Screening of candidate tumor-suppressor genes in 3p21.3 and investigation of the methylation of gene promoters in oral squamous cell carcinoma

  • Authors:
    • Kai Wang
    • Tianyou Ling
    • Hanjiang Wu
    • Jie Zhang
  • View Affiliations

  • Published online on: December 28, 2012     https://doi.org/10.3892/or.2012.2213
  • Pages: 1175-1182
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Abstract

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck malignant tumor. however, its pathological mechanisms have not yet been elucidated. In the present study, we screened for candidate tumor-suppressor genes (TSGs) related to OSCC among 10 candidate genes located in 3p21.3, a region abundant with TSGs based on previous studies, using semi-quantitative reverse transcription PCR (RT-PCR). Three genes, GNAT1, SEMA3B and AXUD1, with low or no expression in OSCC tissues and the cell line TCA8113 were selected, and the promoter methylation status was further analyzed by methylation-specific PCR (MS-PCR). Hypermethylation in the promoter regions of SEMA3B was found in OSCC tissues, and a significant difference in the frequency of methylation of SEMA3B was observed between OSCC and non-cancerous tissues. Furthermore, TCA8113 cells treated with 5-Aza-Cdc started to re-express SEMA3B at a concentration of 5 µM or higher. Our study confirmed that three candidate TSGs with low expression may be involved in OSCC and that hypermethylation in promoter regions may contribute to the low expression of SEMA3B. These findings offer novel insights for clarifying the molecular mechanisms of tumorigenesis of OSCC as well as for aiding in its clinical diagnosis and therapeutic strategy.

Introduction

Head and neck tumors are the common malignant tumors worldwide, while oral squamous cell carcinoma (OSCC) is the most prevalent type of head and neck tumor accounting for 2–3% of malignant tumors and 80% of oral and maxillofacial malignant tumors (1,2). Although the techniques of surgical treatment, radiotherapy and chemotherapy for OSCC have progressed in the past few decades, the 5-year survival rate of OSCC patients has shown no noticeable improvement (3,4). Thus, it is crucial to explore the pathological mechanisms of OSCC, and new biomarkers must be identified and used in the detection of precancerous lesions, early diagnosis and therapy of OSCC.

Numerous factors, such as genetic alterations, epigenetic changes and environmental influence, are involved in the development of OSCC. Inactivation and deletion of tumor-suppressor genes (TSGs) play important roles in the development of carcinomas (5). Previous studies have suggested that analyzing the LOH (loss of heterozygosity) in tumor tissues can facilitate the identification of relevant TSGs. For example, RB(6), p53 (7), Wilms tumor (WT)(8), cyclin-dependent kinase number 2 (CDKN2)(9), adenomatous polyposis coli (APC) (10), deleted in pancreatic carcinoma locus 4 (DPC4) (11), fragile histidine triad (FHIT) (12) and putative protein tyrosine phosphatase gene (PTEN) (13,14) have all been identified using LOH analysis. As for OSCC, a high frequency of LOH has been observed on chromosomes 3p, 4q, 5q, 7q, 8p, 9p, 11q, 13q, 18q, 20q, 21q and 22q regions using comparative genomic hybridization and LOH analysis (1518). Thus, these chromosome segments appear to be target regions where TSGs of OSCC may exist.

Roz et al(19) found LOH at one or more loci of chromosome 3p by investigating 30 oral dysplastic lesions, presenting clinically as either erythroplakias or leukoplakias with histopathological features of either severe epithelial dysplasia or carcinoma in situ. Furthermore, three discontinuous deletion regions including -3p13-21.1, -3p21.3-5 and -3p25, were detected in patients with precancerous lesions and OSCC, which suggest that alterations of these three regions may be early events in the development of OSCC. Yamamoto et al(20) found different degrees of LOH at 3p21.3 in OSCC patients from Japan and India. At the same time, LOH at 3p21-22 was present in other common malignant tumors such as renal carcinoma (21), lung cancer (22), squamous cell carcinoma of the head and neck (23), cervical carcinoma (24) and breast cancer (25). Several groups have studied the relevant TSGs for nasopharyngeal carcinoma (NPC) and lung cancer at region 3p21.3 and found that BLU, RASSF1A, SEMA3B and SEMA3F play vital roles in the development and progression of NPC or lung cancer (2630). Similar studies performed in OSCCs revealed that the expression levels of the LTF, LIMD1, CACNA2D2, RASSF1A, CDC25A and SCOTIN genes located at 3p21.3 were decreased by 67.6, 53.2, 23.7, 15.1, 5.3 and 0.58%, respectively. LOH and DNA methylation alterations may account for the abnormal expression of LTF, LIMD1, CACNA2D2 and RASSF1A.

In the present study, based on the above-mentioned research, bioinformatics analysis was carried out to screen candidate TSGs located at 3p21.3, which to date have not been or have been inadequately studied in OSCC. The methylation state in the promoters of TSGs was also analyzed using methylation-specific PCR (MSP) to explore the role of DNA methylation in the downregulation of TSGs in OSCCs.

Materials and methods

Selecting target genes located at 3p21.3

On the basis of a database search (PubMed, Gene Card, OMIM and Gene Map), using ‘3p21.3’ and ‘tumor-suppressor genes’ as search terms, 10 genes were selected for analysis based on the following inclusion criteria: i) they were previously reported to exhibit low expression or no expression in other tumor types; and ii) the functions of these genes were not previously studied or inadequately studied in OSCCs.

Cell line and tissues

TCA8113, a tongue cancer cell line, was provided by the Cancer Research Institute of Central South University. TCA8113 cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum at 37°C in a humidified atmosphere with 5% CO2. Thirty-six pairs of normal and tumor tissues from OSCC patients were obtained from the Hunan Cancer Hospital (Changsha, Hunan, China) and the Second Xiangya Hospital of Central South University (Changsha, Hunan, China) with informed patient consent prior to treatment. All fresh tissues were snap-frozen in liquid nitrogen and stored until required. Clinicopathological information of the patients is presented in Table I.

Table I

Clinicopathological features of the oral squamous cell carcinoma patients.

Table I

Clinicopathological features of the oral squamous cell carcinoma patients.

GenderLymph node status


Tumor subsiteCasesMaleFemaleAge (years)Median age (years)PositiveNegative
Tongue2824417–8042.11315
Gum32140–6150.312
Soft plate1107373.001
Floor of the mouth2205353.010
Jaw bones21148–5953.520
DNA and RNA extraction

gDNA from all specimens as well as from 1×106 cells was extracted using an improved method for extracting high-molecular-weight DNA with phenol/chloroform according to previously reported procedures (31). Total RNA was isolated from a 100 mg sample from each specimen and a total number of 107 cells using TRIzol reagent according to the manufacturer’s protocol (Invitrogen, USA).

Reverse transcription-PCR (RT-PCR)

Total RNA was treated with DNAse I (Roche Diagnostics, USA) and purified in accordance with the manufacturer’s instructions. Reverse transcription was performed with 1 μg of treated total RNA using AMV reverse transcriptase (Promega, Madison, WI, USA). An equal amount of cDNA from each sample was amplified using specific primers for each gene (Table II). The conditions for PCR reactions were as follows: 95°C for 5 min, followed by 30–34 cycles of 94°C for 35 sec, 55–58°C for 35 sec, 72°C for 35 sec, and 72°C for 10 min for the extension. The PCR products were electrophoresed on 1% agarose gel and visualized by ethidium bromide staining.

Table II

Summary of primer sequences, annealing temperatures and product sizes.

Table II

Summary of primer sequences, annealing temperatures and product sizes.

Squence (5′-3′)Product size (bp)Annealing temperature (°C)CyclesRef.
Primer for RT-PCR
GAPDHF: GAGATCCCTCCAAAATCAAGTG
R: GAGTCCTTCCACGATACCAAAG
2825828
AXUD1F: TGCGTAACAGTCTCCCACTG
R: CCTGATTTCAGCCCTGTCTC
3015830
BAP1F: TACGCTACAACCGTGCTGTC
R: TCAGCCTCCACACACTTCAG
2955830
FUS1F: GGATCTGGCTCACGAGTTCT
R: GAAGGTTATGGGCCAACAGA
2965530
GNAT1F: CATCTGCAACCACCGCTAC
R: CGCACGTCATGTGGGAATA
2216232
LARS2F: TTACTGGATGCCTGTGGATT
R: CCCGTTGTGTTTGGACTTAC
R: GCCTGCGACATACTGTGGTC
2955630
NPRL2F: CGCATTGCTATCCAGAACCT
R: CTTCATAAGCCCGAACTGGA
3006030
RASSF1AF: ACACGTGGTGCGACCTCT
R: GATGAAGCCTGTGTAAGAACCGTCCT
3085832
SEMA3BF: GTCCTCTTCATTGGCACAGAC
R: GTCGCCATTCCTTACGTCTT
3455630
SEMA3FF: AGTGTCCGTACGATCCCAAG
R: ATGACAGGGTTCCTCACGTC
4665832
ZNF35F: GCGCACATAGGCAGTACTCA
R: TGTCTGAAGACCCTGCACTG
2965830
Primer for MSP
GNAT1MF: GTAAAAGATATATTTATGGTCGGC
MR: CCTCCCAAATAACTAAAATTACGAA
19859
UF: AAAAGATATATTTATGGTTGGGTGA
UR: CCTCCCAAATAACTAAAATTACAAA
19559
SEMA3BMF: TGGTTAGGCGGGGTATTTTC
MR: TCAACAATAAAAACGAAAACG
13354(52)
UF: GTGGTTAGGTGGGGTATTTTT
UR: ATCAACAATAAAAACAAAAACA
13554

[i] F, forward primer; R, reverse primer; M, methylation; U, unmethylation.

Gray scale scanning and data analysis

The intensity of each electrophoretic band was measured using ImageMaster VDS (Pharmacia Biotech Inc., Piscataway, NJ), and analyzed by VDS software version 3.0 for band quantification with GAPDH as an internal control. The expression levels of target genes in the tumors and normal tissues were investigated after they were normalized by transforming them into two groups according to the ratios of the band intensity of the target genes over that of GAPDH for the same sample as previously described (32). A ratio >2 indicated that the corresponding gene was upregulated in OSCC and a ratio <1/2 indicated that the corresponding gene was downregulated in OSCC and all other ratio values indicated that the corresponding genes were expressed at a normal level. Each RT-PCR reaction was carried out in triplicate.

Methylation-specific PCR (MSP)

MSP was performed to analyze the methylation state in the promoter regions of the target genes both in OSCC tissues and TCA8113 cells. One microgram gDNA from OSCC tissues and TCA8113 cells was treated with bisulfite, similar to previous methods (33,34). Each sample was amplified with two sets of primers, one set for methylated DNA and one set for unmethylated DNA. When gDNA was treated with bisulphite, the unmethylated cytosine was tranformed to uracil while methylated cytosine was unaffected. Thus, the methylated and unmethylated promoters were distinguished by MSP. The primers used for the methylated and unmethylated target gene promoter regions are listed in Table II. The PCR products were analyzed by 1% agarose gel electrophoresis and ethidium bromide staining.

Sequencing

DNA sequencing was carried out to identify the methylated and unmethylated sequences. MSP products were electrophoresed on a 1% agarose gel, and the products were recovered by using the QIAquick Gel Extraction kit (Qiagen, USA). The purified DNA was then cloned into the pMD18-T (Takara, Japan) vector and transformed into Escherichia coli strain Top 10 according to the manufacturer’s instructions. The recombinant plasmid DNA extracted from the 5 randomly selected positive clones was used for sequencing.

Treatment of TCA8113 cells with 5-aza-2′-deoxycytidine (5-Aza-Cdc)

TCA8113 cells were plated in 6-well plates at a density of 1×105 cells/well and cultured overnight. On the morning of the next day, the TCA8113 cells were treated with 5-Aza-Cdc, a type of demethylation reagent, at final concentrations (as control) of 0 1, 5, 10, 50 and 100 μM, respectively. After changing the medium and treating cells with 5-Aza-Cdc once per 24 h for 4 consecutive days, TCA8113 cells were collected for RNA and DNA extraction to perform RT-PCR and MSP analysis.

Statistical analysis

Statistical analysis was performed using the Chi-square test and the Student’s t-test. In all analyses, SPSS 11.5 statistical software (SPSS, Chicago, IL) was used, and a P-value <0.05 was considered to indicate a statistically significant result.

Results

Differential expression of the selected genes in OSCC and normal tissues

We selected 10 genes using ‘3p21.3’ and ‘tumor-suppressor genes’ as the search terms in several databases (PubMed, Gene Card, OMIM and Gene Map). The selected genes are listed in Table II.

Differential expression of the target genes was assessed using semi-quantitative RT-PCR combined with Gray Scale Scanning analysis in 12 pairs of OSCC tissues and contralateral normal tissues. The results revealed that the expression of GNAT1, SEMA3B and AXUD1 was downregulated or deficient in the OSCC tissues with percentages of 58.3 (7/12), 41.7 (5/12) and 41.7% (5/12), respectively (P<0.05). The expression of ZNF35 was upregulated in 33.3% (4/12) of OSCC tissues. No significant difference in expression for NPRL2, BAP1, FUS1, LARS2, RASSF1A and SEMA3F was observed between the OSCC and normal tissues (P>0.05) (Figs. 1 and 2). We selected three downregulated genes, GNAT1, SEMA3B and AXUD1, for further study.

Expression of GNAT1, SEMA3B and AXUD1 in OSCC tissues and the TCA8113 cell line

RT-PCR was performed to verify the low expression of GNAT1, SEMA3B and AXUD1 in 36 OSCC tissues, corresponding adjacent normal tissues and TCA8113 cells. The results confirmed that the expression of GNAT1, SEMA3B and AXUD1 was downregulated or undetectable in 44.4 (16/36) 50 (18/36) and 47.2% (17/36) of the OSCC tissues, respectively, compared with the normal expression of these genes in adjacent normal tissues (P<0.05) (Fig. 3B and C). No expression of GNAT1 and SEMA3B was detected and AXUD1 was expressed at a normal level in the TCA8113 cells (Fig. 4A) as compared with the expression in OSCC tissues. No significant correlation was observed between the downregulated genes and OSCC metastasis and patient gender when compared with the clinical data (Table III).

Table III

Statistical analysis of the correlation between the downregulated genes and tumor metastasis.

Table III

Statistical analysis of the correlation between the downregulated genes and tumor metastasis.

Genes

AXUD1GNAT1SEMA3B



GroupsCasesN (%)U (%)N (%)U (%)N (%)U (%)
Metastasis1710 (27.8)7 (19.4)10 (27.8)7 (19.4)10 (27.8)7 (19.4)
Non-metastasis199 (25.0)10 (27.8)9 (25.0)10 (27.8)8 (22.2)11 (30.1)
χ20.4720.1391.003
P-value0.4920.7090.317

[i] N, normal expression; U, underexpression.

Hypermethylation of GNAT1 and SEMA3B in OSCC tissues

We analyzed the methylation status of CpG islands in the promoter region of GNAT1 and SEMA3B in several OSSC tissue samples and adjacent non-cancerous tissues. Hypermethylation of GNAT1 and SEMA3B was found in 75 (12/16) and 77.8% (14/18) of OSSC tissues, respectively. In the non-cancerous tissues, different levels of methylation were also detected with a percentage of 56.25% (9/16) for GNAT1 and 38.9% (8/18) for SEMA3B (Fig. 4A). The results were also confirmed by the sequencing of the MSP products (Fig. 4D and E). Statistical analysis indicated that there was a significant difference in the frequency of methylation of SEMA3B between the OSCC and non-cancerous tissues (χ2 test, P=0.018 <0.05) (Table IV), while no statistical significance was found in the frequency of methylation of GNAT1 between the OSCC and non-cancerous tissues (χ2 test, P=0.709 >0.05), suggesting that methylation of the promoter plays an important role in the downregulation of the SEMA3B gene in OSSC tissues.

Table IV

Statistical analysis of the gene promoter methylation status in OSCC tissues.

Table IV

Statistical analysis of the gene promoter methylation status in OSCC tissues.

Genes

GNAT1SEMA3B


GroupsnM (%)U (%)nM (%)U (%)
Control169 (56.25)7 (43.75)187 (38.9)11 (61.1)
Tumor1612 (75.0)4 (25.0)1814 (77.8)4 (22.2)
χ21.2475.60
P-value0.2640.018

[i] Tumor, OSCC tissues; Control, non-cancerous tissues; M, methylated product; U, unmethylated product.

5-Aza-Cdc partially recovers the expression of SEMA3B in TCA8113 cells

RNA and DNA were extracted from TCA8113 cells treated with different concentrations of 5-Aza-Cdc for 4 days for expression and methylation analysis of SEMA3B by RT-PCR and MSP. The results demonstrated that SEMA3B began to be expressed in the TCA8113 cells when treated with a concentration of 5-Aza-Cdc at 5 μM (Fig. 4C). More unmethylated products were amplified from the DNA samples of TCA8113 cells when the higher concentration of 5-Aza-Cdc was administered (Fig. 4B). This suggests that methylation of the promoter plays a critical role in the downregulation of this gene in TCA8113 cells.

Discussion

Inactivation of TSGs and overexpression of oncogenes are the main causes for tumorigenesis. LOH and hypermethylation in promoter regions are the common reasons accounting for the inactivation of TSGs. The short arm of human chromosome 3, a region with a high frequency of LOH, is considered to be rich in TSG-containing sites as indicated by previous studies on lung cancer, breast cancer, ovarian cancer and other malignant tumors (35). Screening tumor-related TSG candidates is an available method based on loss of a chromosome region. Numerous candidate TSGs have been identified in high frequency chromosome-deleted regions such as DLC-1, a potential TSG at 8p21, a region frequently deleted in various types of cancers (33,36) and DMRT1, DMRT3 and DOCK8 located at 9p24.3, a deleted region in squamous cell lung carcinoma (37). In this study, we selected 10 genes including AXUD1, BAP1, FUS1, GNAT1, LARS2, NPRL2, RASSF1A, SEMA3F, SEMA3B and ZNF35 which were located on region 3p21.3 and analyzed the expression of these genes using RT-PCR to find candidate TSGs in OSCC. Three downregulated genes, SEMA3B, AXUD1 and GNAT1, were identified and both SEMA3B and GNAT1 were downregulated in TCA8113 cells.

AXUD1, also termed cysteine-serine-rich nuclear protein 1 gene (CSRNP1), was cloned by Ishiguro et al(38) while studying the functions of AXIN1 which encodes one of the apoptotic proteins induced by TGF-β in hepatoma carcinoma cells. The full length AXUD1 cDNA is 3188 bp and encodes a 64-kDa protein containing 596 amino acids and is located mainly in the nucleus. AXUD1, a downstream responsive protein for AXIN1, negatively regulates the Wnt pathway which plays an important role in early embryonic development. Studies have shown that the abnormal regulation of the Wnt pathway is related to tumorigenesis. The expression of AXUD1, which is high in normal tissues such as lung, placenta, skeletal muscle, pancreas and leukocytes, was found to be downregulated in lung cancer hepatoma and colorectal carcinoma, suggesting that AXUD1 may be a candidate TSG (38). Simultaneously, knockdown of the expression of DAXUD1, a orthologue of the AXUD1/CSRNP family in Drosophila, was found to induce imaginal cells to proliferate. Conversely, overexpression of DAXUD1 retarded cell cycle progression at mitosis behaving similar to a TSG (39). Our study showed that the expression of AXUD1 was downregualted in 47.2% (17/36) of the OSCC tissues. However, this downregulation had no correlation with patient gender and tumor metastasis, indicating that AXUD1 may be involed in the early malignant transformation of the oral mucous membrane. The function of this gene in OSCC warrants further study.

Guanine nucleotide-binding protein G (G protein) subunit α-1a encoded by GNAT1 is an intracellular protein involved in light signal transduction in the retina (40). G protein, as an energy regulator, participates in the regulation of different transmembrane signal transduction pathways (40). Yi et al(41) found that expression of GNAT1 was downregulated in 72.7% (24/33) of nasopharyngeal carcinoma tissues, which was markedly lower than that in chronic nasopharyngitis tissues (100%, 15/15). The study also demonstrated that downregulation of GNAT1 was associated with LOH, but not with abnormal methylation of the promoter region. In our study, in comparison with the expression in normal control tissues, the expression of GNAT1 was significantly downregulated in 44.4% of the OSCC tissues (P=0.037 <0.05) and in TCA8113 cells. However, the downregulation of GNAT1 had no correlation with patient gender and tumor metastasis, suggesting that GNAT1 may be involved in the early development of OSCC.

SEMA3B, a member of the human semaphoring family is involved in apoptosis (42,43) and in antitumor pathways regulated by p53 (44). SEMA3B was cloned by Sekido et al(45). SEMA3B cDNA encodes an 83-kDa protein of 749 amino acids. Using Northern blot analysis, Lerman and Minna (46) detected wide expression of a 3.4-kb SEMA3B transcript that was strongest in placenta, weaker in other tissues including lung and testis and not detectable in small-cell lung cancer cell lines. Missense mutations were found in non-small cell lung cancer cell lines. The authors also showed that SEMA3B is likely to be an extracellular secreted protein. Lerman and Minna concluded that SEMA3B is an attractive candidate tumor-suppressor gene for methylation and functional analysis. In the present study, we detected the expression of SEMA3B mRNA in OSCC tissues and adjacent control tissues. The results showed that the expression level of SEMA3B was significantly lower in OSCC tissues than that in control tissues, which was confirmed by undetectable expression of SEMA3B in TCA8113 cells. The χ2 test confirmed that no significant correlation existed between the downregulated expression of SEMA3B and patient gender and tumor metastasis.

DNA hypermethylation, one of the most common epigenetic alterations, is involved in inactivation of the expression of many TSGs (4749). MSP is a technique that has facilitated the detection of promoter hypermethylation at CpG islands in cell lines and clinical samples, including fresh and frozen tissues (50). Riquelme et al(51) found that the methylation ratio of the promoter region of SEMA3B is 92% (46/50) in gallbladder carcinoma, suggesting that the methylation of SEMA3B may participate in the carcinogenesis and progression of gallbladder carcinoma. The hypermethylation status of SEMA3B was also recorded in non-small cell lung cancer decreasing the expression of SEMA3B(52,53) and in nasopharyngeal carcinoma for GNAT1, however, it did not account for the low expression of GNAT1(41).

Thus, MSP was performed with DNA samples from OSCC tissues, control tissues and 5-Aza-Cdc-treated TCA8113 cells. Hypermethylation status of SEMA3B in promoter regions was assessed in OSCC tissues, and the expression of SEMA3B was partly recovered in TCA8113 cells treated with 5-Aza-Cdc, demonstrating that the hypermethylation status in the promoter region led to, at least partially, low expression of SEMA3B in OSCC in accordance with studies in other cancer types (5153). As for GNAT1, although hypermethylation in the promoter regions was observed in OSSC tissues, there was no significant difference in expression between OSSC tissues and control samples. Futher studies should be conducted to explore the reasons for the low expression of GNAT1.

In conclusion, our study demonstrated that AXUD1, GNAT1 and SEMA3B, three candidate TSGs located at 3p21.3, may be involved in the development of OSCC, and methylation in the promoter region plays a critical role in the low expression of SEMA3B. These findings may lead to new avenues for the early diagnosis and therapy of OSCC.

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March 2013
Volume 29 Issue 3

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Online ISSN:1791-2431

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Spandidos Publications style
Wang K, Ling T, Wu H and Zhang J: Screening of candidate tumor-suppressor genes in 3p21.3 and investigation of the methylation of gene promoters in oral squamous cell carcinoma. Oncol Rep 29: 1175-1182, 2013
APA
Wang, K., Ling, T., Wu, H., & Zhang, J. (2013). Screening of candidate tumor-suppressor genes in 3p21.3 and investigation of the methylation of gene promoters in oral squamous cell carcinoma. Oncology Reports, 29, 1175-1182. https://doi.org/10.3892/or.2012.2213
MLA
Wang, K., Ling, T., Wu, H., Zhang, J."Screening of candidate tumor-suppressor genes in 3p21.3 and investigation of the methylation of gene promoters in oral squamous cell carcinoma". Oncology Reports 29.3 (2013): 1175-1182.
Chicago
Wang, K., Ling, T., Wu, H., Zhang, J."Screening of candidate tumor-suppressor genes in 3p21.3 and investigation of the methylation of gene promoters in oral squamous cell carcinoma". Oncology Reports 29, no. 3 (2013): 1175-1182. https://doi.org/10.3892/or.2012.2213