Open Access

Identification of differentially expressed genes in oral squamous cell carcinoma TCA8113 cells

  • Authors:
    • Jun Wang
    • Lifeng Li
    • Lina Gao
    • Chao Guan
    • Kexin Su
    • Linlin Li
    • Wenping Luo
    • Hongying Chen
    • Ping Ji
  • View Affiliations

  • Published online on: September 29, 2017     https://doi.org/10.3892/ol.2017.7108
  • Pages:7055-7068
  • Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Previous studies have demonstrated that cancer cells with increased levels of aldehyde dehydrogenase ‘bright’ activity (ALDHbr) exhibit stem cell properties compared with cells exhibiting decreased ALDH activity (ALDHlow). To screen possible biomarkers of cancer stem cells in tongue squamous cell carcinoma, ALDHbr and ALDHlow cells were isolated from the tongue squamous cell carcinoma TCA8113 cell line, and suppression subtractive hybridization was performed to identify differentially expressed genes in the two subpopulations. A total of 240 positive clones were randomly selected for sequencing and were functionally characterized using bioinformatical tools. The results of the present study identified the differential expression of 104 clones, 62 of which corresponded to known genes and 42 of which corresponded to unknown genes. Cluster analysis revealed that the known genes were involved in the regulation of the cell cycle and cell differentiation. In addition, analysis of 10 signaling pathways revealed that genes were markedly altered in the ALDHbr cell subpopulation. Additional study is required to identify the function that these genes serve in the biomolecular regulatory mechanisms of cancer stem cells and to assist in explaining the biological behavior of oral squamous cell carcinoma.

Introduction

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck squamous cell carcinoma (HNSCC), and is among the 10 most prevalent cancer types worldwide (1,2). In spite of improvements in the diagnosis and prognosis of OSCC, long-term survival rates have not improved in the past decade (3). To develop effective therapies, an improved understanding of the biological features and underlying molecular mechanisms of OSCC are required.

In previous studies, it has been suggested that the cancer stem cell (CSC) hypothesis may be applied to a number of types of cancer (4,5). According to the hypothesis, a tumor may be viewed as an aberrant organ initiated by a subpopulation of cells, termed CSCs, which exhibit self-renewing capacities and are responsible for tumor maintenance and metastasis (6). The hypothesis provides a novel insight into the understanding of tumorigenesis and since then, the isolation and identification of CSCs have been studied in depth. Previous studies have supported the validity of this hypothesis in a number of malignant diseases, including breast cancer, brain tumor, colon cancer, melanomas and prostate cancer (711). In addition, the existence of CSCs has been identified in HNSCC and has been associated with the expression of aldehyde dehydrogenase (ALDH) (12). Cells with increased ADLH ‘bright’ activity (ALDHbr) exhibit CSC-associated properties, including radio-resistance and the ability to produce tumors with a limited number of cells, which is in contrast to cells with decreased ALDH activity (ALDHlow) (13,14). However, the gene expression profile of the two cell subpopulations remains unknown, which is required to understand the underlying molecular mechanisms of CSCs in HNSCC.

In the present study, ALDHbr and ALDHlow cells were isolated from the OSCC TCA8113 cell line and suppression subtractive hybridization (SSH) was subsequently performed to identify differentially expressed genes in the two subpopulations. Known and unknown differentially expressed genes were identified in subtracted clones, and the known genes were functionally characterized using bioinformatical tools. The results of the present study suggested that the identified genes may be biomarkers for the identification of CSCs in OSCC.

Materials and methods

Cells and cell culture

The tongue squamous cell carcinoma TCA8113 cell line was obtained from the West China College of Stomatology of Sichuan University (Sichuan, China). Cells were maintained in RPMI 1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.), 1% glutamine and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO2.

ALDH assay and cell sorting

An Aldefluor kit (Stemcell Technologies, Inc., Vancouver, BC, Canada) was used to determine ALDH activity in TCA8113 cells, according to the manufacturer's protocol. Cells were suspended in Aldefluor assay buffer, which contained an activated Aldefluor substrate (BAAA, 1 µmol/1×106 cells), as recommended by the manufacturer. As a negative control for all samples, an aliquot of ‘Aldefluor-exposed’ cells (1×108 cells) was transfused into the control tube, which contained 5 µl diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor. Following incubation at 37°C for 40 min, the cells were centrifuged at 250 × g for 5 min and the supernatant was removed. Subsequently, the cell pellets were resuspended in 0.5 ml ice-cold Aldefluor Assay Buffer, and flow cytometric analysis was performed using FACSAria (BD Biosciences, Franklin Lakes, NJ, USA). Aldefluor staining was determined using a green fluorescence channel. Samples treated with DEAB were used as controls and set the threshold that defined the ALDHbr region.

Tumorsphere formation

Since CSCs typically form tumorspheres and non-CSCs die in serum-free medium (15,16), tumorsphere formation in ALDHbr and ALDHlow cells was investigated in the present study. Cells were plated at a low density (1,000 cells/ml) in RPMI1 640 serum-free medium, supplemented with human recombinant epidermal growth factor (20 ng/ml; PeproTech, Inc., Rocky Hill, NJ, USA), basic fibroblast growth factor (20 ng/ml; PeproTech, Inc.) and B27 serum-free supplements (20 µl/ml; Invitrogen; Thermo Fisher Scientific, Inc.). The formation of tumorspheres was observed daily using an inverted phase contrast microscope (magnification, ×100).

Preparation of total RNA

Total RNA was isolated from ALDHbr and ALDHlow cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was quantified using a Unico UV-2000 spectrophotometer (Unico Technologies Co., Ltd., Jiangsu, China). The A260/A280 ratio was between 1.8 and 2.0. Total RNA (~1 µg) was separated on denaturing agarose (1.2% gel) to confirm integrity.

cDNA synthesis

cDNA was synthesized using the SMART™ cDNA Synthesis kit (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer's protocol. Total RNA (1 µg) was reverse transcribed (42°C, 1.5 h) in a 10-µl reaction mixture containing PowerScript™ reverse-transcriptase. Sterile H2O (24.2 µl), 5X Second-Strand Buffer (8.0 µl), dNTP mix (10 mM; 0.8 µl) and 20X Second-Strand Enzyme Cocktail (2.0 µl) were added to the 10 µl first-strand synthesis reaction tubes and incubated at 16°C for 2 h in water. T4 DNA polymerase (Clontech Laboratories, Inc.) 2 µl was then added followed by incubation at 16°C for 30 min in a water bath. Subsequently, 4 µl of 20X EDTA/glycogen mix was added to terminate second-strand synthesis, followed by addition of 100 µl of phenol:chloroform:isoamyl alcohol (25:24:1). Centrifugation was then performed at 2,191.28 × g for 10 min at room temperature. The top aqueous layer was collected and placed in a fresh 0.5-ml microcentrifuge tube. The inter and lower phases were discarded and disposed appropriately. Next, 100 µl of chloroform:isoamyl alcohol (24:1) was added, followed by addition of 40 µl of 4 M NH4OAc and 300 µl of 95% ethanol. Subsequently, centrifugation was performed at 2,191.28 × g for 20 min at room temperature, and the supernatant was collected. The pellet was overlayed with 500 µl of 80% ethanol, and then centrifuged at 2,191.28 × g for 10 min at room temperature. The supernatant was removed and the pellet was air-dried for ~10 min to evaporate residual ethanol. Precipitate was dissolved in 50 µl of sterile H2O, and 6 µl was transferred to a fresh microcentrifuge tube. This sample was stored at −20°C until after RsaI digestion (for agarose gel electrophoresis) to estimate yield and size range of ds cDNA products synthesized.

SSH

Synthesized two-target cDNA was used for SSH, performed with the PCR-select™ cDNA Subtraction kit (Clontech Laboratories, Inc.), according to the manufacturer's protocol. cDNA from ALDHbr and ALDHlow cells was used as the ‘tester’ and ‘driver’, respectively, in the forward subtraction and vice versa for the reverse subtraction. For each subtraction, the ‘tester’ was ligated to adaptor 1 and adaptor 2R in separate ligation reactions, whereas the ‘driver’ was not ligated to adaptors. Following ligation, two samples were subjected to hybridization. For the first hybridization, an excess of ‘driver’ cDNA was added to each adaptor-ligated ‘tester’ cDNA in the hybridization buffer, heat-denatured (98°C, 1.5 min) and subsequently annealed (68°C, 8 h). The two samples from the first hybridization were mixed and fresh denatured ‘driver’ cDNA was added and annealed at 68°C overnight. Following the second hybridization, the sample was diluted in 200 µl dilution buffer and incubated at 68°C for 7 min in a thermal cycler. Subsequently, PCR was performed using the subtracted cDNAs to amplify the desired differentially expressed sequences. The first-round PCR was performed using PCR primer 1 (5′-CTAATACGACTCACTATAGGGC-3′) and the cycling parameters were 72°C for 10 min and 95°C for 2 min, followed by 25 cycles of 94°C for 30 sec, 62°C for 45 sec, 72°C for 1 min and 72°C for 6 min. The second-round PCR reaction was performed using nested primer 1 (5′-TCGAGCGGCCGCCCGGGCAGGT-3′) and nested primer 2R (5′-AGCGTGGTCGCGGCCGAGGT-3′), and the cycling parameters were 95°C for 2 min, followed by 29 cycles of 94°C for 30 sec, 65°C for 45 sec, 72°C for 1 min and 72°C for 6 min.

Cloning of SSH-PCR products

The purified secondary SSH-PCR products were cloned into PMD-18T vector (Takara Bio, Inc., Otsu, Japan) and the ligated products were transformed into E. coli DH5α competent cells. Transformed colonies were selected on Luria-Bertani (LB) agar medium (MP Biomedicals, Santa Ana, CA, USA) containing ampicillin (100 mg/l) at 37°C and ~1,000 positive colonies were obtained, which represented subtraction libraries enriched with differentially expressed genes. A total of 240 positive colonies were selected randomly. A single clone was inoculated in 2 ml LB-ampicillin (100 mg/l) and incubated overnight at 37°C with gentle agitation at 44.72 × g.

PCR amplification of cDNA inserts

To assess the size of inserts, colony PCR was performed in a 50-µl reaction system containing 12.5 µl 10X buffer (Takara Bio, Inc.), 1 µl 10 mM dNTP (Shanghai CPG Biotechnology Co., Ltd., Shanghai, China), 5 µl MgCl2 (Takara Bio, Inc.), 1 µl 50 pM/µl Nested primer 1 (Clontech Laboratories, Inc.), 1 µl 50 pM/µl 2R primer (Clontech Laboratories, Inc.), and 2.5 U Taq DNA polymerase (Takara Bio, Inc.). The PCR parameters were: 95°C for 2 min, followed by 35 cycles of 95°C for 30 sec, 62°C for 45 sec and 72°C for 1 min. Colony PCR products (2 µl) were separated using agarose (1.2% gel) to identify the presence and the size of the inserts prior to sequencing. The controls for this protocol included the unsubtracted tester control for the forward subtraction, the unsubtracted tester control for the reverse subtraction and the unsubtracted tester control for the control skeletal muscle tester cDNA [made from the Control Poly A+ RNA (from human skeletal muscle) provided with the kit (the SMART™ cDNA Synthesis kit (Clontech Laboratories, Inc.)]. It serves as control driver cDNA subtraction. All protocols were repeated 3 times.

Expressed sequenced tag (EST) sequencing and bioinformatical analysis

The selected positive clones were sequenced at the Beijing Genomics Institute (Beijing, China) and the sequences were edited to remove the adaptor-primer and vector DNA sequences. ESTs were compared with non-redundant public databases using the Basic Local Alignment Search Tool (BLAST) (blast.ncbi.nlm.nih.gov/Blast.cgi) nucleotide to retrieve data from GenBank (www.ncbi.nlm.nih.gov/nucleotide) and BLASTX (blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) algorithms of the National Center for Biotechnology Information (NCBI; blast.ncbi.nlm.nih.gov/Blast.cgi). ESTs with E<0.01 were deemed to exhibit significant homology. Homologies >50 nucleotides that exhibited >90% identity to sequences in the database were considered to have significant homologies, as previously described (17). The physiological functions of these ESTs were classified according to Gene Ontology (www.geneontology.org). Pathway analysis was performed using the Gene Set Analysis Toolkit V2 online system (www.webgestalt.org/option.php).

Results

Isolation of ALDHbr cells in tongue squamous cell carcinoma TCA8113 cells

Using the ALDEFLUOR assay and fluorescence-activated cell sorting analysis, the ALDH enzymatic activity in the tongue squamous cell carcinoma TCA8113 cell line was identified to be heterogeneous. As presented in Fig. 1, only a limited proportion (1.3%) of the cells displayed increased ALDH activity (ALDHbr; Fig. 1A), whereas the remaining cells expressed decreased levels of ALDH activity (ALDHlow). DEAB, the specific inhibitor of ALDH, resulted in a decreased proportion of sorted ALDHbr cells (0.1%; Fig. 1B), suggesting the effective isolation of ALDHbr cells. The results of the present study revealed that cancer stem cells with ALDHbr were successfully isolated. Subpopulation cells were selected for additional analysis.

ALDHbr cells form spheres

CSCs may be effectively enriched in serum-free medium (1820). The majority of cells die in serum-free medium due to a lack of nutritive materials; however, CSCs may survive, proliferate and form three-dimensional spheres. In the present study, ALDHbr cells maintained in serum-free medium proliferated and formed spheres within 5 days, whereas ALDHlow cells, maintained in the same medium, did not form spheres and were apoptotic (Fig. 2). The results of the present study indicated that the isolated ALDHbr cells exhibited typical CSC features.

Constructing the SSH library

Using cDNA from ALDHbr cells as ‘testers’ and that of ALDHlow cells as ‘drivers’ and vice versa, PCR-selected cDNA subtraction for forward and reverse libraries, respectively, was performed. Following subtraction, a pool of putative differentially expressed cDNA fragments was obtained. The cDNA fragments ranged between 200 bp and 1 kb, with the majority distributed between 400 and 600 bp (Fig. 3A). Subtracted amplicons were ligated into the PMD-18T plasmid vector and transformed into E. coli DH5α competent cells. In total, 240 white colonies were randomly selected and 48 of these clones were subjected to colony PCR, using nested primers. All the recombinants determined revealed amplicons ranging between 200 and 800 bp (Fig. 3B).

Identification of differentially expressed ESTs

All 240 clones were selected and sequenced. Sequences were not obtained for 14 clones and those were omitted from the present study. Comparison of the unique sequences obtained from each library against the GenBank databases identified 104 unique clones, 62 of which corresponded to known genes and 42 of which were unknown genes, while the remaining 122 clones were redundant. Of the known genes, 28 and 34 genes were upregulated and downregulated in ALDHbr cells, respectively (Tables I and II). The unknown clones were divided into two groups in the NCBI databases, 28 represented human genomic sequences and 14 were present in the human EST database.

Table I.

Characteristics of overexpressed known genes in aldehyde dehydrogenase-positive subpopulation cells.

Table I.

Characteristics of overexpressed known genes in aldehyde dehydrogenase-positive subpopulation cells.

No.Length, bpGeneAccession no.Identities (%)E-valueGene IDGene symbolChromosomal location
1206Drosophila melanogaster CG4699 (CG4699), transcript variant J ref|NM_001170153.1|163/166 (99) 2.00×10−7641911 CG4699CG4699 (WAH)Unknown
2402Homo sapiens solute carrier family 25, member 13 (citrin) (SLC25A13), RefSeqGene on chromosome 7 ref|NG_012247.1|388/389 (99)010165SLC25A137q21.3
3303Homo sapiens kelch-like 2, Mayven (Drosophila) (KLHL2), transcript variant 3, mRNA ref|NM_001161522.1|284/285 (99) 8.00×10−14511275 KLHL2KLHL24q21.2
4225Homo sapiens Niemann-Pick disease, type C1 (NPC1), RefSeqGene on chromosome 18 ref|NG_012795.1|174/175 (99) 3.00×10−834864NPC1   18q11-q12
5369Homo sapiens EP300 interacting inhibitor of differentiation 1 (EID1), mRNA ref|NM_014335.2|353/354 (99)023741 EID1EID115q21.1-q21.2
6289Homo sapiens notch 2 (NOTCH2), RefSeqGene on chromosome 1 ref|NG_008163.1|512/514 (99)04853NOTCH21p13-p11
7234Homo sapiens BRCA1 associated RING domain 1 (BARD1), RefSeqGene ref|NG_012047.1|215/216 (99) 1.00×10−106157266327BARD12q34-q35
8272Homo sapiens inositol 1,4,5-triphosphatereceptor, type 1 (ITPR1), RefSeqGene on chromosome 3 ref|NG_016144.1|253/254 (99) 1.00Ex10−127269954693ITPR13p26-p25
9289Homo sapiens FRG1 (FRG1) gene, complete cds; gb|AF146191.1|AF146191264/272 (98) 2.00×10−125AAD46768.1FRG14q35
10280Homo sapiens methylcrotonoyl-CoA carboxylase 2 (beta) (MCCC2), RefSeqGene on chromosome 5 ref|NG_008882.1|263/263 (100) 3.00×10−13464087MCCC25q12-q13
11410Homo sapiens ribosomal protein, large, P0 (RPLP0), transcript variant 1, mRNA ref|NM_001002.3|388/390 (99)06175 RPLP0RPLP012q24.2
12468Homo sapiens dedicator of cytokinesis 8 (DOCK8), transcript variant 3, mRNA ref|NM_001193536.1|449/450 (99)081704 DOCK8DOCK89p24.3
13365Homo sapiens GLIS family zinc finger 3 (GLIS3), RefSeqGene on chromosome 9 ref|NG_011782.1|342/347 (99) 1.00×10−172169792GLIS39p24.2
14412Homo sapiens PARK2 co-regulated (PACRG) on chromosome 6 ref|NG_011525.1|352/392 (90) 5.00×10−138135138PACRG6q26
15153Homo sapiens collagen, type VII, alpha 1(COL7A1), mRNA ref|NM_000094.3|134/135 (99) 7.00×10−631294 COL7A1COL7A13p21.1
16226Homo sapiens epidermal growth factor receptor(EGFR), RefSeqGene on chromosome 7 ref|NG_007726.1|207/208 (99) 3.00×10−1021956EGFR7p12
17450Homo sapiens neuron navigator 2 (NAV2), transcript variant 4, mRNA ref|NM_001111019.1|391/399 (98)089797 NAV2NAV211p15.1
18288Homo sapiens retinoblastoma 1 (RB1), mRNA ref|NM_000321.2|269/270 (99) 1.00×10−1375925 RB1RB113q14.2
19274Homo sapiens tetratricopeptide repeat protein 12 (TTC12) gene, complete cds, alternatively splicedgb|EF445041.1|214/257 (84) 4.00×10−58ACA06092.1TTC1211q23.1
20232Homo sapiens nuclear receptor corepressor 1 (NCOR1), transcript variant 3, mRNA ref|NM_001190440.1|213/214 (99) 1.00×10−106  9611 NCOR1NCOR117p11.2
21283Homo sapiens SMAD family member 1 (SMAD1), transcript variant 2, mRNA ref|NM_001003688.1|262/263 (99) 1.00×10−1334086 SMAD1SMAD17p15
22646Homo sapiens kelch-like 13 (Drosophila) (KLHL13), RefSeqGene on chromosome X ref|NG_016759.1|628/630 (99)090293KLHL13Xq23-q24
23418Homo sapiens PRP39 pre-mRNA processing factor 39 homolog (S. cerevisiae) (PRPF39), mRNA ref|NM_017922.3|395/399 (99)055015 PRPF39PRPF3914q21.3
24398Homo sapiens septin 9 (SEPT9), transcript variant 4, mRNA ref|NM_001113495.1|379/380 (99)010801 SEPT9SEPT17q25
25537Homo sapiens ATPase, Ca++ transporting, plasma membrane 4 (ATP2B4), transcript variant 1, mRNA ref|NM_001001396.1|186/188 (99) 5.00×10−90493 ATP2B4ATP2B41q32.1
26392PREDICTED: Homo sapiens hypothetical LOC441072 (FLJ31104), partial miscRNA ref|XR_113742.1|203/225 (91) 3.00×10−76441072 FLJ31104FLJ311045q11.2
27227Homo sapiens CD44 molecule (Indian blood group) (CD44), RefSeqGene on chromosome 11 ref|NG_008937.1|207/207 (100) 3.00×10−103960CD4411p13
28364Pongo abelii probable methyltransferase TARBP1-like (LOC100447859), mRNA ref|XM_002809289.1|120/124 (97) 3.00×10−51100447859 LOC100447859LOC100447859Unknown

Table II.

Characteristics of downregulated known genes in aldehyde dehydrogenase-positive subpopulation cells.

Table II.

Characteristics of downregulated known genes in aldehyde dehydrogenase-positive subpopulation cells.

No.Length, bpGeneAccession no.Identities (%)E-valueGene IDGene symbolChromosomal location
1410Pan troglodytes hypothetical protein LOC736141 (LOC736141), mRNA ref|XM_001135501.1|322/364 (89) 3.00×10−117  736141 LOC736141LOC736141X
2505Homo sapiens claudin domain containing 1 (CLDND1), transcript variant 6, mRNA ref|NM_001040199.1|318/320 (99) 9.00×10−16356650 CLDND1CLDND13q12.1
3383Homo sapiens ankyrin repeat domain 36B (ANKRD36B), mRNA ref|NM_025190.3|297/368 (81) 4.00×10−7057730 ANKRD36BANKRD36B2q11.2
4357Homo sapiens catenin (cadherin-associated protein), alpha 1, 102 kDa (CTNNA1), mRNA ref|NM_001903.2|336/337 (99) 1.00×10−1741495 CTNNA1 |CTNNA15q31
5364Homo sapiens electron-transfer-flavoprotein, alpha polypeptide (ETFA), RefSeqGene on chromosome 15 ref|NG_007077.2|  344/346 (99) 5.00×10−1772108ETFA15q23-q25
6493Pan troglodytes RAB7, member RAS oncogene family-like 1, transcript variant 1 (RAB7L1), mRNA ref|XM_001162387.1|264/296 (90) 9.00×10−98469654 RAB7L1RAB7L11q32
7635Homo sapiens wings apart-like homolog (Drosophila) (WAPAL), mRNA ref|NM_015045.2|617/618 (99)023063 WAPALWAPAL/WAPL10q23.2
8459Homo sapiens KIAA0101 (KIAA0101), transcript variant 2, mRNA ref|NM_001029989.1|441/442 (99)09768 KIAA0101KIAA0101/PAF15q22.31
9299Homo sapiens RAN binding protein 10 (RANBP10), mRNA ref|NM_020850.1|  281/281 (100) 2.00×10−14557610 RANBP10RANBP1016q22.1
10224Homo sapiens mitochondrial ribosomal protein S27 (MRPS27), nuclear gene encoding mitochondrial protein, mRNA ref|NM_015084.2|202/204 (99) 2.00×10−9923107 MRPS27MRPS275q13.2
11426Homo sapiens vacuolar protein sorting 13 homolog A (S. cerevisiae) (VPS13A), RefSeqGene on chromosome 9 ref|NG_008931.1|408/410 (99)023230VPS13A9q21
12413Homo sapiens SET binding factor 2 (SBF2), RefSeqGene on chromosome 11 ref|NG_008074.1|392/396 (99)081846SBF211p15.4
13613Homo sapiens MT-RNR2-like 2 (MTRNR2L2), mRNA ref|NM_001190470.1|562/597 (95)0100462981MTRNR2L2Unknown
14423Homo sapiens MT-RNR2-like 8 (MTRNR2L8), mRNA ref|NM_001190702.1|373/399 (94) 9.00×10−167   100463486 MTRNR2L8MTRNR2L8Unknown
15588Homo sapiens WD repeat domain 7 (WDR7), transcript variant 2, mRNA ref|NM_052834.2|133/133 (100) 2.00×10−6323335 WDR7WDR718q21.1-q22
16261Pan troglodytes similar to ORF1; putative (LOC745921), mRNA ref|XR_021946.1|276/326 (85) 6.00×10−83745921 LOC745921LOC745921Unknown
17459Homo sapiens ornithine decarboxylase 1 (ODC1), mRNA ref|NM_002539.1|570/570 (100)04953 ODC1 |ODC12p25
18411Homo sapiens ataxin 7 (ATXN7), RefSeqGene on chromosome 3 ref|NG_008227.1|393/394 (99)080145ATXN73p21.1-p12
19494Homo sapiens zinc finger protein 573 (ZNF573), transcript variant 5, mRNA ref|NM_001172692.1|476/476 (100)0126231 ZNF573ZNF57319q13.12
20538Homo sapiens bromodomain containing 2 (BRD2), transcript variant 3, mRNA ref|NM_001199455.1|297/299 (99) 4.00×10−1516046 BRD2BRD26p21.3
21353PREDICTED: Pan troglodytes similar to uracil DNA glycosylase (LOC743143), mRNA ref|XR_021793.1|326/332 (98) 2.00×10−163743143 LOC743143LOC743143Unknown
22380Homo sapiens SET nuclear oncogene (SET), transcript variant 1, mRNA ref|NM_001122821.1|360/360 (100)06418 SETSET9q34
23205Homo sapiens peroxisomal biogenesis factor 19 (PEX19), transcript variant 4, mRNA ref|NM_001193644.1|184/185 (99) 2.00×10−905824 PEX19PEX191q22
24386Homo sapiens microphthalmia-associated transcription factor (MITF), RefSeqGene on chromosome 3 ref|NG_011631.1|369/369 (100)04286MITF3p14.2-p14.1
25449Homo sapiens fatty acid desaturase 1 (FADS1), mRNA ref|NM_013402.4|431/432 (99)03992 FADS1FADS111q12.2-q13.1
26352Homo sapiens protein tyrosine phosphatase type IVA, member 1 (PTP4A1), mRNA ref|NM_003463.3|332/335 (99) 3.00×10−1707803 PTP4A1PTP4A16q12
27323Homo sapiens chromosome X open reading frame 57 (CXorf57), transcript variant 2, mRNA ref|NM_001184782.1|303/305 (99) 2.00×10−15555086 CXorf57CXorf57Xq22.3
28214Homo sapiens CREB binding protein (CREBBP), RefSeqGene on chromosome ref|NG_009873.1|  197/198 (99) 1.00×10−961387CREBBP16p13.3
29436Homo sapiens mesencephalic astrocyte-derived neurotrophic factor (MANF), mRNA ref|NM_006010.4|411/415 (99)07873 MANFMANFUnknown
30263Homo sapiens ADP-ribosylation factor-like 6 interacting protein 1 (ARL6IP1), mRNA ref|NM_015161.1|243/243 (100) 3.00×10−12423204 ARL6IP1ARL6IP116p12-p11.2
31501Homo sapiens RNA, 18S ribosomal 1 (RN18S1), ribosomal RNA ref|NR_003286.2|479/480 (99)0100008588 RN18S1RN18S1Unknown
32248Homo sapiens eukaryotic elongation factor-2 kinase (EEF2K), mRNA ref|NM_013302.3|227/227 (100) 3.00×10−11529904 EEF2KEEF2K16p12.1
33486Homo sapiens myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila) (MLL), transcript variant 2, mRNA ref|NM_005933.3|466/469 (99)04297 MLLMLL11q23
34499PREDICTED: Macaca mulatta ATP synthase subunit a-like (LOC100426362), mRNA ref|XM_002806045.1|359/441 (81) 3.00×10−93100426362 LOC100426362LOC100426362Unknown
Functional classification of differentially expressed ESTs

On the basis of the functional annotation using Gene Ontology (GO) software, 62 differentially expressed genes were grouped into a number of categories (Fig. 4). In the GO category of biological processes, the highly enriched categories included those associated with metabolic processes (28 genes), biological regulation (21 genes) and developmental processes (16 genes). The molecular functions with those highly enriched genes were associated with protein binding (32 genes) and cellular components in the nucleus (24 genes).

Pathway analysis of differentially expressed ESTs

Signal pathway analysis was performed based on the Wikipathways database and the Pathway Commons database, using the Gene Set Analysis Toolkit V2. The 10 signaling pathways with the most marked alterations in each database are presented in Tables III and IV, and included the transforming growth factor (TGF-) β signaling pathway, the Notch signaling pathway and the c-kit pathway. Typically, ~10 genes were enriched in these pathways and each gene may participate in a number of pathways.

Table III.

Pathway analysis, on the basis of the pathway commons database.

Table III.

Pathway analysis, on the basis of the pathway commons database.

Signaling pathwayEntrez IDsEnrichment statistics
Notch-HLH transcription1387, 4853C=6; O=2; E=0.01; R=290.63; rawP=1.93×10−5; adjP=0.0004
TGF-β receptor1387, 5925, 960C=126; O=3; E=0.14; R=20.76; rawP=0.0004; adjP=0.0032
Generic transcription1387, 4853C=28; O=2; E=0.03; R=62.28; rawP=0.0005; adjP=0.0032
Microphthalmia-associated transcription factor1387, 4286C=51; O=2; E=0.06; R=34.19; rawP=0.0016; adjP=0.0051
Signaling events mediated by stem cell factor receptor (c-Kit)1387, 9611, 4286, 29904C=436; O=4; E=0.50; R=8.00; rawP=0.0016; adjP=0.0051
BMP receptor4086, 4286, 29904C=189; O=3; E=0.22; R=13.84; rawP=0.0014; adjP=0.0051
NOTCH4853, 9611C=58; O=2; E=0.07; R=30.07; rawP=0.0020; adjP=0.0054
Regulation of cytoplasmic and nuclear SMAD2/39611, 4286, 29904C=265; O=3; E=0.30; R=9.87; rawP=0.0035; adjP=0.0066
TGF-β receptor9611, 4286, 29904C=265; O=3; E=0.30; R=9.87; rawP=0.0035; adjP=0.0066
Androgen receptor1387, 5925C=79; O=2; E=0.09; R=22.07; rawP=0.0038; adjP=0.0066

Table IV.

Pathway analysis, on the basis of the Wikipathways database.

Table IV.

Pathway analysis, on the basis of the Wikipathways database.

Signaling pathwayEntrez IDsEnrichment statistics
∆-Notch9611, 4853, 4086, 1956C=86; O=4; E=0.10; R=40.55; rawP=3.05×10−6; adjP=3.97×10−5
Senescence and autophagy5925, 960, 4297C=60; O=3; E=0.07; R=43.60; rawP=4.65×10−5; adjP=0.0003
Androgen receptor1387, 5925, 1956C=115; O=3; E=0.13; R=22.75; rawP=0.0003; adjP=0.0013
B cell receptor5925, 3708, 493C=158; O=3; E=0.18; R=16.56; rawP=0.0008; adjP=0.0021
TGF-β receptor1387, 5925, 960C=155; O=3; E=0.18; R=16.88; rawP=0.0008; adjP=0.0021
Notch1387, 4853C=46; O=2; E=0.05; R=37.91; rawP=0.0013; adjP=0.0026
Id5925, 4086C=51; O=2; E=0.06; R=34.19; rawP=0.0016; adjP=0.0026
TGF-β1387, 4086C=52; O=2; E=0.06; R=33.53; rawP=0.0016; adjP=0.0026
Estrogen1387, 9611C=76; O=2; E=0.09; R=22.94; rawP=0.0035; adjP=0.0051
Wnt and pluripotency1387, 960C=98; O=2; E=0.11; R=17.79; rawP=0.0057; adjP=0.0067

Discussion

CSCs refer to a subset of tumor cells that exhibit the capability to self-renew and generate diverse cells that comprise the tumor (4,21), and have been termed CSCs to reflect the ‘stem-like’ properties and the ability to sustain tumorigenesis. CSCs share important properties with healthy tissue stem cells, including the capacity for self-renewal and differentiation. An implication of the CSC hypothesis is that cancer cells are hierarchically arranged with CSCs located at the apex of the hierarchy (22). CSCs are the only cells that may maintain tumor viability indefinitely. The remaining cells, although actively proliferating and comprising the majority of the tumor, are differentiating and destined to die. The identification of CSCs has marked implications in the study of cancer biology. Previous studies (711) have indicated the existence of CSCs in a number of solid tumors and a variety of cell surface makers have been used to isolate CSC subpopulations, including cluster of differentiation (CD)24, CD133 and CD24; however, none of these markers are exclusively expressed by CSCs in solid tumors.

ALDH is a member of the family of NAD(P)+-dependent enzymes involved in detoxifying a variety of aldehydes to the corresponding weak carboxylic acids (23). The use of ALDH activity in flow cytometry-based methods has enabled the isolation of viable CSC subpopulations in a number of cancer types (2426). In the present study, CSCs were enriched from the tongue squamous cell carcinoma TCA8113 cell line, according to the overexpression of ALDHbr. ALDHbr cells comprised 1.3% of the total cell population, which is consistent with previous studies (13,14). Therefore, ALDHbr-associated CSCs were successfully isolated for additional investigation.

In order to identify stem cell associated genes differentially expressed in ALDHbr and ALDHlow cells, SSH was performed. SSH is advantageous compared with other PCR-based techniques as it selectively amplifies target cDNA fragments (differentially expressed), and simultaneously suppresses non-target DNA amplification, to generate a library of differentially expressed sequences (27). The normalization step equalizes the abundance of cDNAs within a target population and the subtraction step excludes the common sequences between the driver and tester populations (27). In addition, the advantage compared with microarrays is that SSH may isolate novel differentially expressed genes (28). In the present study, two SSH libraries were constructed from cDNAs obtained from ALDHbr and ALDHlow cells, and a total of 240 clones were selected and sequenced. Using GenBank databases, 28 and 34 known genes were identified from the forward and reverse libraries, respectively. A total of 28 of clones revealed homology with chromosome sequences and 14 clones demonstrated homology with ESTs. The known genes were grouped into functional categories on the basis of GO.

In the GO category of biological process, the highly enriched categories included those associated with metabolic processes (28 genes), biological regulation (21 genes) and developmental processes (16 genes). The results of the present study suggested that abnormal stem cell homeostasis associated with the aforementioned processes would result in malignant changes in stem cells.

Signaling pathway analysis identified the 10 pathways that exhibited marked alterations in the Wikipathways database and Pathway Commons database, which included Notch and TGF-β signaling pathways, which have been identified to serve important roles in the regulation of stem cell self-renewal, multi-potency and cell-fate determination (29,30). In addition, one gene may participate in different signaling pathways at the same time; for example, the gene encoding cAMP response element-binding protein (CREB) binding protein (CREBBP/CBP) was involved in 7 of the aforementioned signaling pathways and notably interacted with Wnt signaling to maintain the pluriporency of murine embryonic stem cells in long-term culture (31). A previous study demonstrated that CBP was critical in maintaining an adequate pool of murine hematopoietic stem cells through self-renewal and was important for preventing hematological tumor formation (32), suggesting that CBP was associated with the biological regulation of normal stem cells. There have been a limited number of studies on the expression and function of CBP in CSCs, therefore, whether CBP is a marker of CSCs in tongue squamous cell carcinoma remains unknown. Additionally, nuclear receptor corepressor 1 (NCOR1) was involved in a number of signaling pathways and was initially defined as a regulator of nuclear receptor-mediated repression. NCOR is expressed in the nucleus of neural stem cells (NSCs) and is a regulator of neural stem cells. Following phosphorylation, NCOR translocates to the cytoplasm and induces the astrocytic differentiation of NSCs (33). Furthermore, NCOR has been identified to maintain normal intestinal epithelial cell viability, and silencing of NCOR1 expression in proliferating cells of crypt origin resulted in a rapid viability arrest without associated cell death (34). In glioblastoma multiforme (GBM), NCOR was expressed in the nucleus of undifferentiated CSCs and the nuclear localization of NCOR may function as a marker of GBM stem cells (35).

Differentially expressed genes in tongue squamous carcinoma stem-like cells were profiled using the SSH technique. A total of 62 genes were identified as upregulated or downregulated in tongue squamous carcinoma stem-like cells (termed ALDHbr cells), suggesting that distinct gene expression profiles are present in CSCs. CBP and NCOR1 genes were involved in a number of signaling pathways in ALDHbr cells. The results of a literature review suggested that CBP and NcoR1 may be CSCs markers (3235), which is consistent with the results of the present study. Although the results of the present study are preliminary, a group of candidate genes have been identified, which require additional study.

Acknowledgements

The present study was supported by the ChonQing Science and Technology Commission Project (grant no. 2013-1-030). The authors thank Medjaden Bioscience Ltd. (Hong Kong, China) for assisting in the preparation of the original manuscript.

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December 2017
Volume 14 Issue 6

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APA
Wang, J., Li, L., Gao, L., Guan, C., Su, K., Li, L. ... Ji, P. (2017). Identification of differentially expressed genes in oral squamous cell carcinoma TCA8113 cells. Oncology Letters, 14, 7055-7068. https://doi.org/10.3892/ol.2017.7108
MLA
Wang, J., Li, L., Gao, L., Guan, C., Su, K., Li, L., Luo, W., Chen, H., Ji, P."Identification of differentially expressed genes in oral squamous cell carcinoma TCA8113 cells". Oncology Letters 14.6 (2017): 7055-7068.
Chicago
Wang, J., Li, L., Gao, L., Guan, C., Su, K., Li, L., Luo, W., Chen, H., Ji, P."Identification of differentially expressed genes in oral squamous cell carcinoma TCA8113 cells". Oncology Letters 14, no. 6 (2017): 7055-7068. https://doi.org/10.3892/ol.2017.7108