Identification of novel molecular targets regulated by tumor suppressive miR-375 induced by histone acetylation in esophageal squamous cell carcinoma

  • Authors:
    • Yuka Isozaki
    • Isamu Hoshino
    • Nijiro Nohata
    • Takashi  Kinoshita
    • Yasunori Akutsu
    • Naoyuki Hanari
    • Mikito Mori
    • Yasuo  Yoneyama
    • Naoki  Akanuma
    • Nobuyoshi Takeshita
    • Tetsuro Maruyama
    • Naohiko  Seki
    • Norikazu Nishino
    • Minoru Yoshida
    • Hisahiro Matsubara
  • View Affiliations

  • Published online on: June 28, 2012     https://doi.org/10.3892/ijo.2012.1537
  • Pages: 985-994
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

The aim of this study was to determine whether histone acetylation regulates tumor suppressive microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC) and to identify genes which are regulated by these miRNAs. We identified a miRNA that was highly upregulated in an ESCC cell line by cyclic hydroxamic acid-containing peptide 31 (CHAP31), one of the histone deacetylase inhibitors (HDACIs), using a miRNA array analysis. miR-375 was strongly upregulated by CHAP31 treatment in an ESCC cell line. The expression levels of the most upregulated miRNA, miR-375 were analyzed by quantitative real-time PCR in human ESCC specimens. The tumor suppressive function of miR-375 was revealed by restoration of miR-375 in ESCC cell lines. We performed a microarray analysis to identify target genes of miR-375. The mRNA and protein expression levels of these genes were verified in ESCC clinical specimens. LDHB and AEG-1/MTDH were detected as miR‑375-targeted genes. The restoration of miR-375 suppressed the expression of LDHB and AEG-1/MTDH. The ESCC clinical specimens exhibited a high level of LDHB expression at both the mRNA and protein levels. A loss-of-function assay using a siRNA analysis was performed to examine the oncogenic function of the gene. Knockdown of LDHB by RNAi showed a tumor suppressive function in the ESCC cells. The correlation between gene expression and clinicopathological features was investigated by immunohistochemistry for 94 cases of ESCC. The positive staining of LDHB correlated significantly with lymph node metastasis and tumor stage. It also had a tendency to be associated with a poor prognosis. Our results indicate that HDACIs upregulate miRNAs, at least some of which act as tumor suppressors. LDHB, which is regulated by the tumor suppressive miR-375, may therefore act as an oncogene in ESCC.

Introduction

Esophageal cancer occurs in humans worldwide with a variable geographic distribution, and it ranks eighth among cancers in the order of occurrence (1). Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal cancer in Japan. Despite recent advances in cancer therapy, esophageal cancer remains one of the least responsive malignancies (2). The overall 5-year survival rate for esophageal cancer is approximately 20–25% for all stages (3), therefore, the development of a molecular oncogenic therapy that can provide a higher response rate than the current combinations of chemotherapy and radiotherapy is urgently required.

Epigenetics is a rapidly expanding field that focuses on stable changes in gene expression that are not accompanied by any changes in the DNA sequence, and that are mediated primarily by DNA methylation, histone modifications and small non-coding RNA molecules (4). Histone deacetylation is known to correlate with transcriptional silencing and with the downregulation of the expression of proapoptotic genes, especially in cancer cells. The histone deacetylase inhibitors (HDACIs) were mainly thought to act by modulating the gene expression patterns, including those of genes associated with cell cycle arrest and apoptosis, by inhibiting the activity of histone deacetylases (HDACs)(5). Previous we reported that depsipeptide (FK228) and cyclic hydroxamic acid-containing peptide 31 (CHAP31) have potent antitumor effects against ESCC in vitro and in vivo (68).

Numerous studies have demonstrated that microRNAs (miRNAs), non-coding RNAs 21–25 nucleotides in length, control gene expression by targeting mRNAs for cleavage or translational repression (9). These miRNAs are associated with important biological processes, including development, differentiation, apoptosis, and proliferation (9,10). A growing body of evidence indicates that the miRNA expression profiles associated with particular types of cancer could serve as useful biomarkers for both disease prognosis and diagnosis (11,12).

The purpose of this study was to determine whether histone acetylation is associated with the regulation of the expression of tumor-suppressive miRNAs in ESCC, and to identify the target genes that are regulated by these miRNAs.

Materials and methods

Clinical ESCC specimens

RNA extraction was performed for 19 pairs of primary ESCC and corresponding normal esophageal epithelium. All specimens were obtained from patients who underwent surgical treatment at the Department of Frontier Surgery, Graduate School of Medicine, Chiba University, Japan from 2004 to 2005. The clinicopathological characteristics of the patients and samples are listed in (Table I). The staging of the tumors was carried out according to the TNM classification. The tissues were frozen in liquid nitrogen immediately, and stored at −80°C.

Table I

The clinicopathological features of patients with ESCC.

Table I

The clinicopathological features of patients with ESCC.

No.GenderAgeLocationaUICCb TUICCb NUICCb Stage
1F48Mt444a
2M69Mt1b01
3M75Mt333
4M73Mt323
5M67Lt414a
6F70Mt1b01
7M65Mt1b01
8M53Ae1b33
9M65Mt313
10M67Lt223
11M71Mt323
12M77Mt302
13M70Lt313
14M66Lt1b01
15M47Mt302
16M68Lt333
17M53Ae414a
18M60Lt403
19M71Lt313

{ label (or @symbol) needed for fn[@id='tfn1-ijo-41-03-0985'] } RNU48 was used as an internal control.

a Mt, middle thoracic esophagus; Lt, lower thoracic esophagus; Ae, abdominal esophagus.

b UICC, the N and stage are described according to UICC (International Union Against Cancer) TNM Classification (Sixth Edition, 2002).

Immunohistochemical staining was performed for 94 patients wh underwent surgical resection from 1997 to 2005. Normal esophageal epithelial tissue specimens were obtained far from the cancer in the specimens. All patients gave their informed consent for tissue donation. Surgical treatments were performed without any preoperative radiotherapy or chemotherapy.

ESCC cell culture and reagents

The human ESCC cell lines were cultured in DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS in a humidified incubator containing 5% CO2 at 37°C. The T.Tn cells were provided by the Japanese Cancer Research Resources Bank. TE2 cells were provided by Tohoku University. CHAP31 was provided by Dr M. Yoshida (RIKEN Advanced Science Institute, Wako, Saitama, Japan), and was dissolved in dimethyl sulfoxide.

Total RNA preparation and miRNA analysis

The cells were seeded into 225 cm2 flasks and incubated for 48 h, then treated with or without an IC50 concentration of CHAP31 and harvested after 12 h of treatment. The cells were washed with PBS and processed for RNA extraction with TRIzol. The miRNA expression patterns were evaluated using the TaqMan Low Density Array Human MicroRNA Panel v1.0 (Applied Biosystems, Foster City, CA). The assay was conducted in 2 steps: generation of cDNA by reverse transcription, and a TaqMan real-time PCR assay. Briefly, the miRNAs in the samples were converted into cDNA using 365 specific stem-loop reverse transcription primers. The quantity of mature miRNAs was evaluated using specific TaqMan real-time PCR primers and probes. Real-time PCR was performed in duplicate using the GeneAmp Fast PCR Master Mix (Applied Biosystems) and the ABI 7900HT Real-Time PCR System. The comparative CT method was used to determine the expression levels. The relative miRNA expression data were analyzed using the GeneSpring GX version 7.3.1 software package (Agilent Technologies), as previously described (13). Normalization to an endogenous gene (RNA48) was used to normalize the expression data.

RNA isolation

The tissue specimens and cells were treated with the TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol, for total RNA extraction. The RNA concentrations were determined spectrophotometrically, and the molecular integrity was checked by gel electrophoresis. The RNA quality was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

Mature miRNA transfection and small interfering RNA treatment

The RNA sequences used in this study included mature miR-375, Pre-miR™ miRNA precursors (hsa-miR-375; Pre-miR ID: PM10327), miRNA-control (P/N: AM17111; Applied Biosystems), small interfering RNA [Stealth Select RNAi™ siRNA; si-LDHB Cat#; HSS106003 and HSS106005 (Invitrogen)], and siRNA-control (Stealth™ RNAi negative control medium GC Duplex; 12935-300). The RNA sequences were incubated with Opti-MEM (Invitrogen) and Lipofectamine™ RNAiMax reagent (Invitrogen) as previously described (14).

Cell proliferation assay

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

Cell migration assay

Cell migration was evaluated using modified Boyden Chambers (Transwells; Corning/Costar #3422, NY, USA) containing uncoated transwell-polycarbonate membrane filters with 8 μm pores in 24-well tissue culture plates. Cells were transfected with 10 nM miRNA or siRNA by reverse transfection and plated in 10 cm dishes at 8×105 cells. The cells were cultured for 48 h, and then 5×104 cells were added to the upper chamber of each well and allowed to migrate for 48 h. The non-migratory cells were gently removed from the filter surface of the upper chamber, and the cells that migrated to the lower side were fixed and stained with Diff-Quick (Sysmex Corporation, Tokyo, Japan). The number of cells migrating to the lower surface was determined microscopically by counting 4 constant areas per well. Triplicate wells were measured for cell migration in each treatment group.

Cell invasion assays

The cell invasion assays were carried out using modified Boyden Chambers containing transwell-precoated Matrigel membrane filter inserts with 8 μm pores in 24-well tissue culture plates (BD Biosciences, Bedford, MA) as previously described (13). All experiments were performed in triplicate.

Screening for miRNA-375 target genes by a microarray analysis

The expression profiles of TE2 and T.Tn cells transfected with miRNA-375 were displayed and compared against miRNA-negative control transfectants using the Oligo-microarray human 44K platform (Agilent Technologies) as previously described (15). The hybridization and washing steps were performed as previously described (16). The arrays were scanned using a Packard GSI Lumonics ScanArray 4000 (Perkin Elmer, Boston, MA, USA) and the data were analyzed. Data from each microarray study were subjected to global normalization (16).

The predicted target genes and their target miRNA site seed regions were explored using the TargetScan software program (release 5.1, http://www.targetscan.org/). The sequences of predicted mature miRNAs were confirmed using the miRBase software program, release 13.0 (http://microrna.sanger.ac.uk/).

Real-time quantitative RT-PCR

First-strand cDNA was synthesized from 1 μg of total RNA using a High Capacity cDNA reverse transcription kit (Applied Biosystems). The gene-specific PCR products were assayed continuously using a 7900-HT Real-Time PCR system according to the manufacturer’s protocol. The first PCR step was a 10 min denaturation at 95°C, followed by 40 cycles of a 15 sec denaturation at 95°C and a 1 min annealing/extension at 63°C. The TaqMan probes and primers used to amplify LDHB (Hs00929956_m1), MTDH (Hs00757841_m1), PRDX1 (Hs03044567_g1), CXCL1 (Hs00236937_m1), MAL2 (Hs00294541_m1), CHSY1 (Hs00208704_m1) and GAPDH (Hs02758991_g1) were Assay-On-Demand Gene Expression Products (Applied Biosystems). All reactions were performed in triplicate and included a negative control lacking cDNA. The expression levels of miRNA-375 (Assay ID: 000564) were analyzed by TaqMan quantitative real-time PCR (TaqMan MicroRNA Assay; Applied Biosystems) and normalized to RNU48 (Assay ID, 001006).

Western blot analysis

The cells were harvested and lysed 72 h after transfection. Each cell lysate (50 μg of protein) was separated by electrophoresis using Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes. Immunoblotting was performed with a monoclonal antibody against LDHB (1:10000; 2090-1; Epitomics, Burlingame, CA, USA) and a polyclonal antibody against MTDH (1:200; HPA015104; Sigma-Aldrich). A GAPDH antibody (1:1000; ab8245; AbCam, Cambridge, UK) was used as an internal loading control. The membranes were washed and incubated with a goat anti-rabbit IgG (H+L)-HRP conjugate (Bio-Rad). The complexes were visualized with the Immun-Star WesternC chemiluminescence kit (Bio-Rad), and the expression levels of these proteins were evaluated using the ImageJ software package (version 1.44; http://rsbweb.nih.gov/ij/index.html).

Expression of LDHB and MTDH determined by immunohistochemistry in clinical esophageal squamous cell carcinoma specimens

Immunohistochemical staining was performed to detect the expression of LDHB and MTDH in the cancerous and normal epithelial regions in 19 ESCC clinical specimens. The LDHB expression was also evaluated for 94 cases to assess the immunohistochemical features of LDHB during the progression of ESCC. Paraffin blocks were cut into 3 μm-thick sections, and mounted after staining with hematoxylin and eosin.

Immunohistochemical staining was performed with a monoclonal LDHB antibody (1:250; 2090-1; Epitomics, Burlingame, CA, USA) and a polyclonal AEG-1/MTDH antibody (1:350; HPA015104; Sigma-Aldrich). Secondary antibodies (biotinylated rabbit anti-rabbit immunoglobulins, Dako K4003) were applied to all slides for 60 min at 37°. The color was developed by 2 min of incubation with DAB chromogen on the slides. The slides were all counterstained with hematoxylin.

The proportion of the specimen showing positive staining for LDHB in five representative fields at magnification ×100 was evaluated independently by two observers who were blinded to the clinicopathological characteristics and prognosis of the patients. The LDHB expression was graded according to the percentage of LDHB positive cells using the scale: 0–10% (−), 10–50% (1+), 50–100% (2+).

Comparisons between the groups were performed using the chi-square test. The overall survival was calculated from the time of the surgical treatment until mortality or the last follow-up date. The correlation between the overall survival and LDHB expression was computed by the log-rank test and presented as curves determined using the Kaplan-Meier method.

Results

Identification of upregulated miRNAs in ESCC cell lines treated with CHAP31

The raw data were normalized using an internal reference, RNU44, and 11 upregulated and 9 downregulated miRNAs we identified using a cutoff p-value of <0.05 and a fold-change of the with/without CHAP31 treatment value <0.5 (Log2 ratio; Table II). The expression of miR-375 in CHAP31-treated cells was upregulated more than 400-fold in both cell lines, and was then identified to be a potential tumor suppressor.

Table II

Upregulated and downregulated miRNAs in ESCC cell lines treated with an IC50 concentration of CHAP31.

Table II

Upregulated and downregulated miRNAs in ESCC cell lines treated with an IC50 concentration of CHAP31.

No.microRNAAccession no.Fold change (CHAP31/control)
Average
T.TnTE2
1miR-375MIMAT00007281724.872473.2171099.045
2miR-449aMIMAT0001541184.6079.23896.922
3miR-449bMIMAT000332727.04519.51423.280
4miR-192MIMAT000022217.48315.14316.313
5miR-497MIMAT000282028.6232.56915.596
6miR-132MIMAT000042613.07710.88511.981
7miR-194MIMAT000046012.2538.50210.378
8miR-146b-5pMIMAT00028092.8133.3943.103
9miR-183MIMAT00002613.2222.6972.959
Expression of miR-375 in ESCC clinical specimens

The expression levels of miR-375 were significantly downregulated in clinical ESCC specimens in comparison to neighboring normal tissue sections (Fig. 1).

Effect of miR-375 transfection on the proliferation, migration and invasion in ESCC cells

The functional significance of miR-375 was evaluated with a gain-of-function assay using miR-375 transfectants. The XTT assay showed significant inhibition of cell proliferation in miR-375 transfectants in comparison with mock and miRNA-control transfectants after a 72-h treatment (% cell proliferation, T.Tn; 43.7±1.4, 100.0±1.0 and 120.0±3.2, respectively, P<0.0001, TE2; 75.5±2.3, 100.0±1.5 and 101.5±1.7, respectively, P<0.0001; Fig. 2A).

The migration assay demonstrated that the number of cells that migrated was significantly decreased in miR-375 transfectants in comparison to mock and miRNA-control transfectants (% cell migration, T.Tn; 36.9±4.3, 100.0±11.1 and 125.2±14.8, respectively, P<0.0001, TE2; 44.1±2.4, 100.0±2.7 and 93.7±5.3, P<0.0001; Fig. 2B).

Similarly, the Matrigel invasion assay demonstrated that the number of invading cells was significantly decreased in miR-375 transfectants in comparison to mock and miRNA-control transfectants (% cell invasion, T.Tn; 24.8±3.4, 100.0±13.8 and 112.2±5.9, respectively, P<0.0001, TE2; 70.4±6.1, 100.0±10.3 and 96.1±6.0, respectively, P=0.0132; Fig. 2C).

Screening of miR-375 target genes by a genome-wide gene expression analysis

The effect of miR-375 on protein-coding genes was examined to identify candidate molecular targets of miR-375 in ESCC cells. A comprehensive gene expression analysis was performed with miR-375 transfectants in both the T.Tn and TE2 cell lines. MiR-control transfectants that produced raw signal values of <3,000 were excluded before comparisons were made. Sixteen genes were downregulated by <−1.0 (Log2 ratio) in the miR-375 transfectants in both the T.Tn and TE2 cell lines (Table III). The 3′UTR of these genes were screened for miR-375 target sites using the TargetScan database. Six of these 16 genes had miR-375 target sites in their 3′UTR, and were identified as putative target genes of miR-375.

Table III

Genes downregulated by miR-375 treatment in ESCC cell lines.

Table III

Genes downregulated by miR-375 treatment in ESCC cell lines.

No.Entrez gene IDGene nameGene symbolLog2 ratio
miR-375
T.TnTE2AverageTarget
18000Prostate stem cell antigenPSCA−1.50−1.95−1.72-
2642587NPC-A-5LOC642587−1.58−1.60−1.59-
33945Lactate dehydrogenase BLDHB−1.48−1.60−1.541
48581Lymphocyte antigen 6 complex, locus DLY6D−1.59−1.33−1.46-
592140MetadherinMTDH−1.24−1.63−1.431
65052Peroxiredoxin 1PRDX1−1.43−1.35−1.391
7218Aldehyde dehydrogenase 3 family, memberA1ALDH3A1−1.36−1.32−1.34-
82919Chemokine (C-X-C motif) ligand 1 (melanoma Growth stimulating activity, α)CXCL1−1.24−1.37−1.301
91789DNA (cytosine-5-)-methyltransferase 3 βDNMT3B−1.28−1.31−1.29-
101475Cystatin A (stefin A)CSTA−1.02−1.57−1.29-
11114569Mal, T-cell differentiation protein 2MAL2−1.23−1.34−1.291
12445Argininosuccinate synthetase 1ASS1−1.26−1.18−1.22-
13216Aldehyde dehydrogenase 1 family, member A1ALDH1A1−1.11−1.31−1.21-
1484958Synaptotagmin-like 1SYTL1−1.20−1.21−1.20-
1510857Progesterone receptor membrane component 1PGRMC1−1.32−1.02−1.17-
1622856Chondroitin sulfate synthase 1CHSY1−1.09−1.01−1.051
Expression levels of candidate miR-375 target genes in ESCC clinical specimens

The mRNA expression levels of the six candidate genes were measured in clinical specimens of ESCC 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=0.0289 and P=0.0246, respectively). The other four genes (PRDX1, CXCL1, MAL2 and CHSY1) were not significantly upregulated in the specimens of ESCC (Fig. 3).

LDHB and MTDH mRNA and protein levels are repressed by miR-375

Gain-of-function studies were conducted using miR-375-transfected T.Tn and TE2 cells, and the mRNA and protein expression levels of LDHB (Fig. 4A) and MTDH (Fig. 4B) were found to be markedly downregulated in the transfectants in comparison to the mock controls.

The expression levels of LDHB and MTDH by IHC in ESCC clinical specimens

The expression of LDHB and MTDH was observed in all the specimens examined, but the expression in tumors was much higher in comparison to that in the corresponding normal epithelium (Fig. 5).

The correlation between LDHB expression and the clinicopathological characteristics

Positive staining for LDHB was found in 68% of the cases. The correlation between LDHB expression and the clinicopathological features, including patient age, gender, tumor depth, lymph node metastasis, distant metastasis, tumor stage and tumor differentiation was investigated (Table IV).

Table IV

Correlation between LDHB expression and clinicopathological characteristics.

Table IV

Correlation between LDHB expression and clinicopathological characteristics.

Clinicopathological features LDHB LDHB+P-value
Age
  ≤65 years18390.9309
  >65 years1225
Gender
  Male27540.6775
  Female310
Tumor depth
  Tis/T115200.0796
  T2/T3/T41544
Lymph node metastasis
  N01923<0.05
  N11141
Distant metastasis
  M025560.8219
  M158
Stage
  0/I1310<0.005
  II/III/IV1655
Tumor differentation
  Well10160.3558
  Moderate1137
  Poor910
  Other01

The level of LDHB staining correlated significantly with lymph node metastasis (P<0.05) and the tumor stage (P<0.005). There was no significant correlation between LDHB staining and other factors.

Relationship between LDHB expression and patient prognosis

No significant differences in survival were observed according to the LDHB expression levels, although there was a tendency for the patients with high immunoreactivity for LDHB to have a poorer prognosis (Fig. 6A and B).

Effect of LDHB loss-of-function in ESCC cell lines

A loss-of function assay using a siRNA analysis was performed to examine the oncogenic function of LDHB. The effects of si-LDHB on the mRNA and protein expression levels were evaluated 72 h after transfection into both T.Tn and TE2 cells. The LDHB mRNA and protein levels were both reduced after transfection. The XTT assay revealed significant inhibition of cell proliferation in si-LDHB transfectants in comparison to mock and si-control transfectants after 72 h. The Matrigel invasion assay demonstrated that the number of invading cells was significantly lower in the si-LDHB transfectants compared to mock and si-control transfectants (Fig. 7).

Discussion

This study showed that HDACIs induced miR-375 overexpression in ESCC cell lines, and that miR-375 downregulated LDHB and AEG-1/MTDH in ESCC cell lines. HDACs are associated with numerous types of cancer and regulate cancer development (5). Histone deacetylation correlates with transcriptional silencing and with the downregulation of the expression of proapoptotic genes, especially in cancer cells (58). HDACIs cause changes in the acetylation status of chromatin, resulting in changes in gene expression, induction of apoptosis, cell cycle arrest, and inhibition of angiogenesis and metastasis (17,18).

Dysregulation of miRNAs is associated with dysregulated gene expression of tumor suppressors and oncogenes in several types of cancer (9). miRNAs are differentially expressed in several cancers, including ESCC, as indicated by their expression signatures (1315,1921). A previous study analyzed the function of miR-375 as a tumor suppressor in head and neck squamous cell carcinoma and maxillary sinus squamous cell carcinoma, and investigated the target genes and their function (15,22). Another study showed significantly lower expression of miR-375 in ESCC (19).

The correlation(s) between DNA demethylation or histone acetylation and miRNAs has not been fully elucidated, and few reports exist on this correlation regarding miR-375 (2325). The downregulation of miR-375 is caused by promoter hypermethylation.

A computational analysis revealed that miR-375 is located in a CpG island on chromosome 2q35 (National Center for Biotechnology Information) (24). The acetylation of lysine residues in the N-terminal histone tail of the unmethylated CpG island induces an open structure of the chromatin and increased the transcription of that region (5,26). Although histone acetylation might directly upregulate miR-375, further experiments are required to confirm this.

We showed that the reinstatement of miR-375 could inhibit cancer cell proliferation and invasion in ESCC cell lines. In a recent study, it was shown that miR-375 inhibits tumor growth and metastasis in ESCC in vivo and in vitro. That study also revealed that the downregulation of miR-375 significantly correlated with a poor prognosis in ESCC (25).

In the present study, the genome-wide gene expression analysis revealed six candidate genes that were regulated by miR-375(LDHB, MTDH, PRDX1, CXCL1, MAL2 and CHSY1). In this analysis, the criterion used for selection was upregulation in cancer tissues. Two genes, LDHB and MTDH, were of particular interest as they had also been identified in a search for miR-375 targets in HNSCC, an indication that these genes may have a role in the oncogenesis of human squamous cell carcinoma (15,22).

LDHB is known to convert lactase to pyruvate, which is then further oxidized (27). A correlation between LDHB expression and cancer has been reported (27). It was also revealed that the serum levels of LDHB are specifically elevated in non-small cell lung carcinoma patients, and are progressively increased with clinical stage (28). Kinoshita et al reported that the mRNA expression of LDHB might serve as a predictor of a poor prognosis in maxillary sinus squamous cell carcinoma (22). In our study, the knockdown of LDHB by RNAi showed a tumor suppressive effect in ESCC cells. In addition, ESCC clinical specimens exhibited a high level of LDHB expression at both the mRNA and protein levels compared with the normal esophageal epithelium. Kaplan-Meier curves and log-rank tests revealed that positive immunoreactivity for the LDHB protein had a tendency to indicate a poor prognosis. The current results indicate that LDHB plays an important role in cancer signaling pathways in ESCC.

Recent studies have shown that Metadherin (MTDH)/Astrocyte Elevated Gene 1 (AEG-1) plays a key role in tumor progression, invasion, metastasis, and resistance to chemotherapies (29). There is overexpression of AEG-1/MTDH in ESCC, and a multivariate analysis indicated that AEG-1/MTDH expression is a valuable marker of ESCC progression (30).

The current study suggested the possibility that histone deacetylase inhibition, the downregulation of miR-375, and the upregulation of LDHB and AEG-1/LDHB are involved in the initiation and development of ESCC. Further studies are required to elucidate the additional roles of miR-375-regulated molecular networks and to characterize the epigenetic crosstalk between histone acetylation and miRNAs, and also to determine the mechanism underlying the involvement of LDHB and MTDH in human oncogenesis.

Acknowledgements

This work was supported by Grant-in-Aid for Scientific Research (KAKENHI) no. 23890031.

References

1. 

Enzinger PC and Mayer RJ: Esophageal cancer. N Engl J Med. 349:2241–2252. 2003. View Article : Google Scholar : PubMed/NCBI

2. 

Akutsu Y and Matsubara H: The significance of lymph node status as a prognostic factor for esophageal cancer. Surg Today. 41:1190–1195. 2011. View Article : Google Scholar : PubMed/NCBI

3. 

National Cancer Institute (Bethesda, MD, USA). The Surveillance, Epidemiology and End Results (SEER) Program. Cancer Statistics Review. 2007.

4. 

Boumber Y and Issa JP: Epigenetics in cancer: what’s the future? Oncology (Williston Park). 25:220–226. 2282011.

5. 

Hoshino I and Matsubara H: Recent advances in histone deacetylase targeted cancer therapy. Surg Today. 40:809–815. 2010. View Article : Google Scholar : PubMed/NCBI

6. 

Hoshino I, Matsubara H, Ochiai T, et al: Histone deacetylase inhibitor FK228 activates tumor suppressor Prdx1 with apoptosis induction in esophageal cancer cells. Clin Cancer Res. 11:7945–7952. 2005. View Article : Google Scholar : PubMed/NCBI

7. 

Murakami K, Matsubara H, Hoshino I, et al: CHAP31 induces apoptosis only via the intrinsic pathway in human esophageal cancer cells. Oncology. 78:62–74. 2010. View Article : Google Scholar : PubMed/NCBI

8. 

Hoshino I, Matsubara H, Ochiai T, et al: Gene expression profiling induced by histone deacetylase inhibitor, FK228, in human esophageal squamous cancer cells. Oncol Rep. 18:85–92. 2007.PubMed/NCBI

9. 

Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 116:281–297. 2004. View Article : Google Scholar : PubMed/NCBI

10. 

Kloosterman WP and Plasterk RH: The diverse functions of microRNAs in animal development and disease. Dev Cell. 11:441–450. 2006. View Article : Google Scholar : PubMed/NCBI

11. 

Lu J, Getz G, Miska EA, et al: MicroRNA expression profiles classify human cancers. Nature. 435:834–838. 2005. View Article : Google Scholar : PubMed/NCBI

12. 

Calin GA and Croce CM: MicroRNA signatures in human cancers. Nat Rev Cancer. 6:857–866. 2006. View Article : Google Scholar : PubMed/NCBI

13. 

Chiyomaru T, Enokida H, Nakagawa M, et al: miR-145 and miR-133a function as tumour suppressors and directly regulate FSCN1 expression in bladder cancer. Br J Cancer. 102:883–891. 2010. View Article : Google Scholar : PubMed/NCBI

14. 

Ichimi T, Enokida H, Seki N, et al: Identification of novel microRNA targets based on microRNA signatures in bladder cancer. Int J Cancer. 125:345–352. 2009. View Article : Google Scholar : PubMed/NCBI

15. 

Nohata N, Hanazawa T, Seki N, et al: Tumor suppressive microRNA-375 regulates oncogene AEG-1/MTDH in head and neck squamous cell carcinoma (HNSCC). J Hum Genet. 56:595–601. 2011. View Article : Google Scholar : PubMed/NCBI

16. 

Sugimoto T, Seki N, Hata A, et al: The galanin signaling cascade is a candidate pathway regulating oncogenesis in human squamous cell carcinoma. Genes Chromosomes Cancer. 48:132–142. 2009. View Article : Google Scholar : PubMed/NCBI

17. 

Wagner JM, Hackanson B, Lübbert M, et al: Histone deacetylase (HDAC) inhibitors in recent clinical trials for cancer therapy. Clin Epigenetics. 1:117–136. 2010. View Article : Google Scholar : PubMed/NCBI

18. 

Ma X, Ezzeldin HH and Diasio RB: Histone deacetylase inhibitors: current status and overview of recent clinical trials. Drugs. 69:1911–1934. 2009. View Article : Google Scholar : PubMed/NCBI

19. 

Kano M, Seki N, Matsubara H, et al: miR-145, miR-133a and miR-133b: Tumor-suppressive miRNAs target FSCN1 in esophageal squamous cell carcinoma. Int J Cancer. 127:2804–2814. 2010. View Article : Google Scholar : PubMed/NCBI

20. 

Kikkawa N, Hanazawa T, Seki N, et al: miR-489 is a tumour-suppressive miRNA target PTPN11 in hypopharyngeal squamous cell carcinoma (HSCC). Br J Cancer. 103:877–884. 2010. View Article : Google Scholar : PubMed/NCBI

21. 

Yoshino H, Chiyomaru T, Nakagawa M, et al: The tumour-suppressive function of miR-1 and miR-133a targeting TAGLN2 in bladder cancer. Br J Cancer. 104:808–818. 2011. View Article : Google Scholar : PubMed/NCBI

22. 

Kinoshita T, Nohata N, Yoshino H, et al: Tumor suppressive microRNA-375 regulates lactate dehydrogenase B in maxillary sinus squamous cell carcinoma. Int J Oncol. 40:185–193. 2012.PubMed/NCBI

23. 

Li X, Lin R and Li J: Epigenetic silencing of microRNA-375 regulates PDK1 expression in esophageal cancer. Dig Dis Sci. 56:2849–2856. 2011. View Article : Google Scholar : PubMed/NCBI

24. 

Tsukamoto Y, Nakada C, Moriyama M, et al: MicroRNA-375 is downregulated in gastric carcinomas and regulates cell survival by targeting PDK1 and 14-3-3zeta. Cancer Res. 70:2339–2349. 2010. View Article : Google Scholar : PubMed/NCBI

25. 

Kong KL, Kwong DL, Guan XY, et al: MicroRNA-375 inhibits tumour growth and metastasis in oesophageal squamous cell carcinoma through repressing insulin-like growth factor 1 receptor. Gut. 61:33–42. 2012. View Article : Google Scholar : PubMed/NCBI

26. 

Rountree MR, Bachman KE, Baylin SB, et al: DNA methylation, chromatin inheritance, and cancer. Oncogene. 20:3156–3165. 2001. View Article : Google Scholar : PubMed/NCBI

27. 

Zha X, Wang F, Zhang H, et al: Lactate dehydrogenase B is critical for hyperactive mTOR-mediated tumorigenesis. Cancer Res. 71:13–18. 2011. View Article : Google Scholar : PubMed/NCBI

28. 

Chen Y, Zhang H, Xiao X, et al: Elevation of serum l-lactate dehydrogenase B correlated with the clinical stage of lung cancer. Lung Cancer. 54:95–102. 2006. View Article : Google Scholar : PubMed/NCBI

29. 

Hu G, Wei Y and Kang Y: The multifaceted role of MTDH/AEG-1 in cancer progression. Clin Cancer Res. 15:5615–5620. 2009. View Article : Google Scholar : PubMed/NCBI

30. 

Yu C, Chen K, Song L, et al: Overexpression of astrocyte elevated gene-1 (AEG-1) is associated with esophageal squamous cell carcinoma (ESCC) progression and pathogenesis. Carcinogenesis. 30:894–901. 2009. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

September 2012
Volume 41 Issue 3

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Isozaki Y, Hoshino I, Nohata N, Kinoshita T, Akutsu Y, Hanari N, Mori M, Yoneyama Y, Akanuma N, Takeshita N, Takeshita N, et al: Identification of novel molecular targets regulated by tumor suppressive miR-375 induced by histone acetylation in esophageal squamous cell carcinoma. Int J Oncol 41: 985-994, 2012
APA
Isozaki, Y., Hoshino, I., Nohata, N., Kinoshita, T., Akutsu, Y., Hanari, N. ... Matsubara, H. (2012). Identification of novel molecular targets regulated by tumor suppressive miR-375 induced by histone acetylation in esophageal squamous cell carcinoma. International Journal of Oncology, 41, 985-994. https://doi.org/10.3892/ijo.2012.1537
MLA
Isozaki, Y., Hoshino, I., Nohata, N., Kinoshita, T., Akutsu, Y., Hanari, N., Mori, M., Yoneyama, Y., Akanuma, N., Takeshita, N., Maruyama, T., Seki, N., Nishino, N., Yoshida, M., Matsubara, H."Identification of novel molecular targets regulated by tumor suppressive miR-375 induced by histone acetylation in esophageal squamous cell carcinoma". International Journal of Oncology 41.3 (2012): 985-994.
Chicago
Isozaki, Y., Hoshino, I., Nohata, N., Kinoshita, T., Akutsu, Y., Hanari, N., Mori, M., Yoneyama, Y., Akanuma, N., Takeshita, N., Maruyama, T., Seki, N., Nishino, N., Yoshida, M., Matsubara, H."Identification of novel molecular targets regulated by tumor suppressive miR-375 induced by histone acetylation in esophageal squamous cell carcinoma". International Journal of Oncology 41, no. 3 (2012): 985-994. https://doi.org/10.3892/ijo.2012.1537