vector (Promega, Madison, WI, USA). Full-length exons 1 and 2 of the SOX21-AS1 PCR products were amplified using individual primer pairs (exon1-F: ATGGAATTCTCTTCTTGGCTCCGGGCAGGGTG and exon1-R: ATGAAGCTTCTGAGCCGGTGCAGAGGGCG; exon2-F: ATGGAATTCGTTTAGGCGAGTGGAGAGTCCG, and exon2-R: ATGCTCGAGAATCTTTAGGACAAAACTGAGC). The PCR products were digested with the restriction enzyme EcoRI and cloned into the pCMV-Tag 2A vector (Stratagene, La Jolla, CA, USA).
In vitro methylation
We sequentially methylated pGL4-SV40, pGL4-SRE promoter, pGL4-CRE promoter, and pGL4-SOX21-AS1-P in vitro by using M. SssI, M. HpaII, and M. HhaI methyltransferase enzymes (Invitrogen, Grand Island, NY, USA). A luciferase assay was performed in SAS cells by using a Dual-Glo luciferase reporter assay system kit (Promega, Madison, WI, USA) 24 h after transfection.
Combined bisulfite restriction and sequencing analyses
DNA was subjected to bisulfite conversion by using the EZ DNA Methylation-Gold Kit (Zymo Research Corporation, Orange, CA, USA). The bisulfite-converted genomic DNA was used for methylation analysis of the promoter with the specific methylation primers. The methylation status of the genomic DNA of individual samples was examined using TaqI or BstUI digestion (New England Biolabs, MA, USA). Furthermore, the PCR products were cloned into the pJET1.2 vector (Promega, Madison, WI, USA), and several clones were used for sequencing. The individual primers used in the present study were as follows: CpG1-F: ATGAAATTTTTAATAAAATTGGAAAGGT and CpG1-R: CCAAATAAAA ACAAA AAAACCAAAC; CpG2-F: GGTTGTTTTTGGGATATTTTAATTTT and CpG2-R: CTAAAAACCCCCTTTAACACTTAAC; CpG3-F: GGAGGAGGTGGAGTTTAGGATT and CpG3-R: AAAACCACAACCAAAACAACTACA.
SAS cells were transfected with pCMV-SOX21-AS1, pCMV-SOX21-AS1-exon1, pCMV-SOX21-AS1-exon2, and empty vectors by using Lipofectamine® 2000 Reagent (Invitrogen, Grand Island, NY, USA). For the clonogenic assay, 2000 transfecting cells were plated onto 6-well plates for 2 weeks until substantial-sized colonies were formed. Subsequently, the colonies were analyzed using crystal violet staining.
Cell migration and invasion assays
The migration and invasion abilities of cells were assessed in vitro by using a transwell assay. In brief, cells were resuspended at a density of 4.5 × 105 in 2% fetal bovine serum and then added to the upper chamber of the transwells (Falcon, Corning Incorporated, USA) without Matrigel (BD Biosciences, MA, USA) for the migration assay or with a Matrigel coating for the invasion assay. Chambers were incubated in a CO2 incubator at 37 °C for 36–48 h; the remaining cells in the upper chamber were removed using cotton swabs, and the cells on the undersurface of the transwells were fixed in 10% formaldehyde solution. Cells were stained with crystal violet solution, and the numbers of cancer cells in three fields were counted under a phase-contrast microscope.
The chi-square test, Fisher exact test, Student t test, ANOVA, Mann–Whitney U test, and Kruskal–Wallis one-way ANOVA were used to evaluate the correlation of each lncRNA expression with different oral tissues or clinicopathological parameters. For clinicopathologic outcome and survival analysis, the RNA expression levels (-delta Ct) of SOX21-AS1 were dichotomized as low expression and high expression with the cutoff value (−9.8647) based on a receiver operating characteristic (ROC) curve analysis. The clinicopathologic outcome is usually defined as the time of initial diagnosis or surgery. Disease-specific survival was measured from the time of the initial resection of the primary tumor to the date of cancer-specific death or the final follow-up. Disease-free survival was calculated from the date of the initial resection of the primary tumor to the date of recurrence or the final follow-up. In this study, the median time of follow-up was 25.68 (range, 1.00–68.27) months. The cumulative survival curves were estimated using the Kaplan–Meier method, and the survival curves were compared using the log-rank test. A Cox proportional hazards model was used for determining independent predictors of survival by using factors significant in univariate analysis as covariates; P < 0.05 was considered statistically significant.
The authors would like to thank Department of Medical Education and Research, Kaohsiung Veterans General Hospital, for providing the laser capture microdissection system and Genomics and Proteomics Core Libratory, Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital, for the help with NGS data analysis.
This work was supported by grants from Kaohsiung Veterans General Hospital (VGHKS 104-051, VGHKS 104-092, VGHKS 104-103, and VGHKS104-G01-2).
Availability of data and materials
Next-generation sequencing data, methylation data, and analytical conditions can be provided upon request.
CMY and THW executed this study and wrote the draft of this manuscript. SCL performed analysis of NGS data. HCC and MCL performed clinical sample collection. HHL and YKT performed the statistical analysis. PFL and YLS helped to prepare manuscript. LPG and KWT supervised the study and edited the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study protocol was approved by the institutional review board of Kaohsiung Veterans General Hospital (Kaohsiung, Taiwan; IRB number VGHKS14-CT6-18).
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