Introduction

Head and neck squamous cell carcinoma (HNSCC), the sixth most common malignancy worldwide, arises in the oral cavity, oropharynx, larynx or hypopharynx.1 In spite of considerable advances in multimodality therapy including surgery, radiotherapy and chemotherapy, the overall 5-year survival rate for patients with HNSCC is only 40–50%. It is among the lowest of the major cancer types and has not improved dramatically for decades.2 HNSCC is a disease characterized by genetic and biological heterogeneity. Aberrant signal pathways have been identified in HNSCC and inhibition of epidermal growth factor receptor has proven to be a successful therapeutic strategy.3 However, most studies on the cancer genome have focused mainly on protein-coding genes and our understanding of alterations of non-protein-coding sequences in cancer is largely unclear.

MicroRNAs (miRNAs) constitute a class of small non-protein-coding RNA molecules, 19–22 nucleotides in length.4 They negatively regulate gene expression at the post-transcriptional level through translational repression and/or mRNA cleavage. They have important roles in a variety of biological processes including proliferation, differentiation and apoptosis.4 They bind to the 3′-untranslated region (UTR) of target mRNAs by imperfect 7–8 mer base pairing, so one miRNA can regulate multiple target genes. Bioinformatic predictions indicate that miRNAs regulate more than 30% of the protein-coding genes.5 miRNAs contribute to the initiation and development of various types of cancers.6 miRNAs are aberrantly expressed in many human cancers, and can function either as tumor suppressors or oncogenes. Downregulated miRNAs in cancer cells could function as tumor suppressors by negatively regulating oncogenes, alternatively overexpressed miRNAs could function as oncogenes by repressing tumor suppressor genes.7 We previously reported aberrantly expressed miRNAs based on miRNA expression signatures in several cancers, and identified tumor suppressive miRNAs and their target oncogenes.8, 9, 10, 11, 12 In HNSCC, we found that miRNAs such as miR-489, miR-1 and miR-133a were downregulated in cancer cells and potentially had tumor suppressive function through direct repression of several oncogenes.11, 13, 14, 15

miR-375 was significantly downregulated in hypopharyngeal squamous cell carcinoma, esophageal squamous cell carcinoma and maxillary sinus squamous cell carcinoma (unpublished data) in our miRNA expression signatures.10, 11 Interestingly, several reports found that miR-375 is one of the most downregulated miRNAs in HNSCC.16, 17 In other cancers, miR-375 is also known to function as a tumor suppressor by targeting certain oncogenes, including PDK1, YWHAZ/14-3-3ζ, YAP and JAK2.18, 19, 20, 21 Thus miR-375 is a potential tumor suppressor candidate in HNSCC. However, the function of miR-375 and its target genes in HNSCC remains uncertain.

The aim of this study was to clarify the functional significance of miR-375 in HNSCC and to identify cancer pathways regulated by miR-375. We analyzed the effect of miR-375 on cell proliferation and cell apoptosis in HNSCC cell lines. For target genes searches of miR-375, we performed genome-wide gene expression analysis. We focused on AEG-1/MTDH as a candidate target oncogene of miR-375. The identification of a novel tumor suppressive miR-375-regulated oncogene pathway could further unravel the molecular mechanism of HNSCC oncogenesis.

Materials and methods

HNSCC cell culture

Human HNSCC cell lines (SAS, derived from a primary lesion of tongue squamous cell carcinoma; and FaDu, derived from a primary lesion of hypopharyngeal squamous cell carcinoma) were used. Both cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37 °C.

RNA isolation

Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. RNA concentrations were determined spectrophotometrically, and molecular integrity was checked by gel electrophoresis. RNA quality was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

Mature miRNA transfection and small-interfering RNA treatment

The following RNA species were used in this study: mature miRNAs, Pre-miR miRNA precursors (hsa-miR-375; Pre-miR ID: PM10327), miRNA-control (P/N: AM17111) (Applied Biosystems, Foster City, CA, USA), small interfering RNA (Stealth Select RNAi siRNA; si-AEG-1/MTDH Cat#; HSS150644 and HSS150645) (Invitrogen) and siRNA-control (Stealth RNAi Negative Control Medium GC Duplex; 12935-300). RNAs were incubated with Opti-MEM (Invitrogen) and Lipofectamine RNAiMax reagent (Invitrogen) as described previously.9 Transfection efficiency of Pre-miR in cell lines was confirmed based on downregulation of TWF1 (PTK9) mRNA following transfection with miR-1 as previously reported.8, 10

Cell proliferation and cell apoptosis assays

Cells were transfected with 10 nM miRNA or siRNA by reverse transfection and plated in 96-well plates at 3 × 103 cells per well. After 72 h, cell proliferation was determined by the XTT assay, using the Cell Proliferation Kit II (Roche Molecular Biochemicals, Mannheim, Germany).10, 11 Triplicate wells were measured for cell viability in each treatment group.

SAS and FaDu cells were transiently transfected with miRNA-control or miR-375 and were harvested after 72 h by trypsinization. Double staining with FITC-annexin V and propidium iodide (PI) was carried out using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Bedford, MA, USA) according to the manufacturer's recommendations. Cells were analyzed by flow cytometry (FACScan, BD Biosciences) equipped with CellQuest software (BD Biosciences). Cells were discriminated into viable cells, dead cells, early apoptotic cells and apoptotic cells, and then the relative ratios of early apoptotic cells to miRNA-control transfectants from each experiment were compared. Experiments were conducted in triplicate.

Target gene search for miR-375

Genome-wide screens using miR-375 transfectants were performed to identify target genes of miR-375 in two HNSCC cell lines, SAS and FaDu. Oligo-microarray human 44 K (Agilent Technologies) was used for expression profiling of the transfectants in comparison with a miRNA-negative-control transfectant. Hybridization and wash steps were performed as previously described.22 The arrays were scanned using a Packard GSI Lumonics ScanArray 4000 (Perkin Elmer, Boston, MA, USA). The data were analyzed by means of DNASIS array software (Hitachi Software Engineering, Tokyo, Japan), which converted the signal intensity for each spot into text format. The log2 ratios of the median-subtracted background intensities were analyzed. Data from each microarray study were normalized by a global normalization method.22

Predicted target genes and their target miRNA-binding site seed regions were investigated using TargetScan (release 5.1, http://www.targetscan.org/). The sequences of the predicted mature miRNAs were confirmed using miRBase (release 17.0, http://microrna.sanger.ac.uk/).

Real-time quantitative reverse-transcription PCR

First-strand of complementary DNA was synthesized from 1 μg of total RNA using a High Capacity complementary DNA Reverse Transcription Kit (Applied Biosystems). Gene-specific PCR products were assayed continuously using a 7900-HT Real-Time PCR System according to the manufacturer's protocol. The initial PCR step consisted of a 10-min hold at 95 °C, followed by 40 cycles consisting of a 15-sec denaturation at 95 °C and a 1-min annealing/extension at 63 °C. TaqMan probes and primers for HERPUD1 (P/N: Hs00206652_m1), LDHB (P/N: Hs00929956_m1), PSIP1 (P/N: Hs00253515_m1), AEG-1/MTDH (P/N: Hs00757841_m1), SLC7A11 (Hs00204928_m1) and GAPDH (A/N: NM_002046) internal control were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products). The expression levels of miR-375 (Assay ID: 000564) were analyzed by TaqMan quantitative real-time PCR (TaqMan MicroRNA Assay; Applied Biosystems) and normalized to RNU48 (A/N: X96648). All reactions were performed in triplicate, and included negative control reactions that lacked complementary DNA.

Western blot

Cells were harvested 72 h after transfection and lysates were prepared. A measure of 50 μg protein from each lysate was separated by NuPAGE on 4–12% bis-tris gels (Invitrogen) and transferred to polyvinylidene difluoride membranes. Immunoblotting was performed with diluted (1:200) polyclonal AEG-1/MTDH antibody (HPA015104; Sigma-Aldrich, St Louis, MO, USA), with β-actin antibody (sc-1615; Santa Cruz Biotechnology, Santa Cruz, CA, USA) used as an internal control. The membrane was washed and incubated with goat anti-rabbit IgG (H+L)-HRP conjugate (Bio-Rad, Hercules, CA, USA). Specific complexes were visualized by echochemiluminescence (GE Healthcare Bio-Sciences, Princeton, NJ, USA), and the expression level of each protein was evaluated by ImageJ software (ver.1.44; http://rsbweb.nih.gov/ij/index.html).

Plasmid construction and dual-luciferase assay

The wild-type sequences of AEG-1/MTDH 3′-UTR (WT-3′-UTR) and those with deleted miR-375 target sites (DEL-3′-UTR) were inserted between the XhoI and PmeI restriction sites in the 3′-UTR of the hRluc gene in the psiCHECK-2 vector (Promega, Madison, WI, USA). Sequences of oligonucleotides were described in Supplementary Information. The synthesized DNA was cloned into the psiCHECK-2 vector. FaDu cells were then transfected with 5 ng vector, 10 nM mature miRNA molecules, Pre-miRNA miR-375 (Applied Biosystems), and 1 μl Lipofectamine 2000 (Invitrogen) in 100 μl Opti-MEM (Invitrogen). Firefly and Renilla luciferase activities in cell lysates were determined using a dual-luciferase assay system (E1910; Promega). Normalized data were calculated as the quotient of Renilla/firefly luciferase activities.

Clinical HNSCC specimens

A total of 20 pairs of primary HNSCC (oral cavity, six; larynx, three; oropharynx, five; hypopharynx, six) and corresponding normal epithelial samples were obtained from patients at Chiba University Hospital (Chiba, Japan) from 2007 to 2011. All tissue specimens were obtained from patients undergoing surgical treatment. Normal tissues were obtained far from the center of the cancer in surgical specimens. No cancer cells were detected in neighboring formalin-fixed paraffin-embedded tissues. Written consent for tissue donation for research purposes was obtained from each patient before tissue collection. The protocol was approved by the Institutional Review Board of Chiba University. The specimens were immersed in RNAlater (Qiagen, Valencia, CA, USA) and stored at −20 °C until RNA was extracted.

Statistical analysis

The relationships between two groups and the numerical values obtained by real-time reverse-transcription PCR were analyzed using the non-parametric Mann–Whitney U test or the paired t-test. The relationships among more than three variables and numerical values were analyzed using the Bonferroni's-adjusted Mann–Whitney U test. All analyses were carried out using Expert StatView (version 4, SAS Institute, Cary, NC, USA).

Results

Expression of miR-375 in HNSCC clinical specimens

Data characterizing 20 HNSCC patients are presented in Table 1. The expression levels of miR-375 were significantly downregulated in clinical HNSCC specimen compared with adjacent normal tissues (Figure 1a).

Table 1 Clinical features of 20 patients with HNSCC
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Figure 1
figure 1

miR-375 function as a tumor supressor in HNSCC. (a) The expression levels of miR-375 were significantly downregulated in tumor tissues compared with normal tissues. RNU48 was used as an internal control. (b) Cell proliferation was determined by the XTT assay in the SAS and FaDu cell lines 72 h after transfection of 10 nM miR-375 or miRNA-control. The XTT assay showed significant inhibition of cell proliferation in miR-375 transfectants in comparison with mock cultures. (c) The data for the early apoptotic cell fraction at 72 h after transfection are expressed as the relative value of the average expression of the mock cultures. The apoptosis assay showed significant promotion of early cell apoptosis in miR-375 transfectants in comparison with mock cultures. *P<0.0001.

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Effect of miR-375 transfection of HNSCC cell lines

To determine the function of miR-375, we performed gain-of-function analysis using miR-375 or miRNA-control transfectants. The XTT assay showed statistically significant inhibition of cell proliferation in miR-375 transfectants in comparison with mock controls after 72 h. Both miR-375-transfected cultures grew only 70.5% in SAS and 74.0% in FaDu as much as mock cultures (both P<0.0001, Figure 1b).

Cell apoptosis in miR-375 transfected cells was assessed by flow cytometry. The fraction of early apoptotic cells significantly increased in miR-375 transfectants, ∼1.7-fold in SAS and 2.0-fold in FaDu compared with mock controls (both P<0.0001; Figure 1c).

Gene expression profiling identifies downregulated genes in miR-375 transfectants

To investigate candidate molecular targets of miR-375 in HNSCC cells, we examined the effect of miR-375 on protein-coding genes. Mature miR-375 was transiently transfected into SAS and FaDu cells, with miRNA-control transfectants. Comprehensive gene expression analysis (see Methods section) clearly showed changes in gene expression patterns between miR-375 and negative-control transfectants. To identify candidate miR-375 target genes, first, signal values of raw data in miR-control transfectants less than 5000 were cut off. Thereafter, a cut-off of value <−2.00-fold was applied to the array data. This filter resulted in the identification of 55 genes that were significantly downregulated upon miR-375 transfection in both SAS and FaDu cells (55 genes are shown in Table 2). Entries from the microarray data were approved by the Gene Expression Omnibus (GEO), and were assigned GEO accession number GSE26032. The 3′-UTR of these downregulated genes were examined for miR-375 target sites using the TargetScan database. Of the 55 putative gene targets, sixteen genes contained miR-375 target sites. We selected five genes that were downregulated more than YWHAZ/14-3-3ζ, a reported target of miR-375.18

Table 2 A total of 55 downregulated genes by miR-375
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Expression levels of candidate target genes of miR-375 in HNSCC clinical specimens

We measured the mRNA expression levels of five candidate genes in HNSCC clinical specimens by quantitative real-time reverse-transcription-PCR. Two genes, lactate dehydrogenase B (LDHB) and astrocyte elevated gene-1/metadherin (AEG-1/MTDH) were significantly upregulated in cancer tissues (P=0.0143 and P=0.0154, respectively, Figure 2). The other three genes (HERPUD1, PSIP1 and SLC7A11) were not significantly upregulated in the tumor tissues of HNSCC (Supplementary Figure 1).

Figure 2
figure 2

Expression levels of LDHB and AEG-1/MTDH mRNA in HNSCC clinical specimens. The expression levels of LDHB and AEG-1/MTDH mRNA were significantly upregulated in tumor tissues compared with normal tissues. GAPDH was used as an internal control.

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AEG-1/MTDH is directly regulated by miR-375

We focused on AEG-1/MTDH, because AEG-1/MTDH has been reported to be an oncogene in several cancers.23, 24 The expression levels of AEG-1/MTDH mRNA were significantly decreased in both HNSCC cell lines (SAS and FaDu) transfected with miR-375 compared with mocks (Figure 3a). The protein expression levels were also markedly reduced in miR-375 transfectants compared with mocks (Figure 3b).

Figure 3
figure 3

AEG-1/MTDH mRNA and protein expression in the HNSCC cell lines. (a) AEG-1/MTDH mRNA expression 48 h after transfection with 10 nM miR-375. AEG-1/MTDH mRNA expression was significantly repressed in miR-375 transfectants. GAPDH was used as an internal control. *P<0.0001 (b) AEG-1/MTDH protein expression 72 h after transfection with miR-375. β-Actin was used as a loading control. The protein expression level of AEG-1/MTDH was also repressed in miR-375 transfectants.

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FaDu cells were used to determine the mechanism of miR-375 suppression of AEG-1/MTDH expression. The TargetScan database identified one putative target site in the 3′-UTR of AEG-1/MTDH (Figure 4, upper). We performed a luciferase reporter assay to determine whether AEG-1/MTDH mRNA had a target site for miR-375. We used a vector encoding either the partial sequence of the 3′-UTR of AEG-1/MTDH mRNA, including the predicted miR-375 target site, positions 1454–1460 (WT-3′-UTR) or a vector lacking the miR-375 target site (DEL-3′-UTR). We found that the luminescence intensity was significantly reduced by transfection of the WT-3′-UTR while DEL-3′-UTR blocked the decrease in luminescence (Figure 4, lower).

Figure 4
figure 4

Interaction of miR-375 with AEG-1/MTDH 3′-UTR. At 24 h after transfection with 10 nM miR-375, miRNA-control or mocks, a reporter plasmid containing AEG-1/MTDH WT-3′-UTR or DEL-3′-UTR and a plasmid expressing Renilla luciferase (hRluc) were co-transfected into FaDu cells. Firefly luciferase activity was normalized to Renilla luciferase activity. Relative luciferase activity in miR-375 transfectants was compared with that in mock cultures, which was set at 1, in cells transfected with WT-3′-UTR or DEL-3′-UTR. *P<0.0001.

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Effects of AEG-1/MTDH silencing in HNSCC cell lines

Loss-of-function assays using si-AEG-1/MTDH were performed to examine the oncogenic function of AEG-1/MTDH. We asked whether si-AEG-1/MTDH reduced both mRNA and protein expression levels of AEG-1/MTDH in SAS and FaDu. Both AEG-1/MTDH mRNA and protein were reduced in two si-AEG-1/MTDH transfectants (Figure 5). The XTT assay revealed significant cell growth inhibition in two si-AEG-1/MTDH-transfected cells at 72 h after transfection. Specifically, with si-AEG-1/MTDH_1 and si-AEG-1/MTDH_2, SAS grew only 38.7 and 39.8%, respectively, while FaDu proliferated 75.1 and 78.1%, respectively, compared with the mock cultures (Figure 6).

Figure 5
figure 5

Knockdown of AEG-1/MTDH in HNSCC cell lines by siRNAs. (a) AEG-1/MTDH mRNA expression 48 h after transfection with 10 nM si-AEG-1/MTDH_1, si-AEG-1/MTDH_2 or si-control. AEG-1/MTDH mRNA expression was repressed in si-AEG-1/MTDH_1 and si-AEG-1/MTDH_2 transfectants. GAPDH was used as an internal control. *P<0.0001. (b) AEG-1/MTDH protein expression 72 h after transfection of the siRNAs. β-Actin was used a loading control. The protein expression level of AEG-1/MTDH was also repressed in si-AEG-1/MTDH_1 and si-AEG-1/MTDH_2 transfectants.

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Figure 6
figure 6

Effect of si-AEG-1/MTDH on cell proliferation in HNSCC cell lines. Cell proliferation was determined with the XTT assay in the SAS and FaDu cell lines 72 h after transfection of 10 nM si-AEG-1/MTDH_1, si-AEG-1/MTDH_2 or si-control. The XTT assay showed significant inhibition of cell proliferation in si-AEG-1/MTDH_1 and si-AEG-1/MTDH_2 transfectants in comparison with mock cultures. *P<0.0001.

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Discussion

Numerical articles have reported that miRNAs are aberrantly expressed in many types of human cancers.25 Moreover, miRNA expression signatures of HNSCC have been reported by many researchers.26, 27 Recently, we also reported differentially expressed miRNAs in hypopharyngeal squamous cell carcinoma and esophageal squamous cell carcinoma based on expression signatures.8, 9, 10, 11, 12 When we compared our expression data with past reports from other researchers, miR-375 was frequently observed to be downregulated in many types of cancers including HNSCC.16, 17, 18, 19, 20 DNA methylation and histone deacetylation might be involved in downregulation of miR-375 in gastric cancer.18 According to one of those studies of HNSCC, an expression ratio of miR-221:miR-375 provided a useful biomarker (92% sensitivity and 93% specificity) in distinguishing tumor from normal tissues.16 Another report of 51 formalin-fixed HNSCC specimens showed that miR-375 was significantly reduced, and exogenous overexpression of miR-375 in HNSCC cell lines reduced both proliferation and clonogenicity.17

In this study, we asked whether miR-375 had a tumor suppressive function in HNSCC cells. Our data revealed that restoration of miR-375 suppressed cancer cell proliferation and induced cell apoptosis in SAS and FaDu cells, confirming past results. Tumor suppressive function of miR-375 was reported with other cancer cells, such as esophageal, gastric and liver cancer.18, 19, 20, 21 Our data and those in past articles strongly indicate that miR-375 is an important miRNA functioning as a tumor suppressor in human cancers.

Of particular interest is the fact that one miRNA regulates many protein-coding genes. However, the nature of mRNA—microRNA networks in the human genome is unclear. The elucidation of new regulatory networks in cancer is important for understanding oncogenesis. For that reason, we are continuing our investigation of tumor suppressive miRNA-regulated oncogenic targets in various cancer cells.13, 14, 15, 28, 29, 30 In this study, we adopted a method of genome-wide gene expression analysis of SAS and FaDu cells, using miR-375 transfectants to search target genes regulated by miR-375. Our miR-375-regulated gene expression profile showed that the expression of several genes was reduced in miR-375 transfectants. Among them was YWHAZ/14-3-3ζ, a potent antiapoptotic gene. It had already been reported that this gene was repressed by ectopic expression of miR-375, and luciferase reporter assays demonstrated that it was a direct target of miR-375 in gastric cancer.18 Because this previously known target of miR-375 appeared in our expression list, our profile appeared to be an effective approach to identify new targets of miR-375.

We focused on five downregulated genes (HERPUD1, LDHB, PSIP1, AEG-1/MTDH and SLC7A11), which contained miR-375 target sites and were significantly downregulated more than YWHAZ/14-3-3ζ in our expression profile. Our criterion for selection in this analysis was that candidate miR-375-regulated oncogenes should be upregulated in cancer tissues. We validated the expression levels of candidate genes using HNSCC clinical specimens, and two genes (LDHB and AEG-1/MTDH) were overexpressed in cancer tissues compared with adjacent non-cancerous tissues. Among them, we focused on AEG-1/MTDH to study the functional significance of HNSCC. Although AEG-1/MTDH has been interpreted as an oncogene in several cancers, its functional role in HNSCC is not understood. Interestingly, in breast cancer, miR-26a directly controlled AEG-1/MTDH in the cell line MCF7.31 However, no relationship between AEG-1/MTDH and miR-375 was known.

AEG-1/MTDH was initially cloned as a gene induced by HIV-1 infection or tumor necrosis factor-alpha treatment in human fetal astrocytes.32 Overexpression of AEG-1/MTDH was observed in many types of cancers, such as malignant astrocytoma,33 neuroblastoma,34 and cancers of esophagus,35 breast,36 prostate37 and liver.38 Many reports have indicated that AEG-1/MTDH contributes to several steps in human oncogenesis, including cancer-cell progression, invasion, metastasis and chemoresistance. Thus, AEG-1/MTDH can be considered an oncogene.23, 24 Our findings in this study support the oncogenic nature of AEG-1/MTDH in HNSCC. It is well known that AEG-1/MTDH functions as a downstream mediator in signaling pathways such as PI3K/AKT,39 NFκB40 and Wnt/β-catenin.38 We hypothesized that tumor suppressor miR-375 may regulate these oncogenic pathways, and its downregulation is a key step in human HNSCC oncogenesis.

It is reported that AEG-1/MTDH expression indicated prognosis of survival of patients with esophageal squamous cell carcinoma and breast cancer.35, 36 However, in our cohort, there was no significant relationship between miR-375 or AEG-1/MTDH expression and clinico-pathological parameters such as lymph node metastasis, distant metastasis and the TNM classification. Our sample number was too small to test for a relationship. The cohort analysis is an important problem, which should be prepared for the large number of clinical specimens. It will be necessary to challenge this problem in future.

In conclusion, the reduction of miR-375 and the increased expression of AEG-1/MTDH are frequent events in HNSCC cancer cells. miR-375 may function as a tumor suppressor and may directly regulate AEG-1/MTDH. miR-375 regulates novel cancer pathways and could provide new insights into molecular mechanisms in HNSCC and might contribute to the development of new therapeutic strategies for the disease.