EBPα-induced miR-100 expression suppresses tumor metastasis and growth by targeting ZBTB7A in gastric cancer

ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

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Original Articles

FC/EBPα-induced miR-100 expression suppresses tumor metastasis and growth by targeting ZBTB7A in gastric cancer Duan-Bo Shi, Ya-Wen Wang, Ai-Yan Xing, Ji-Wei Gao, Hui Zhang, Xiang-Yu Guo, Q1 Peng Gao * Department of Pathology, School of Medicine, Shandong University, Jinan 250012, China

A R T I C L E

I N F O

Article history: Received 16 April 2015 Received in revised form 28 August 2015 Accepted 29 August 2015 Keywords: Gastric cancer miR-100 Metastasis ZBTB7A C/EBPα

A B S T R A C T

MicroRNAs have been reported to play key roles in various human cancers, including gastric cancer. However, understanding of the expression of miR-100 and its regulatory mechanisms in human gastric cancer remains elusive. In this study, we reveal that miR-100 is downregulated in gastric cancer samples and gastric cancer cell lines. Furthermore, lower miR-100 expression was found in primary gastric cancer samples with lymphatic metastasis compared to those without lymphatic metastasis. Overexpression of miR-100 suppressed tumor growth in vivo and inhibited gastric cancer invasion and metastasis in vitro and in vivo. Furthermore, we demonstrated that miR-100 reduced gastric cancer aggressiveness by directly targeting ZBTB7A. Knockdown of ZBTB7A by siRNA disrupted gastric cancer progression by impairing tumor invasion and metastasis. High expression of ZBTB7A was significantly correlated with poorer prognosis in gastric cancer patients. Our results also showed that the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) could induce the expression of miR-100 by binding to the putative promoter region of miR100. This study demonstrated that miR-100 could be induced by C/EBPα and may act as a tumor suppressor gene by inhibiting ZBTB7A. © 2015 Published by Elsevier Ireland Ltd.

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Introduction MicroRNAs (miRNAs) are a class of non-coding, 20–25 nucleotidelong RNAs that can regulate target gene expression at the posttranscriptional level by inhibiting translation or by increasing messenger RNA (mRNA) degradation through imperfect pairing with the 3′-untranslated regions (3′-UTRs) of mRNA [1]. Recent evidence indicates that miRNA levels are closely correlated with tumor metastasis and proliferation. For example, the highly expressed miR10b in metastatic breast cancer cells promotes cell invasion and migration by targeting the HOXD10 gene [2]. Depletion of miR191 up-regulated the expression of the target genes SOX4, IL1A and TMC7 in hepatocellular carcinoma, decreased cell proliferation and induced apoptosis [3]. The finding that miRNAs act as tumor suppressors and oncogenes by targeting specific genes sheds new light on anticancer treatment. Moreover, miRNA expression could be considered a prognosis marker, as dysregulation of miRNAs has been reported to be associated with the clinical outcome of human cancers [4–6]. Gastric cancer is still a major public health problem worldwide. It is the second most common cause of cancer-related mortality [7]. Fortunately, research on miRNAs and their molecular mecha-

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* Corresponding author. Tel.: +86 531 88382045; fax: +86 531 88383168. E-mail address: [email protected] (P. Gao).

nisms in gastric cancer have opened a new door for therapeutic strategies. For example, miR-145 and miR-218 suppress gastric cancer cell metastasis by inhibiting the expression of N-cadherin (CDH2) and Robo1, respectively [8,9]. One miRNA aberrantly expressed in human cancer, miR-100, is known to target mTOR in childhood adrenocortical tumors and in clear cell ovarian cancer [10,11]. In acute myeloid leukemia, miR100 promotes cancer cell proliferation by negatively regulating RBSP3 [12]. However, the role of miR-100 and its underlying mechanism in gastric cancer remains unknown. Recently, some mechanisms underlying the aberrant expression of miRNAs in cancer have been reported. For instance, transcription factors and DNA methylation of CpG islands in promoter regions could play an important role in regulating miRNAs [2,13]. Therefore, it is of great interest to investigate the mechanism underlying the regulation of miR-100 expression in gastric cancer. Materials and methods

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Tissue samples

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Primary gastric cancers, lymph node metastatic loci of gastric cancers and nontumorous gastric mucosa taken 3 cm from the tumor site were obtained from patients who underwent gastric cancer radical resection at the Qi Lu Hospital of Shandong University from 2004 to 2006. All tissues were obtained with patients’ informed consent. None of the patients received preoperative chemotherapy or radiation therapy. This study was approved and conducted in accordance with the policies of the Ethics Committee of Shandong University. All cases of gastric cancer were evaluated

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http://dx.doi.org/10.1016/j.canlet.2015.08.029 0304-3835/© 2015 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Duan-Bo Shi, et al., FC/EBPα-induced miR-100 expression suppresses tumor metastasis and growth by targeting ZBTB7A in gastric cancer, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.08.029

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histologically according to Lauren’s and the World Health Organization (WHO)’s classifications (IARC Press, Lyon, 2000) and staged usings the TNM staging of the International Union Against Cancer (UICC 2002).

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Human gastric cancer cell lines MKN-45 (poorly differentiated), MKN-28 (well differentiated), SGC-7901 (poorly differentiated, metastatic) and BGC-823 (poorly differentiated) were used in this study. The first two cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), and the other two cell lines were purchased from Shanghai Cancer Institute (Shanghai, China). All cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified cell incubator with an atmosphere of 5% CO2.

Cell culture

RNA extraction and real-time quantitative PCR

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Gastric cancer samples and non-tumorous gastric mucosa were microdissected under a dissecting microscope to avoid cross contamination between stromal and necrotic tissues (Leica ASLMD, Witts Baden, Germany). Total RNA from the tumor samples and non-tumor samples was extracted using a miRNeasy FFPE kit (Qiagen, Valencia, CA, USA), following the manufacturer’s protocol. Total RNA of cell lines was isolated using Trizol reagent (TaKaRa, Dalian, China) according to the manufacturer’s instruction. For miRNA and ZBTB7A mRNA expression analysis, real-time quantitative PCR (RT-qPCR) was performed as previously described [14]. The relative expression of miR-100 or ZBTB7A to its control was assessed using the 2−ΔCT method. Each sample was analyzed in triplicate.

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Cell transfection was performed as previously described [8]. Precursor miR100 (Ambion, Austin, TX, USA) or miR-100 inhibitor and the negative control (GenePharma, Shanghai, China) were used to investigate the effects of miR-100 on gastric cancer cells. The siRNA against ZBTB7A (si-ZBTB7A, GenePharma, Shanghai, China) were as follows: si-ZBTB7A#1, 5′-GCUGGACCUUGUAGAUCAAtt-3′, 5′UUGAUCUACAAGGUCCAGCtt-3′; si-ZBTB7A#2, 5′-UCAAGAAAGACGGCUGCAAtt-3′, 5′-UUGCAGCCGUCUUUCUUGAtt-3′.

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Cell migration and invasion assay in vitro was performed using Transwells with 8 μm pore size (24-well insert, Corning, New York, USA) as previously described [15].

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Twenty-four hours after transfection, the transfected cells were seeded in triplicate into 96-well plates at a density of 4 × 103 cells per well. Then MTS and EDU assays were performed to test the proliferation ability of the cells with CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, San Luis Obispo, CA, USA) and Cell-lightTM EdU Apollo 567 In Vitro Kit (Ribobio, Guangzhou, China) at 24, 48 and 72 h. For three-dimensional (3D) cell culture and alamarBlue® assay, the 96 well plates were first coated with MaxGelTM ECM (extracellular matrix, SigmaAldrich, St. Louis, USA), then the transfected cells were seeded as described above and the proliferation ability of the cells was analyzed with alamarBlue® assay kit (Invitrogen, Carlsbad, USA), following the manufacturer’s protocol. Independent proliferation assays were performed three times.

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Cells were harvested 48 h after transfection and were stained using Annexin V-FITC/PI Apoptosis Detection Kit (BestBio, Shanghai, China) following the manufacturer’s instructions. Briefly, transfected cells were trypsinized, collected and incubated with Annexin V-FITC and PI. Stained cells were detected by flow cytometry for early and late apoptosis analysis. Independent experiments for apoptosis were performed three times.

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The orthotropic tumor model was established as previously described [8]. In brief, SGC7901 cells were transfected with lentivirus vector LV3-GFP-miR-100 or LV3GFP-miR-negative control (Genecopoeia, Rockville, MD, USA), and then cells were injected into the lateral tail vein or the axillary fossa of the mice. The xenograft tumors in living mice were measured by observing GFP expression using an in vivo Carestream Molecular Imaging system (Carestream Health, Inc., New York, USA). Eight weeks after injection, the mice were sacrificed, and the tumor nodules, lungs and liver were extracted for assessment. The tumor volume (V) was calculated as V = a × b2/2, where a and b are the long and short axes of the tumor nodule, respectively. Tumor nodules and metastatic loci were confirmed histologically.

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Plasmid construction and dual-luciferase reporter assay The pmirGLO-3′UTR vectors were constructed as described previously [8]. The transcription factor binding sites were first predicted by PROMO (http:// alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3), and then the putative promoter regions of miR-100 harboring the binding sites were amplified by PCR and subcloned into the upstream region of the PGL3-basic vector (Promega). The constructs were named PGL3-P100 (promoter region from −445 bp to +299 bp), PGL3-P100-1 (promoter region from −341 bp to −63 bp) and PGL3P100-1M (promoter region from −341 bp to −63 bp, the two C/EBPα binding sites were mutated). Then 100 ng of the transcription factor vectors pCMV-p53 or pcDNA3.1-C/EBPα was cotransfected with 50 ng of PRL-TK vectors (Promega, as an internal control) and 100 ng of PGL3-P100, PGL3-P100-1 or PGL3-P100-1M. The relative expression of firefly luciferase normalized to Renilla luciferase activity was obtained. Independent experiments for the luciferase reporter assay were performed three times.

Q2

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Western-blot

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In brief, proteins from cell lysates were resolved by electrophoresis, transferred to a PVDF membrane and blotted with antibodies for ZBTB7A (1:1000, Abcam, Cambridge, UK), C/EBPα (1:1000, Abcam) and β-actin (1:1000, Abcam). Independent experiments for western-blot assay were performed three times.

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Immunohistochemistry

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Immunohistochemistry (IHC) was performed as described previously [8].

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Cell transfection

Cell migration and invasion assay in vitro

Cell proliferation assay

Cell apoptosis assay

Orthotropic tumor model

5′-Rapid amplification of cDNA ends (5′-RACE) method to identify the transcription start site (TSS) of miR-100

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To identify the TSS of miR-100, a 5′-RACE assay was carried out using the 5′Full RACE Kit (TaKaRa) according to the manufacturer’s protocol. In brief, total RNA was extracted from SGC7901 cells and reverse transcribed into cDNA using Reverse Transcriptase and Random 9-mers. The first round of PCR amplification was performed with 5′-RACE Outer Primer and nested PCR primer GSP1, followed by the second round of PCR with 5′-RACE Inner Primer and nested PCR primer GSP2. Amplified products were sequenced for TSS analysis. Independent experiments for 5′RACE were performed in triplicate.

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Statistical analysis

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All of the results were analyzed using Prism 5 software (GraphPad Software, San Diego, CA, USA) and data are expressed as mean ± SE. The significance of the differences was determined with the Student’s t-test between two groups or with oneway ANOVA among three groups. The correlation between miR-100 expression level and tumor size or ZBTB7A level was calculated by Spearman’s correlation. For patient survival assessment, Kaplan–Meier survival analysis and log-rank test were used. The Cox proportional hazards regression was used to evaluate the effect of tumor variables on patient survival. χ2 test was used to analyze spontaneous invasion/ metastasis formation in vivo and the C/EBPα protein expression in gastric cancer/ non-tumorous tissues. P values less than 0.05 were considered to be statistically significant.

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Results The relative expression of miR-100 in gastric cancer tissues and cell lines, and its correlation with clinicopathological features We analyzed the expression of miR-100 in 112 cases of gastric cancers and 18 cases of non-tumorous gastric mucosa by RTqPCR. Compared with human non-tumorous gastric mucosa, miR100 in gastric cancer samples was found to be significantly downregulated (Fig. 1A). Intriguingly, miR-100 expression in primary gastric cancer samples with lymph node metastasis (LNM) was much lower than those without LNM (Fig. 1B). This result was validated in four gastric cancer cell lines: SGC7901, MKN45, BGC823 and MKN28 (Fig. 1C). Similarly, miR-100 was under-expressed in the metastatic gastric cancer cell line SGC7901 compared with other three gastric cancer cell lines (Fig. 1C). However, for the patients with LNM, no significant difference was observed between primary gastric cancer and their metastatic loci (data not shown), indicating that the downregulation of miR-100 expression occurred in the early stages of LNM development and remained stable during the

Please cite this article in press as: Duan-Bo Shi, et al., FC/EBPα-induced miR-100 expression suppresses tumor metastasis and growth by targeting ZBTB7A in gastric cancer, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.08.029

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Fig. 1. The expression of miR-100 in gastric cancers and its effect on cancer cell aggressiveness in vitro and in vivo. (A) The expression of miR-100 was significantly downregulated in 112 cases of primary gastric cancer tissues compared with 18 cases of non-tumorous gastric mucosa tissues analyzed by RT-qPCR (t-test, P < 0.001). (B) miR-100 level was even lower in primary gastric cancer tissues with lymph node metastasis (LNM) in comparison with primary gastric cancer tissues without LNM (t-test, P = 0.018). (C) miR-100 expression was markedly attenuated in four gastric cancer cell lines and was especially underexpressed in metastatic cell line SGC7901 compared to the other three non-metastatic cell lines (t-test, P < 0.001). (D) miR-100 expression was inversely correlated with the tumor size of primary gastric cancer by Spearman’s correlation test (n = 112, P = 0.009, r = −0.247). (E and F) In vitro migration and invasion assay was performed with a transwell chamber and migrated cells were counted. The result showed that miR-100 significantly suppressed the migration and invasion ability of SGC7901 and BGC823 in vitro (t-test, **P < 0.01, *P < 0.05). (G) miR-100 suppressed cancer cell invasion and metastasis in vivo. SGC7901 cells were transfected with LV3-GFP-miR-100 vector or LV3-GFP-miR-negative control vector and then 2.5 × 106 cells were injected into nude mice via the tail vein with GFP as a marker protein. Representative H&E staining of lungs and tumor nodules isolated from the mice. In miR-negative control group, the tumor nodules were well-encapsulated by intact fibrotic capsules and no lung metastasis was found. In contrast, blood vessel invasion (the red arrow), stroma invasion and lung metastasis (the black arrow) were observed in the miR-100 group (magnification ×200). (H) Eight weeks post implantation, GFP imaging of the living mice was measured.

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subsequent metastasis process. The correlations between miR100 expression level and clinicopathologic characteristics of gastric cancers are summarized in Table 1. Lower expression of miR-100 was correlated with positive LNM and lager tumor size (≥50 mm) (Table 1). Consistently, we further verified that miR-100 expression was inversely correlated with tumor

size by Spearman’s correlation test (Fig. 1D). Expression of miR100 was also found to be correlated with WHO histological classification (Table 1). No statistically significant association was observed between miR-100 expression and patient age, gender, clinical stage or Lauren’s classification (Table 1). Moreover, log-rank test showed no significant difference between the low expression group

Please cite this article in press as: Duan-Bo Shi, et al., FC/EBPα-induced miR-100 expression suppresses tumor metastasis and growth by targeting ZBTB7A in gastric cancer, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.08.029

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Table 1 Clinicopathologic characteristics of gastric cancers associated with miR-100 and ZBTB7A expression. Variables

No.

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Age (y) ≤60 >60 Gender Male Female Tumour size (mm) <50 ≥50 Clinical stage I/II III/IV Lymph node metastasis Yes No WHO histological classification Well-differentiated Moderately differentiated Poorly differentiated Mucinous adenocarcinoma Lauren’s classification Intestinal type Diffuse type

miR-100

ZBTB7A

miR-100 suppresses proliferation of gastric cancer in vivo

Median

P-value

Median

P-value

50 62

0.0054 0.0044

0.283

34.0 53.5

0.214

96 16

0.0046 0.0073

0.482

45.5 49.75

0.741

53 59

0.0063 0.0041

0.028

47. 5 47.0

0.368

56 56

0.0048 0.0058

0.124

29.5 55.25

0.033

66 46

0.0066 0.0041

0.021

55.5 29.5

0.027

6 35 52 19

0.0061 0.0030 0.0051 0.0086

0.032

10.0 32.0 52.5 49.0

0.267

41 71

0.0040 0.0058

0.229

31.0 52.0

0.037

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press gastric cancer invasion and metastasis, thereby acting as a potential anti-metastatic agent.

and high expression group in overall survival (OS) or disease free survival (DFS) (Fig. S1). miR-100 suppresses gastric cancer invasion and metastasis in vitro and in vivo Regarding the downregulation of miR-100 in metastatic gastric cancer tissues and cell lines, we wondered whether miR-100 could play the role of a tumor metastatic suppressor in gastric cancer. miR100 was efficiently overexpressed in SGC7901 and BGC823 cell lines after transfection with the miR-100 precursor (Fig. S2A and B). The migration and invasion capability of transfected cells was reduced significantly compared to the miR-negative control group (Fig. 1E and F). In addition, when miR-100 was knocked down (Fig. S2C and D), enhanced migration and invasion capability of gastric cancer cells were observed both in SGC7901 and BGC823 cells (Fig. S4E and F). To further investigate whether overexpression of miR-100 has any effect on the metastatic capability of gastric cancer cells in vivo, SGC7901 cells transfected with LV3-GFP-miR-100 or LV3-GFP-miRnegative control were injected into the axillary fossa of the mice. miR-100 was overexpressed stably in SGC7901 cells during the in vivo experimental period, as detected by RT-qPCR (Fig. S3B and C) and GFP observation (Fig. 1H, Fig. S3A). We found local invasion, with cancer cells invading the stroma surrounding the tumors in six of the eight injected mice in the LV3-GFP-miR-negative control group. In stark contrast, stromal invasion was only found in two of the nine injected mice in the LV3-GFP-miR-100 group, with tumor nodules well-encapsulated by intact fibrotic capsules in the remaining seven mice (Fig. 1G and Table S1). As stroma invasion is a critical step in the invasion-metastasis cascade, these data suggested that miR100 suppressed metastasis of ectopic tumors and that this metastatic suppression is due at least in part to its ability to impair local invasion. Furthermore, we injected miR-100-expressing cells or negative control cells into the lateral tail vein of the mice. As expected, metastasis in the lungs was observed in 3 of the 6 injected mice in the LV3-GFP-miR-negative control group. In contrast, no metastasis loci were found in the LV3-GFP-miR-100 group (Fig. 1G and Table S1). The results indicated that ectopic expression of miR-100 could sup-

To explore the role of miR-100 on cell proliferation, EDU, MTS and alamarBlue® assays were performed in SGC7901 and BGC823 cell lines. However, no significant difference in cell growth was observed between the precursor miR-100 or miR-100 inhibitor transfected cells and the negative control group (Fig. 2A, Fig. S4A–D). Flow cytometry further demonstrated that miR-100 had no effect on cell apoptosis (Fig. 2B). Intriguingly, we found that miR-100 played an important role in inhibiting tumor growth in vivo. Tumor nodules derived from miR-100 overexpressing cells grew substantially slower compared with the control group (Fig. 2C–E). Given that the expression level of miR-100 was inversely correlated with tumor size in human gastric cancers, our results indicated that miR-100 may suppress the growth of gastric cancer in vivo. Zbtb7a is a direct functional target of miR-100 in gastric cancer To explore the molecular mechanism underlying the tumor suppressing function of miR-100 in further detail, we first searched for potential miR-100 target genes using three algorithms (Targetscan, Pictar and Miranda). Among hundreds of predicted genes, ZBTB7A, F11R, FZD5 and CLDN4 were selected because they were reported to be overexpressed in human cancers and were also implicated in tumor metastasis and growth [16–20]. Luciferase reporter assays revealed that the relative luciferase activity of the ZBTB7A pmirGLO3′UTR vector was significantly reduced in miR-100 overexpressing SGC7901 and BGC823 cells, respectively (Fig. 3A). As expect, when miR-100 was knocked down, the upregulated luciferase activity of the ZBTB7A pmirGLO-3′UTR vector was observed (Fig. 3A). No significant suppression or activation of luciferase activity was observed in the other three predicted genes (Fig. 3B–D). On the contrary, when the binding sites of miR-100 in the ZBTB7A pmirGLO-3′UTR were mutated, its responsiveness to miR-100 regulation was abrogated (Fig. S5A). Furthermore, western blot experiments showed that overexpression of miR-100 reduced the protein level of ZBTB7A in SGC7901 and BGC823 cells by 81.8 ± 3.8% and 62.9 ± 1.3%, respectively (Fig. 3E). Upregulated protein level of ZBTB7A was also observed when miR-100 was knocked down (Fig. 3F). In addition, miR-100 significantly suppressed the protein expression of ZBTB7A in the xenograft tumors of the mice (Fig. 3G). This result was further confirmed by IHC in gastric cancer cell lines (Fig. S5B). On the other hand, miR-100 expression level was found to be inversely correlated with the expression of ZBTB7A protein in human gastric cancers (Fig. 4E-d). These results demonstrated that ZBTB7A was a direct target of miR-100. In order to investigate the effect of down-regulated ZBTB7A on gastric cancer cells, we inhibited ZBTB7A expression using siRNA (Fig. 4A and B and Fig. S5E and F). As expected, similar to the miR100 overexpressing cells, the migration and invasion capability of the ZBTB7A knockdown cells was suppressed (Fig. 4C and D and Fig. S5G and H). However, downregulation of ZBTB7A had no influence on cell’s proliferation activity or apoptosis (Fig. S5C and D). ZBTB7A expression is correlated with patient survival and clinicopathological variables The expression of ZBTB7A protein was examined by IHC in human gastric cancer samples and was analyzed in combination with patient clinicopathological parameters and survival. ZBTB7A expression was much higher in primary gastric cancers with LNM when compared to the cases without LNM, and higher ZBTB7A expression was also observed in the cancer tissue of metastatic loci (Table 1, t-test,

Please cite this article in press as: Duan-Bo Shi, et al., FC/EBPα-induced miR-100 expression suppresses tumor metastasis and growth by targeting ZBTB7A in gastric cancer, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.08.029

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Fig. 2. miR-100 suppresses proliferation of gastric cancer in vivo, but not in vitro. (A and B) The BGC823 and SGC7901 cells were transfected with 30 nm miR-100 precursor or negative control. 24 h after transfection, there was no significant difference between the proliferation activity between the miR-100 group and the negative control group by EDU assay (A, t-test, P > 0.05). Consistent with these results, 48 h after transfection, flow cytometry assay showed that miR-100 had no effect on cell apoptosis in the two cell lines (B, t-test, P > 0.05). (C) SGC7901 cells were transfected with LV3-GFP-miR-100 vector or LV3-GFP-miR-negative control vector, and then 2 × 106 cells were injected into the axillary fossa of the mice. Eight weeks after the injection, the mice were killed and the tumor nodules were harvested. (D and E) Tumor volume (V) was calculated every week as V = a × b2/2, where a and b are the long and short axes of the tumor nodule, respectively. Tumor nodules derived from miR-100 overexpressing cells grew substantially slower compared with the control group during the whole tumor growth period (D). Tumor nodules were found in all the eight nude mice in LV3-GFP-miRnegative control group at the end of the eighth week, while they were found in only five out of the nine nude mice in the LV3-GFP-miR-100 group (χ2 test, P = 0.031). Moreover, the mean tumor volume of the tumor nodules of the miR-100 group (916 ± 453 mm3) was smaller than that of the negative group (2295 ± 934 mm3) (E).

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P = 0.027, Fig. 4E-a, E-b and E-c). ZBTB7A expression was also found to be correlated with clinical stage and Lauren’s classification (Table 1). No significant association was found between ZBTB7A expression and patient age, gender, tumor size or WHO histological classification (Table 1). Kaplan–Meier survival analysis and log-rank test showed that the OS and DFS rate of patients with high ZBTB7A expression were markedly poorer than those with low ZBTB7A expression (Fig. 4F and G). In multivariate analysis, high ZBTB7A expression was an independent unfavorable predictor of overall survival (Table 2).

Our results indicated that ZBTB7A might play an important role in the pathogenesis of gastric cancer and could potentially be used as a prognostic bio-marker for gastric cancer patients. C/EBPα induces the expression of miR-100 and is downregulated in gastric cancer To examine the mechanism underlying the regulation of miR100 expression, the TSS was identified by the 5′-RACE method. We analyzed the transcription factor binding sites in the 2.0 kb

Please cite this article in press as: Duan-Bo Shi, et al., FC/EBPα-induced miR-100 expression suppresses tumor metastasis and growth by targeting ZBTB7A in gastric cancer, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.08.029

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Fig. 3. ZBTB7A was identified as a direct target gene of miR-100. (A–D) The 3′-UTR fragments of the putative target genes harboring miR-100 binding sites were subcloned into the downstream of a luciferase reporter vector pmirGLO. The pmirGLO-3′UTR vectors of the four predicted target genes were co-transfected with precursor miR-100 or negative control into SGC7901 and BGC823 cells. The relative luciferase activity of ZBTB7A pmirGLO-3′UTR vector was significantly reduced in the miR-100 overexpressing cells. The upregulated luciferase activity of the ZBTB7A pmirGLO-3′UTR vector was also observed when miR-100 was knocked down (A). The regulation of ZBTB7A by miR100 was much more significant than that of the other three predicted putative genes including F11R (B), FZD5 (C) and CLDN4 (D). (E) The ZBTB7A protein expression of the SGC7901 and BGC823 cells was clearly reduced after the transfection of miR-100 (t-test, **P < 0.01, *P < 0.05). (F) The ZBTB7A protein expression both in SGC7901 and BGC823 cells was upregulated when miR-100 was knocked down (t-test, *P < 0.05). (G) The ZBTB7A protein expression of implanted mice tumors of the miR-100 overexpressing group was significantly decreased compared with the control group (F, t-test, *P < 0.05).

9 fragment upstream of the TSS and found some well known transcription factor binding sites including two C/EBPα binding sites and one P53 binding site in the 0 to −400 bp sequence (Fig. S6A and B). The −445 bp to +299 bp sequence was first analyzed in a luciferase reporter assay and the result showed that the fragment exhibited promoter activity and that the activity could be enhanced significantly by C/EBPα (Fig. 5B), but not P53 (Fig. 5A). Therefore, we further investigated the promoter activity of the −341 bp to −63 bp fragment that harbored the C/EBPα sites and observed a consistent result (Fig. 5C). When the two binding sites (−106 bp to −100 bp and −178 bp to −172 bp) in the region were mutated, the promoter activity enhanced by C/EBPα was attenuated (Fig. 5D). The results

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Table 2 Multivariate analysis of DFS and OS of 112 patients with gastric cancer.

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Variables

26 27 28 29 30 31 32 33 34 35

Q5

Clinical stage I/II III/IV Lymph node metastasis Yes No ZBTB7A Low High

confirmed that the −63 bp to −341 bp fragment had promoter activity and could be activated by C/EBPα. The regulatory function of C/EBPα in inducing miR-100 expression was confirmed by detecting miR-100 level after the transcription factor was successfully overexpressed in the transfected cells (Fig. 5E). The expression of miR-100 was significantly up-regulated in SGC7901 (2.2-fold) and MKN45 (1.6-fold) cells after the transfection of C/EBPα (Fig. 5F). The expression of C/EBPα was investigated by IHC in gastric cancer. The IHC staining for C/EBPα was defined as positive when nuclear staining was observed. As previously described, when >50% of the epithelia were stained weakly or negatively, the cases were classified as downregulation [21]. As expect, the results suggested that the expression of C/EBPα was significantly decreased (Fig. S6C, χ2 test, P = 0.026) in gastric cancer tissues (21/75, 28%) compared with that in non-tumorous mucosa (3/35, 8.6%).

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Disease free survival

Overall survival

HR (95% CI)

P

HR (95% CI)

P

2.398 (1.374–4.186)

0.002

2.412 (1.441–4.037)

0.001

1.366 (0.716–2.606)

0.343

1.597 (0.863–2.955)

0.136

1.66 (0.966–2.854)

0.067

1.7 (1.031–2.803)

0.038

Discussion miRNAs can be oncogenic or tumor suppressive in human cancers by targeting specific genes and therefore play a crucial role in tumorigenesis [22]. Recently, downregulated miR-100 was reported in childhood adrenocortical tumors [10], breast cancer [23] and clear cell ovarian cancer [11]. In contrast, upregulated miR-100 indicated an unfavorable outcome in acute myeloid leukemia [12]. Despite the importance of miR-100 in tumorigenesis, the role of miR100 in gastric cancer has not been sufficiently studied. In the present

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Fig. 4. ZBTB7A promoted gastric cancer cell metastasis and correlates with patient survival. (A and B) ZBTB7A expression knockdown by si-ZBTB7A#1 in SGC7901 and BGC823 cells. mRNA expression and protein expression were detected by RT-qPCR (A) and by western blot (B), respectively. Expression was significantly reduced compared with the negative control (t-test, ***P < 0.001, **P < 0.01, *P < 0.05). (C and D) The migration and invasion ability of BGC823 and SGC7901 was decreased in cells transfected with si-ZBTB7A#1 in comparison with the negative control (t-test, **P < 0.01, *P < 0.05). (E) The expression of ZBTB7A protein in gastric cancer tissues was detected by IHC. To assess ZBTB7A protein expression in human gastric cancer tissues, the percentage of positive cancer cells detected by IHC was evaluated by choosing 500 cancer cells randomly for counting in each case. The gastric cancer tissues with LNM usually showed higher percentage of positive ZBTB7A cells (b, magnification ×200), compared with those without LNM (a, magnification ×200), with stronger positive staining or higher percentage of positivity of ZBTB7A protein observed in the metastatic cancer tissue which had invaded the lymph nodes (c, magnification ×200). The correlation between miR-100 expression and ZBTB7A protein expression in human gastric cancers was calculated by Spearman’s correlation. An inverse correlation was found between miR-100 level and ZBTB7A protein expression in human gastric cancers (d, n = 112, r = −0.210, P = 0.026). (F and G) The association between ZBTB7A expression and patient survival. To investigate the association between ZBTB7A expression and patient survival, the median expression of ZBTB7A was defined as the cutoff point to divide the patients into a low expression group and a high expression group. Kaplan–Meier survival analysis and log-rank test showed that the OS rate (F) and DFS (G) rate of the patients with high ZBTB7A expression were markedly poorer than those with low ZBTB7A expression (log-rank test, P = 0.005 and P = 0.011, respectively).

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Fig. 5. C/EBPα induced miR-100 expression by binding to the putative promoter region. (A and B) Transcriptional factor binding sites were predicted in the 0 to −400 bp fragment of the putative promoter region of miR-100. Thus the −445 bp to +299 bp fragment was subcloned into PGL3-basic vector and was named PGL3-P100. Then the PGL3-P100 vector was analyzed for promoter activity in luciferase reporter assay. The results showed that the −445 bp to +299 bp fragment exhibited promoter activity and that the activity could be enhanced by transcription factor C/EBPα (B, t-test, **P < 0.01, *P < 0.05). However, the promoter activity could not be enhanced by transcription factor P53 (A, t-test, P > 0.05). (C) The −341 bp to −63 bp fragment that harbored the C/EBPα and P53 binding sites was subcloned into PGL3-basic vector for further analysis of promoter activity, which was named PGL3-P100-1. The results suggested that the fragment exhibited promoter activity and could also be enhanced by transcription factor C/EBPα when compared with the control group (t-test,*P < 0.05). (D) Two of the C/EBPα binding sites (−106 bp to −100 bp and −178 bp to 172 bp) in the −341 bp to −63 bp fragment were mutated and subcloned into PGL3-basic vector. The vector was named PGL3-P100-1M. PGL3-P100-1 and PGL3-P100-1M were cotransfected with PcDNA3.1C/EBPα. The results showed that the promoter activity of PGL3-P100-1M was significantly attenuated after the cotransfection compared with PGL3-P100-1 (t-test, *P < 0.05). (E) SGC7901 and MKN45 cells were transfected with PcDNA3.1-C/EBPα plasmid, then the C/EBPα protein level was examined by western-blot assay. The result showed that C/EBPα was successfully expressed in PcDNA3.1-C/EBPα transfected cells (t-test, P < 0.001), compared with the negative control group. (F) The expression of miR-100 could be induced by C/EBPα. SGC7901 and MKN45 cells were transfected with PcDNA3.1-C/EBPα or PcDNA3.1-empty plasmid and the expression of miR-100 was detected by RT-qPCR 48h after the transfection. The results showed that the expression of miR-100 in C/EBPα-expressing cells was significantly upregulated (t-test,*P < 0.05). (G) The transcription start site (TSS) located in the 3.8 kb upstream region of precursor miR-100. Transcription factor C/EBPα may induce miR-100 expression by binding to the two binding sites in the putative promoter region of miR-100. The up-regulation of miR-100 by C/EBPα could decrease ZBTB7A expression and act as a tumor suppressing gene.

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study, we demonstrated that miR-100 was dramatically downregulated in gastric cancer tissues and cell lines. Further analysis revealed that miR-100 levels were even lower in the cases with lymphatic metastasis and that decreased miR-100 was associated with a larger tumor size (≥5 cm). We also verified that miR-100 expression was inversely correlated with tumor size by Spearman’s correlation test. These results suggested that miR-100 may play an important role in regulating gastric cancer metastasis and proliferation. As expected, restoration of miR-100 inhibited the invasion and metastasis ability of gastric cancer cells in vitro and in vivo. Additionally, the growth rate of gastric cancer cells was attenuated significantly in vivo, but not in vitro. It is possible that the growth discrepancy of the cancer cells observed in vitro relative to that in vivo was due to the differences in the growth environment. Despite the discrepancy observed between the in vivo and in vitro growth rate, the tumor microenvironment in vivo is a more reliable testing environment for the evaluation of miR-100’s in vivo function; therefore, miR-100 could be an important tumor-suppressing gene with the ability to suppress metastasis and proliferation in gastric cancers. In subsequent research on miR-100’s tumor suppression mechanism, we confirmed that ZBTB7A was a novel target of miR-100. ZBTB7A, also known as Pokemon, LRF, FBI-1 and OCZF, is a member

of the POK (POZ and Krüppel) protein family of transcriptional repressors. Previous studies have revealed that ZBTB7A is an important proto-oncogene deregulated in various cancers [16,17,24–26]. Maeda et al. [16] reported that ZBTB7A promoted cellular transformation by suppressing ARF, and that ZBTB7A also promoted cell proliferation and lymphoma formation in transgenic mice. Overexpressed ZBTB7A was capable of promoting cell proliferation by regulating p53, p21 and p27 expression [24]. However, to our knowledge, the function of ZBTB7A and its clinicopathological significance in human gastric cancer has not yet been characterized. We identified ZBTB7A as a direct target of miR-100 in gastric cancer by dual-luciferase reporter and western-blot assays. In addition, an inverse correlation between miR-100 and ZBTB7A expression in human gastric cancers was observed. Knockdown of ZBTB7A by siRNA markedly reduced the migration and invasion ability of gastric cancer cells in vitro. However, cancer cell proliferation activity remained unaltered, which was similar to the effect of miR-100 on the gastric cancer cells in vitro. We further investigated the ZBTB7A expression with respect to the clinicopathological variables of human gastric cancers. We found that high ZBTB7A expression was significantly correlated with LNM. This observation supported our previous findings that, after miR100 and siRNA knockdown, downregulation of ZBTB7A significantly

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reduced the invasion and metastasis ability of gastric cancer cells. Highly expressed ZBTB7A was also reported to be correlated with tumor metastasis in breast cancer [26] and ovarian cancer [17]. These data indicate that ZBTB7A plays an important and active role in human cancer metastasis, including gastric cancers. Furthermore, ZBTB7A expression was found to be associated with clinical stage, lymph node metastasis and Lauren’s classification. We also found that high ZBTB7A expression was associated with both poor disease free survival and poor overall survival. Multivariate analysis showed that higher ZBTB7A expression was an independent predictor associated with poor prognosis, indicating that ZBTB7A may serve as a biomarker in predicting the survival of the patients with gastric cancers. A single gene may be targeted by multiple miRNAs, it is therefore likely that ZBTB7A expression level is regulated cooperatively by the accumulative function of several miRNAs, including miR100. Thus, although miR-100 did not itself predict the prognosis of gastric cancer patients, its target ZBTB7A could serve as an independent predictor. Q3 Although in some papers ZBTB7A was described as a tumor suppressor gene in prostate cancer [27] and colon cancer [28], the possibility that a tumor-related gene has inconsistent functions in different human cancers with different regulatory mechanisms exists. Take CTNND1 as an example, it was suggested that reduced CTNND1 expression was related to poor prognosis in patients with bladder cancer [29]. However, we revealed that CTNND1, negatively regulated by miR-145, promoted tumor aggressiveness in gastric cancer [30]. The pro-metastasis role of CTNND1 was also reported in lung cancer and pancreatic adenocarcinoma [31,32]. Furthermore, dual function of p38α was reported in colon cancer: suppression of colitisinduced tumor initiation but requirement for cancer cell survival Q4 [33]. Therefore, the possibility that ZBTB7A acted as a protooncogene in gastric cancers exists with respect to our results. It is of great importance to understand the mechanisms regulating the ectopic expression of miRNAs. Some molecular mechanisms have already been identified. The transcription factor Twist, for instance, induces the expression of miR-10b in breast cancer [2]. miR-492 originates from the coding sequence of keratin 19 (KRT19) and its expression is influenced by PLAG1 [34]. In addition, it has been recently reported that aberrant DNA methylation of CpG islands in the promoter region may play a key role in regulating miRNA expression [13]. We were highly interested in understanding the mechanism underlying abnormal miR-100 expression, which was still undiscovered. In this study, we demonstrated that transcription factor C/EBPα stimulated miR100 expression by binding to the promoter fragment −341 bp to −63 bp. C/EBPα belongs to the C/EBP family of basic leucine zipper transcription factors and is required for the differentiation of multiple cell types [35]. As a tumor suppressor, C/EBPα expression was found to be decreased in a multitude of human tumors, such as head and neck squamous cell cancer [36], lung cancer [37], endometrial cancer [38] and acute myeloid leukemia (AML) [39]. Moreover, C/EBPα expression was also found to be decreased in 30% of gastric cancers and could act as tumor-suppressing gene [21]. Consistently, we also confirmed that C/EBPα was downregulated in gastric cancer tissues compared with that in non-tumorous mucosa. In this study, we first identified the TSS of miR-100 and then analyzed the C/EBPα binding sites within the putative promoter region of miR100. Luciferase reporter assays and RT-qPCR confirmed the role of C/EBPα in inducing the expression of miR-100 in gastric cancer cells by binding to the −63 bp to −341 bp fragment. Consistent with these results, when two of the binding sites of C/EBPα were mutated, no significant induction of miR-100 was observed by luciferase assay. Therefore, up-regulation of miR-100 expression is induced, at least in part, by C/EBPα binding to the putative promoter region of miR-100.

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In conclusion, our experiments suggested that miR-100 functions as a tumor suppressor gene, at least partially, by suppressing its direct target gene ZBTB7A. Importantly, our results revealed for the first time that miR-100 expression could be induced by the transcription factor C/EBPα, providing a mechanism that can induce abnormal expression of miR-100. The accumulation of ZBTB7A that is associated with decreased miR-100 levels promoted tumor invasion and metastasis and contributed to the progression of gastric cancers. Thus, miR-100 expression induced by C/EBPα could downregulate the expression of ZBTB7A and act as tumor suppressing gene in gastric cancer (Fig. 5G). Moreover, ZBTB7A protein expression could be used as an independent prognostic indicator for patients with gastric cancers. Our findings may lead to the discovery of new potential therapies for gastric cancer patients through the upregulation of miR-100 or downregulation of ZBTB7A expression. Funding This work was supported by the National Natural Science Foundation of China (Nos. 81372856, 81172351) and Program for New Century Excellent Talents in University (No. NCET-12-0335). Authors’ contributors Peng Gao conceptualized and designed this manuscript. DuanBo Shi and Ai-Yan Xing collected clinical tumor samples. Duan-Bo Shi and Ya-Wen Wang performed data analysis and interpretation. Peng Gao, Duan-Bo Shi and Ya-Wen Wang wrote the manuscript. Acknowledgments Transcription factor vectors pCMV-p53 and pcDNA3.1-CEBP/α were a gift from Prof. Anli Jiang, Shandong University, China. Conflict of interest All authors promise that there is no conflict to disclose. Ethics approval The study was approved by the Ethics Committee of Shandong University. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.08.029. References [1] E.C. Lai, Micro RNAs are complementary to 3′ UTR sequence motifs that mediate negative post-transcriptional regulation, Nat. Genet. 30 (4) (2002) 363– 364. [2] L. Ma, J. Teruya-Feldstein, R.A. Weinberg, Tumour invasion and metastasis initiated by microRNA-10b in breast cancer, Nature 449 (7163) (2007) 682– 688. [3] E. Elyakim, E. Sitbon, A. Faerman, et al., hsa-miR-191 is a candidate oncogene target for hepatocellular carcinoma therapy, Cancer Res. 70 (20) (2010) 8077–8087. [4] E. Bandres, N. Bitarte, F. Arias, et al., microRNA-451 regulates macrophage migration inhibitory factor production and proliferation of gastrointestinal cancer cells, Clin. Cancer Res. 15 (7) (2009) 2281–2290. [5] Y. Xu, F. Zhao, Z. Wang, et al., MicroRNA-335 acts as a metastasis suppressor in gastric cancer by targeting Bcl-w and specificity protein 1, Oncogene 31 (11) (2012) 1398–1407. [6] Y.W. Wang, D.B. Shi, X. Chen, et al., Clinicopathological significance of microRNA-214 in gastric cancer and its effect on cell biological behaviour, PLoS ONE 9 (3) (2014) e91307.

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