Upregulation of AUF1 is involved in the proliferation of esophageal squamous cell carcinoma through GCH1

  • Authors:
    • Yi Gao
    • Wenjie Wang
    • Jinming Cao
    • Fangjun Wang
    • Yangyang Geng
    • Jianping Cao
    • Xiaohui Xu
    • Jundong Zhou
    • Pengfei Liu
    • Shuyu Zhang
  • View Affiliations

  • Published online on: September 28, 2016     https://doi.org/10.3892/ijo.2016.3713
  • Pages: 2001-2010
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Abstract

Esophageal squamous cell carcinoma (ESCC) has one of the highest mortality rates worldwide. AU-rich element RNA-binding factor 1 (AUF1) is an established RNA-binding protein. AUF1 influences the process of development, apoptosis and tumorigenesis via interacting with adenylate-uridylate rich elements (AREs) bearing mRNAs. However, the clinical relevance of AUF1 and its biological function in ESCC progression have not been reported. In the present study, we first investigated the expression of AUF1 in the ESCC tissue samles and normal samples. We found a significantly higher expression of AUF1 in ESCC tissues than that in normal tissues and tumor adjacent tissues. The expression of AUF1 correlated with ESCC stage (P=0.011) and marginally correlated with lymph node metastasis (P=0.055) of ESCC patients. Silencing of AUF1 by an siRNA inhibited the proliferation and enhanced the apoptosis of ESCC cells. mRNA profiling by microarray analysis revealed that AUF1 knockdown affected 285 genes (fold change ≥2) that function in multiple pathways. GTP cyclohydrolase I (GCH1), the rate limiting enzyme for BH4 synthesis, was found to be downregulated. One of the AU-rich elements in the 3'UTR of GCH1 was found to be responsive to AUF1 expression by luciferase assay. Knockdown of GCH1 suppressed cell proliferation and colony formation of ESCC cells. The expression of AUF1 significantly correlated with that of GCH1 in ESCC tissues. Taken together, we demonstrated the overexpression of AUF1 in esophageal carcinoma and identified GCH1 as AUF1's effector for the proliferation of ESCC cells.

Introduction

Esophageal cancer is the sixth leading cause of cancer death in male and the ninth in female, with an estimated over 400,000 new cases and deaths each year worldwide (1). The two main histological types are esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. ESCC is the most common histological subtype in China with 95% of all esophageal cancer cases. With new methods of diagnosis and treatment, esophageal cancer still has a poor 5-year survival rate (2). Most of their mortality is not due to their primary tumors, but to their invasiveness and metastasis (3). A better understanding of molecular pathogenesis of ESCC is urgent for the diagnosis and treatment of the disease.

Regulation of gene expression is a major control point in organisms to adapt to the environment. The regulation of mRNA stability and translation are the basis of eukaryotes in response to intrinsic or extrinsic stimuli. Many mRNAs encode proto-oncogenes and cytokines, the aberrant mRNA stability is associated with inflammation and carcinogenesis (4,5). It is recognized that approximately 16% of all human protein coding genes are encoded by mRNAs that contain an adenylate-uridylate (AU)-rich element (ARE) motif in their 3′UTR (69). AU-rich element RNA-binding factor 1 (AUF1), also called the heterogeneous nuclear ribonucleoprotein D (hnRNPD), is an established RNA-binding protein. The human AUF1 locus maps to 4q21, and it comprises a family of proteins composed of four isoforms with different molecular masses: p37, p40, p42 and p45 (10). AUF1 isoforms express preferentially in different tissues, which have their distinct biological properties (10). AUF1 influences the process of development, apoptosis and tumorigenesis via interacting with ARE-bearing mRNAs (11).

Altered AUF1 expression level may be a promoter to the development and progression of multiple types of cancers. It was reported that overexpression of AUF1 remarkably affected the stability of ARE containing mRNAs. Kumar et al (12) found that the expression level of nuclear AUF1 was significantly higher in oral squamous cell carcinoma than that in normal cells, and downregulated AUF1 with RNAi resulted in decreased proliferation rates. Overexpression of AUF1 isoform p37 developed sarcomas with deregulated c-Myc, c-Fos and c-Jun and cyclin D1 expression in tumor tissues (13). AUF1 has been shown to facilitate the decay of multiple target mRNAs, but occasionally to modulate the stability and translation of several target mRNAs (14). Several studies have shown that AUF1 plays an anti-tumorigenic role in regulation of the cell cycle and apoptosis. For example, increasing the AUF1 by UVC may promote apoptosis by reducing ARE-containing Bcl-2 mRNA (15). Treatment with postaglandin A2 (PGA2), an anticancer agent, induces expression of AUF1 isoform p45 and leads to the destabilization of cyclin D1 mRNA in non-small cell lung cancer cells (16). The above evidence suggests that AUF1 may be a promoter or inhibitor in the processes of carcinogenesis or proliferation, which vary between different types of cancer cells.

The clinical relevance of AUF1 and its biological function in ESCC progression have not been reported. In the present study, we investigated the expression of AUF1 in the ESCC tissue samles and normal samples. We further illustrated the role and downstream effector of AUF1 in driving cell proliferation of ESCC cells.

Materials and methods

Tissue samples

The ESCC samples were collected from 104 patients who had not received chemotherapy or radiotherapy prior to surgery. For these patients, if preoperative ultrasonography or computed tomography (CT) did not show any enlarged cervical lymph nodes (minor axis <0.5 cm), patients underwent a subtotal esophagectomy with two-field lymphadenectomy through a right thoracotomy, followed by a laparotomy. If enlarged cervical lymph nodes were detected by preoperative ultrasonography or CT, patients underwent radical oesophagectomy with cervico-thoraco-abdominal three-field lymphadenectomy through a right thoracotomy, followed by a laparotomy and a cervical incision. The resection extent includes nodes along the cervical part of the esophagus and deep cervix. Tumor adjacent tissues were obtained 3 cm away from the tumor. Fifteen normal esophageal tissue samples were obtained from surgical resections of trauma patients. These normal esophageal tissue samples were obtained from upper endoscopy of trauma patients, which were performed by the same operator. These patients had a fish bone or a poultry bone stuck. These tissues were obtained postoperatively between 2010 and 2013 from the Gastrointestinal Center, Jiangyin People's Hospital, the Jiangyin Clinical College of Xuzhou Medical University (Jiangyin, China) as previously reported (17). All patients provided signed and informed consents for their tissue samples to be used for scientific research. The ethics approval for the study was obtained from the Jiangyin People's Hospital, the Jiangyin Clinical College of Xuzhou Medical University. All diagnoses were based on pathological and/or cytological evidence. The histological features of the specimens were evaluated by a senior pathologist according to the classification criteria from the World Health Organization (18). Tissue samples were obtained prior to chemotherapy and radiation therapy and were immediately fixed in 10% neutral buffered formalin prior to immunohistochemistry analysis.

Immunohistochemistry staining (IHC)

The tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin. Three-micrometer thick paraffin sections were deparaffinized and heat-treated with citrate buffer (pH 6.0) for 7 min as an epitope retrieval protocol. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 15 min at room temperature and tissue non-specific binding sites were blocked with skim milk powder at 4% applied for 30 min. Sections were then incubated with the AUF1 and GCH1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h and mixed with skim milk powder at 2% again to reduce the non-specific staining. Biotinylated secondary antibody was then added for 30 min. IHC staining was visualized with substrate solution containing diaminobenzidine (DAB) and hydrogen peroxide. The counter-staining was performed with haematoxylin. All steps were performed at room temperature. Negative controls consisted of tissue sections undergoing similar staining procedures in the absence of the primary antibody. The criteria for scoring the stained sections were as follows: negative (0), <10% of the whole tissue section stained positive; weakly positive (+ 1), 10–25% of the whole tissue section stained positive; moderately positive (+ 2), 25–75% of the whole tissue section stained positive; and strongly positive (+ 3), 75% of the tissue section stained positive. Tissues with score 0 or 1 were considered as AUF1-negative, while with score 2 or 3 were considered as AUF1-positive.

Cell culture and transfection

The human esophageal cancer cell line Eca-109 was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (Gibco, Grand Island, NY, USA). Cells were grown in a 37°C incubator with 5% CO2.

The siRNA targeting AUF1 (siRNA-AUF1) and siRNA control (siRNA-NC) used in the present study were obtained from Santa Cruz Biotechnology. The cells were grown in a 6-well culture plate to 70–80% confluence and then transfected with either siRNA-NC, siRNA-AUF1 or siRNA-GCH1 (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After 48 h, the cells were collected for western blot analysis.

Cell viability assay

Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates. The next day, the cells were transfected with plasmids or siRNAs according to the experimental design. The cells were then incubated with 20 μl MTT (5 mg/ml) for 4 h. After the medium was removed, 100 μl DMSO was added and the optical density (OD) at 490 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The viability index was calculated as experimental OD value/control OD value. Three independent experiments were performed in quadruplicate.

Focus formation

Cells transfected with the indicated vectors were plated at low density (1,000 cells/6-cm plate), incubated for 10 days and fixed and stained with crystal violet. Foci and colonies containing >50 cells were counted using a microscope.

Western blot analysis

After transfection, cells were harvested and lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 4°C. Protein (40 μg) from each lysate was fractionated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk in PBS-Tween-20 for 1 h at room temperature, the membranes were blotted with AUF1 and GCH1 primary antibodies (Abcam, Cambridge, UK). GAPDH (Beyotime Institute of Biotechnology, Nantong, China) was used as a loading control. After washing four times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h. The proteins were visualized using the enhanced chemiluminescence (ECL; Beyotime Institute of Biotechnology).

Microarray analysis

Microarray-based mRNA expression profiling was performed using the Roche-NimbleGen (135K array) array (Roche NimbleGen, Madison, WI, USA). The microarrays contained approximately 45,033 assay probes corresponding to all of the annotated human mRNA sequences (NCBI HG18). Total RNA labeling and hybridization were performed using standard conditions according to the manufacturer's instructions. Genes with fold change of 2 or greater were subsequently subjected to pathway analysis using Ingenuity Pathway Analysis (Qiagen, Redwood City, CA, USA).

RNA extraction and real-time PCR

The total RNA from the esophageal tissue samples was extracted with TRIzol (Invitrogen) and reverse transcribed to complementary DNA (cDNA) using an oligo(dT)12 primer and SuperScript II (Invitrogen). The SYBR-Green dye (Takara Bio, Osaka, Japan) was used for the amplification of cDNA. The mRNA levels of transcripts were measured using real-time quantitative PCR in triplicate on a Prism 7500 real-time PCR machine (Applied Biosystems, Foster City, CA, USA). Primers specific for the genes are listed in Table I. PCR was performed in a 20 μl reaction mixture. The thermal cycling conditions included an initial denaturation step of 95°C for 20 sec, followed by 40 cycles at 95°C for 5 sec and annealing at 60°C for 30 sec. The relative mRNA expression levels were normalized to GAPDH.

Table I

Primer sequences for real-time PCR analysis.

Table I

Primer sequences for real-time PCR analysis.

GeneForward primerReverse primer
GAPDH 5′-AAGGTGAAGGTCGGAGTC-3′ 5′-GAAGATGGTGATGGGATTTC-3′
Nalcn 5′-CCATGAAATCTGTCGTGTGC-3′ 5′-TGCTCACTGGACAATCAAGG-3′
UGP2 5′-TCTGGAAACCTTCGCATCTT-3′ 5′-TTGCTCCATCTGTGCTTTTC-3′
BEX2 5′-TAATAAAGGGGAGCCCTTGG-3′ 5′-ACTGCCCGCAAACTATGACT-3′
Prdm16 5′-ACGCTTGGTTGAAGGTCACT-3′ 5′-TGGAAAAGTCGGAATGAACC-3′
Luciferase assays

Two luciferase reporters containing the full-length 3′UTR fragment of the GCH1 3′UTR (+1 to +1063, relative to the stop coden) was constructed by Shanghai Biobuy Co., Ltd. (Shanghai, China). Then, constructs each with a deletion of the two potential AU-rich elements in the GCH1 3′UTR were constructed. For the luciferase assay, the reporter vector with the GCH1 3′UTR was co-transfected with pRL-TK (Promega, Madison, WI, USA) to correct the transfection efficiency. Luciferase activity was measured with the Dual-luciferase reporter assay system (Promega). Promoter activities were expressed as the ratio of firefly luciferase to Renilla luciferase activities.

Intracellular nitric oxide (NO) determination

Total NO concentration in cultured cells was detected by measuring the concentration of nitrate and nitrite by modified Griess reaction method. After lysis of cells, a total Nitric Oxide assay kit (Beyotime Institute of Biotechnology) was used to detect NO. The optical densities at 540 nm wavelength were recorded using a microplate reader (BioTek Instruments, Winooski, VT, USA) and the concentrations of NO were calculated according to the standard curve.

Statistical analysis

Data are expressed as the means ± standard error of the mean (SEM) of at least three independent experiments. Standard error bars were included for all data points. The data were first analyzed with the Kolmogorov-Smirnov test for data distribution normality. The data were then analyzed using Student's t-test when only two groups were present or assessed by one-way analysis of variance (ANOVA) when more than two groups were compared. The P-values for t-tests were performed by 2-tailed t-tests. Correlation analysis of the mRNA expression data was performed using the Pearson test. Statistical analysis was performed using SPSS software (Release 19.0; SPSS, Inc., Chicago, IL, USA). Data were considered significant at P<0.05.

Results

Upregulation of AUF1 is correlated with clinicopathological feature in esophageal squamous cell carcinoma

To investigate the expression of AUF1 in human ESCC tissues, we performed immunohistochemistry analysis on tissue section of normal esophageal tissue specimens, ESCC and tumor adjacent tissues. Fifteen paraffin-embedded normal esophageal tissues and 104 ESCC tissues, 54 of which had corresponding adjacent tissues were collected. The demographic features of patients with ESCC for the immunohistochemistry staining are summarized in Table II. Results from IHC staining showed that a generally stronger staining of AUF1 was observed in the nucleus of ESCC tissues, whereas very weak staining of AUF1 was observed in the normal samples (Fig. 1A). When the samples were scored by a pathologist, as described in Materials and methods, AUF1 expression was similar between normal esophageal tissues and tumor adjacent tissues, while increased significantly among ESCC tissues, and between tumor tissues and tumor adjacent tissues (P<0.05; Fig. 1B). All the above results indicate that AUF1 may be involved in the pathogenesis of ESCC.

Table II

Patient demographic features for the IHC analysis.

Table II

Patient demographic features for the IHC analysis.

NormalGrade IGrade IIGrade IIIGrade IVAll tumors
Number151552334104
Mean age (years)58.7362.8859.2156.5066.0058.90
Age range (years)32–6555–6933–7332–7552–6732–76
Gender
 Male1093723473
 Female561510031

Next, the relationship between clinicopathology of ESCC patients and AUF1 IHC staining score was analyzed. As shown in Table III, the results showed a non-significant correlation between AUF1 expression and age (P=0.610), gender (P=0.637) and lymphatic invasion (P=0.096) of ESCC patients. AUF1 expression correlated with ESCC stage (P=0.011) and marginally correlated with lymph node metastasis (P=0.055). These results suggest AUF1 as a characteristic of this malignancy.

Table III

Relationship between clinicopathological parameters and the expression of AUF1 in esophageal squamous cell carcinoma (ESCC) (n=104).

Table III

Relationship between clinicopathological parameters and the expression of AUF1 in esophageal squamous cell carcinoma (ESCC) (n=104).

Clinicopathological parametersCaseAUF1 expressionP-value

NegativePositive
Age (years)0.610
 <64612239
 ≥64431330
Gender0.637
 Male732548
 Female311021
Stage0.011
 11596
 2521834
 333726
 4413
pN0.055
 (+)41932
 (−)632637
Lymphatic invasion0.096
 (+)491237
 (−)552332

[i] pN, lymph node metastasis.

AUF1 expression affects the proliferation and apoptosis of ESCC cells

Because AUF1 expression was associated with ESCC progression, we sought to explore the role of AUF1 in esophageal cancer cell growth. Eca-109 cells were transfected with a siRNA control (siRNA-NC) or siRNA trageting AUF1 (siRNA-AUF1). As shown in Fig. 2A, the protein level of AUF1 was markedly reduced after transfection of siRNA-AUF1. Next, the effect of AUF1 on cell growth was measured with MTT and colony formation. The results showed that decreased AUF1 expression suppressed cell growth and focus formation (Fig. 2B and C). We next investigated whether reduced cell growth was associated with apoptosis in ESCC cells. As shown in Fig. 2E, silencing of AUF1 significantly increased apoptosis of Eca-109 cells. These results demonstrated that AUF1 modulates the proliferation and apoptotic cell death of ESCC cells.

mRNA microarray screening of AUF1-affected genes

Although many AUF1-regulated genes have been identified, the profile of AUF1-affected genes in ESCC cells remains elusive. To further analyze the underlying mechanisms responsible for AUF1-mediated effects, we screened gene expression between AUF1 silencing (siRNA-AUF1) and the control cells (siRNA-NC) by microarray. Twenty-four hours after the transfection, a total of 285 genes (138 upregulated and 147 downregulated genes) were identified with an expression differential of 2-fold or greater between the two groups (n=2; Fig. 3A and Table IV). The differentially expressed genes included Nalcn, MMP23B, E2F1, BEX2 and GCH1. We then validated the results from microarray analysis by RT-PCR. As shown in Fig. 3B, compared with control cells, several genes dysregulated in microarray data, such as Nalcn, UGP2, BEX2 and Prdm16 showed consistent changes by real-time PCR analysis. As expected, AUF1 appeared to have modulated the Eca-109 cells via complex mechanisms. Pathway analysis revealed that AUF1 affected multiple pathways, including MAPK signaling pathway, transcriptional regulation and adipocytokine signaling pathway (Fig. 3C). These results suggested that AUF1 might be a wide-spectrum regulator of gene expression in esophageal cancer cells.

Table IV

Micorarray analysis of gene expression changes between siRNA-NC- and siRNA-AUF1-transfected cells.

Table IV

Micorarray analysis of gene expression changes between siRNA-NC- and siRNA-AUF1-transfected cells.

Gene nameFold change upregulatedChromosomeDescription
NALCN25.949chr13Sodium leak channel, non selective
UGP220.386chr2UDP-glucose pyrophosphorylase 2
SCAF117.645chr19SR-related CTD-associated factor 1
KRTAP10-1017.180chr21Keratin associated protein 10-10
MMP23B16.273chr1Matrix metallopeptidase 23B
MAP7D215.894chrXMAP7 domain containing 2
TMEM12915.310chr4Transmembrane protein 129, E3 ubiquitin protein ligase
DACT115.130chr14Dishevelled-binding antagonist of β-catenin 1
ARHGAP3013.292chr1Rho GTPase activating protein 30
E2F113.152chr20E2F transcription factor 1
PCNXL212.669chr1Pecanex-like 2 (Drosophila)
NID112.587chr1Nidogen 1
PAK611.838chr15P21 protein (Cdc42/Rac)-activated kinase 6
AMBRA111.740chr11Autophagy/Beclin-1 regulator 1
TAF1111.715chr6TAF11 RNA polymerase II, TATA Box binding protein (TBP)-associated factor, 28 kDa
NCDN11.146chr1Neurochondrin
C14ORF10111.047chr14Transmembrane protein 260
WDR2010.609chr14WD repeat domain 20
CCDC74A10.475chr2Coiled-coil domain containing 74A
TRO10.257chrXTrophinin

Gene nameFold change downregulatedChromosomeDescription

BEX223.010chrXBrain expressed X-linked 2
PRDM1619.479chr1PR domain containing 16
ARX18.122chrXAristaless related homeobox
CPZ17.834chr4Carboxypeptidase Z
GRM417.232chr6Glutamate receptor, metabotropic 4
PHF20L116.919chr8PHD finger protein 20-like 1
ANO913.588chr11Anoctamin 9
SLC24A413.212chr14Solute carrier family 24
BAZ1B12.677chr7Bromodomain adjacent to zinc finger domain, 1B
C1ORF14711.959chr1Chromosome 1 open reading frame 147
SIRT210.788chr19Sirtuin 2
RCBTB110.731chr13Regulator of chromosome condensation (RCC1) and BTB (POZ) domain containing protein 1
ISM110.317chr20Isthmin 1, angiogenesis inhibitor
OAZ210.129chr15Ornithine decarboxylase antizyme 2
SNCAIP10.088chr5Synuclein, α interacting protein
CAMK1G10.031chr1 Calcium/calmodulin-dependent protein kinase IG
MYCT19.578chr6Myc target 1
SEC14L38.865chr22SEC14-like lipid binding 3
SGCB8.732chr4Sarcoglycan, β
GCH18.726chr14GTP cyclohydrolase 1
AUF1 regulated GCH1 expression through 3′UTR

Among AUF1 affected genes, we focused on GCH1, which catalyzes the formation of 5,6,7,8-tetrahydrobiopterin (BH4), an essential co-factor for all nitric oxide synthases (19,20). GCH1 has been reported to contribute to cancer progression (21). Sequence analysis of the GCH1 3′UTR identified two potential AU-rich elements (Fig. 4A), which were likely to be regulated by AUF1. We therefore investigated whether AUF1 modulated the expression of GCH1 and cancer progression through GCH1. The effect of AUF1 on the GCH1 3′UTR was also measured using luciferase constructs containing the GCH1 3′UTR with or without AUF1 binding sites (Fig. 4A). The results revealed that deletion of the ARE1 (pGL3-GCH1 3UTR Del1) showed significantly decreased luciferase activity compared with that of wild-type GCH1 3′UTR in Eca-109 cells (Fig. 4B). However, deletion of the ARE2 (pGL3-GCH1 3UTR Del2) showed similar luciferase activity as compared with that of wild-type GCH1 3′UTR in Eca-109 cells (Fig. 4B). Western blot analysis further confirmed that knockdown of AUF1 decreased the expression of GCH1 (Fig. 4C). Because GCH1 is an essential co-factor for all NOS, as expected, silencing of AUF1 significantly reduced cellular NO level. These results suggested that AUF1 regulated GCH1 through ARE1 region (Fig. 4D).

Expression of AUF1 correlates with GCH1 in ESCC tissues

The above mentioned results led us to investigate whether the expression level of AUF1 correlated with that of GCH1 in tissue samples. Immunohistochemistry was used to detect the expression levels of GCH1 protein in 102 ESCC tissue samples. Representative IHC staining of both AUF1 and GCH1 in the same tissue is shown in Fig. 5A. Tissue samples with strong AUF1 staining in the nucleus exhibited positive GCH1 staining, whereas weak AUF1 expression showed negative GCH1 staining. Statistical analysis revealed that the protein levels of the two genes correlated with each other (Pearson r=0.251, P=0.019; Fig. 5B), indicating that the expression of AUF1 correlates with the expression of GCH1 in human ESCC tissues.

Inhibition of GCH1 suppresses the proliferation and colony formation in ESCC cells

To explore the role of GCH1 in esophageal cancer cell growth, cells were first transfected with a siRNA-NC or siRNA-GCH1, and the resulting growth was measured with MTT and colony formation assay. The results showed that GCH1 silencing caused significantly decreased cell viability (75% of the control group). Moreover, knockdown of GCH1 attenuated the number and the size of colonies of Eca-109 cells. These results suggest that GCH1 promotes esophageal tumor growth in vitro. (Fig. 6).

Discussion

AUF1 plays a central role in various physiological and pathological processes. Excessive and dysregulated AUF1 expression has been implicated in diseases including cancers. Overexpression of AUF1 has been detected in numerous malignancies, including breast, skin, thyroid, liver, oral squamous cell carcinoma and sarcoma (12,13,16,2224). AUF1 has therefore been recognized to modulate different cancer-related events. However, there is considerable controversy in defining its role in cancer progression. Several studies have shown that AUF1 has both tumor promoting and tumoricidal effects depending on cancer types (1216). AUF1 has been suggested to modulate different cancer-related events including apoptosis, tumorigenesis and development (10). However, the role of AUF1 in ESCC progression has not been reported. In the present study, we found that the expression of AUF1 was significantly higher in ESCC tissues than that in normal tissues and tumor adjacent tissues. In this study, we found that AUF1 levels are correlated with tumor stage, indicating that AUF1 may be a predicator of malignant degree, and related with prognosis of the malignancy. We also found that AUF1 silencing resulted in markedly decreased cell proliferation and focus formation and elevated cell apoptosis.

AUF1 has been implicated in regulating the expression of numerous mRNAs containing 3′UTR ARE, including Bcl2, cyclin D1 and SOD1 (10,13,14). However, the regulatory profile of AUF1 may be cell-specific. To comprehensively understand the function of AUF1 in esophageal cancer, we performed mRNA microarray-based screening analysis in Eca-109 cells. Through this analysis, we found that overexpression of AUF1 affected multiple genes, such as Nalcn, UGP2, BEX2 and Prdm16. Among AUF1 affected genes, GCH1 encoded protein is the first and rate-limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis, catalyzing the conversion of GTP into 7,8-dihydroneopterin triphosphate (25). BH4 is an essential element required for nitric oxide (NO) formation by all nitric oxide synthases (NOSs) (26). NO has been reported to modulate many cancer-related events, such as angiogenesis, apoptosis, cell cycle, invasion and metastasis (27), and correlated with the development of several cancers, including breast, cervical, lung, gastric and head and neck cancer (2833). Therefore, GCH1 is likely to play important roles in cancer progression. Chen et al (34) reported that GCH1 promotes cancer angiogenesis in vitro and in vivo. Consistently, another group demonstrates that GCH1 inhibition reduces tumor growth by: i) direct killing of tumor cells; ii) inhibiting angiogenesis; and iii) enhancing the antitumoral immune response. However, little is known about the functional significance of GCH1 in ESCC cancer. Here, we report that GCH1 was regulated by AUF1 and involved in the proliferation and colony formation in ESCC cells. The function of GCH1 in other aspects and regulatory mechnisms merit further investigation.

In summary, we found that AUF1 expression was significantly increased in ESCC tumor tissues, and that its expression correlated with tumor stage in human ESCC tissue samples. Furthermore, knockdown of AUF1 resulted in markedly decreased proliferation and increased cell apoptosis. AUF1 silencing-affected genes were screened by microarray assay. In particular, we demonstrated that AUF1 regulated the expression of GCH1 through its 3′UTR. Silencing of GCH1 inhibited the growth of ESCC cells. In conclusion, AUF1/GCH1 axis may present a novel potential target in the treatment of ESCC.

Acknowledgements

The present study was supported by the Natural Science Foundation of Jiangsu Province (BK20161152), the National Natural Science Foundation of China (31670857, 81672975, 81522039 and 31400720), the Key Research and Development Program of China (2016YFC0904702) and the Key Scientific Research Program of Wuxi Municipal Health Bureau (Z201509).

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November 2016
Volume 49 Issue 5

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Online ISSN:1791-2423

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Copy and paste a formatted citation
APA
Gao, Y., Wang, W., Cao, J., Wang, F., Geng, Y., Cao, J. ... Zhang, S. (2016). Upregulation of AUF1 is involved in the proliferation of esophageal squamous cell carcinoma through GCH1. International Journal of Oncology, 49, 2001-2010. https://doi.org/10.3892/ijo.2016.3713
MLA
Gao, Y., Wang, W., Cao, J., Wang, F., Geng, Y., Cao, J., Xu, X., Zhou, J., Liu, P., Zhang, S."Upregulation of AUF1 is involved in the proliferation of esophageal squamous cell carcinoma through GCH1". International Journal of Oncology 49.5 (2016): 2001-2010.
Chicago
Gao, Y., Wang, W., Cao, J., Wang, F., Geng, Y., Cao, J., Xu, X., Zhou, J., Liu, P., Zhang, S."Upregulation of AUF1 is involved in the proliferation of esophageal squamous cell carcinoma through GCH1". International Journal of Oncology 49, no. 5 (2016): 2001-2010. https://doi.org/10.3892/ijo.2016.3713