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1 IUBMB Life, 64(7): , July 2012 Research Communication MicroRNA-181b Targets camp Responsive Element Binding Protein 1 in Gastric Adenocarcinomas Lin Chen*, Qian Yang*, Wei-Qing Kong*, Tao Liu, Min Liu, Xin Li and Hua Tang Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, Tianjin, China Summary MicroRNAs are a class of small endogenous non-coding RNAs that function as post-transcriptional regulators. In our previous study, we found that mir-181b was significantly downregulated in human gastric adenocarcinoma tissue samples compared to the adjacent normal gastric tissues. In this study, we confirm the down-regulation of mir-181b in human gastric cancer cell lines versus the gastric epithelial cells. Overexpression of mir-181b suppressed the proliferation and colony formation rate of gastric cancer cells. mir-181b downregulated the expression of camp responsive element binding protein 1 (CREB1) by binding its 3 untranslated region. Overexpression of CREB1 counteracted the suppression of growth in gastric cancer cells caused by ectopic expression of mir-181b. These results indicate that mir-181b may function as a tumor suppressor in gastric adenocarcinoma cells through negative regulation of CREB1. Ó 2012 IUBMB IUBMB Life, 64(7): , 2012 Keywords Abbreviations mirna; mir-181b; camp responsive element binding protein 1; cell growth; gastric carcinoma. ASO, antisense oligonucleotide; CREB1, camp responsive element binding protein 1; EGFP, enhanced green fluorescence protein; GAPDH, glyceraldehyde phosphate dehydrogenase; mir-181b, microrna- 181b; mirna, microrna; UTR, untranslated region. INTRODUCTION Gastric cancer is the second leading cause of cancer-related mortality worldwide (1). Gastric adenocarcinoma is derived from the gastric epithelium mucosal cells, occupies 95% of the Additional Supporting Information may be found in the online version of this article. Received 22 November 2011; accepted 24 February 2012 *The authors are contributed equally to this work. Address correspondence to: Hua Tang, Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, 22 Qi- Xiang-Tai Road, Tianjin , China. Tel./Fax: htang2002@yahoo.com gastric malignant tumor, and is the most common form of gastric cancer. Multiple factors are involved in the development of gastric carcinogenesis including inactivation of tumor suppressor genes, activation of oncogenes, and abnormalities of cell cycle regulators and growth factors. MicroRNAs (mirnas) are a class of 22nt non-coding RNAs that regulate gene expression by interacting with complementary sites in the 3 untranslated region (3 UTR) of target genes (2). The genes encoding mirnas are transcribed into primary mirnas in the nucleus, predominantly by RNA polymerase II, and then cleaved by Drosha to produce stem-loop-structured precursor molecules (pre-mirnas). Pre-miRNAs are then exported to the cytoplasm where the RNase III enzyme, Dicer, further processes them into mature mirnas (22 nucleotides) (3). Recent studies have shown that dysregulated expression or mutation of mirnas were associated with various human cancers and have indicated that mirnas can function as tumor suppressor genes and oncogenes (4). Specifically in gastric cancers, mirnas have been shown to function as tumor suppressors or oncogenes (5). For example, mir-375 and mir-142-5p have been identified as classifiers used to recognize recurrence and non-recurrence in gastric cancer; a high frequency recurrence and poor survival were observed in gastric cancer cases with high levels of mir-375 and low levels of mir-142-5p (6). mir-9, which is downregulated in human gastric cancer cells, plays a role in regulating gastric cancer cell growth by targeting NF-jB1 (7). In our previous study, we found that mir-181b displayed prominent and coincident down-regulation in human gastric cancer tissues compared to the matched normal gastric tissues (8). In this study, we focused on the effects of mir-181b on the cellular phenotypes of MGC-803, BGC-823, and SGC-7901 gastric adenocarcinoma cell lines. Previous studies have identified CREB-regulated genes including many types of oncogenes (e.g., bcl-2), cell cycle related genes (e.g., cyclina1, cyclinb1, and cyclind2), signal transduction proteins, activated transcription factor 3, NF-jB, and other growth-related genes (9). camp responsive element binding protein 1(CREB1), a member of the ISSN print/issn online DOI: /iub.1030

2 MIR-181B TARGETS CREB1 IN GASTRIC CANCER 629 CREB family of proteins, is an oncogene that promotes tumor cell growth and proliferation (10), and is found to be a target of mir-181b in this study. Our findings indicate that mir-181b functions as a tumor suppressor in gastric adenocarcinoma. MATERIALS AND METHODS mirna Target Prediction TargetScan, PicTar, and mirbase were used to predict mirna targets. Plasmid Construction and Oligonucleotides In this study, we constructed the following vectors: pcdna3/pri-mir-181b, ASO-miR-181b, pcdna3/ enhanced green fluorescence protein (EGFP)-CREB1-3 UTR, pcdna3/ EGFP-CREB1-3 UTR mutant and psilencer/shrna-creb1. The details of vector construction as well as the sequence of the oligonucleotides can be found in the Supporting Information. Tissue Samples, Cell Lines, and Transfection GES-1 (human gastric epithelial cells), MGC-803, BGC-823, and SGC-7901 cells were maintained in RPMI 1640 (GIBCO) with 10% heat-inactivated fetal bovine serum, 100 IU/mL penicillin, and 0.1 mg/ml streptomycin in a humidified 5% (v/v) atmosphere of CO 2 at 37 8C. Transfection was performed with Lipofectamine 2000 Reagent following the manufacturer s protocol (Invitrogen, Carlsbad, CA). Fluorescent Reporter Assay Cells were transfected with pri-mir-181b or the control vector, pcdna3, in 48-well plates in conjunction with the reporter vector pcdna3/egfp-creb1 3 UTR or pcdna3/egfp- CREB1 3 UTR-mut. Additionally, the ASO-miR-181b or ASO- NC vector was co-transfected into cells with the pcdna3/ EGFP-CREB1 3 UTR or pcdna3/egfp-creb1 3 UTR-mut vector. Also, the mir-181b mimics, mutated mir-181b (mir- 181b-mut) or mimics NC was co-transfected into cells with the pcdna3/egfp-creb1 3 UTR. The pdsred2-n1 vector (Clontech, Mountain View, CA) expressing RFP was spiked in and used for normalization. The intensities of EGFP and RFP fluorescence were detected with a Fluorescence Spectrophotometer F-4500 (Hitachi, Tokyo, Japan). The EGFP expression level was also measured by quantitative RT-PCR and Western blot assays. Quantitative RT-PCR The quantitative RT-PCR assays were performed to detect the relative levels of the CREB1 mrna and mature mir-181b (11). The details of the method are given in the Supporting Information. Western Blot Cultured cells were lysed in RIPA lysis buffer. Lysates were collected and cleared by centrifugation and the protein concentration was determined. Total cell lysates (50 lg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose membranes. The membranes were incubated with an antibody against CREB1, EGFP, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) overnight at 4 8C. Then, the membranes were incubated with goat anti-rabbit peroxidase-conjugated secondary antibody. Protein expression was assessed by enhanced chemiluminescence and exposure to chemiluminescent detection film. Antibodies were purchased from Tianjin Saier Biotech and Sigma-Aldrich. Cell Proliferation Assay MGC-803, BGC-823, and SGC-7901 cells were seeded in a 96-well plate at 6,000, 7,000, and 8,000 cells per well, respectively, one day before transfection. The cells were transfected with pri-mir-181b or the control vector at 0.15 lg per well. The MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to determine cell viability 24, 48, and 72 H after transfection. The absorbance at 570 nm was measured using a lquant Universal Microplate Spectrophotometer (BioTek, Winooski, VT). Colony Formation Assay After transfection, MGC-803, BGC-823, and SGC-7901 cells were counted and seeded in 12-well plates (in triplicate) at 200, 150, and 250 cells per well, respectively. Culture medium was replaced every 3 days. Colonies were counted from the 6th day after seeding only if they contained more than 50 cells. Cells were stained using crystal violet. The rate of colony formation was calculated with the following equation: colony formation rate (%) 5 (number of colonies/number of seeded cells) Statistical Analysis Data are expressed as the means 6 standard deviation (SD), and P \ 0.05 is considered statistically significant by Student s t test. RESULTS Downregulation of mir-181b in Human Gastric Adenocarcinoma and in Gastric Cancer Cell Lines Quantitative real-time PCR (qrt-pcr) was used to detect differential expression of mir-181b in 10 pairs of human gastric adenocarcinomas and corresponding adjacent normal tissues. Results showed that the mir-181b expression level in gastric adenocarcinoma is significantly lower than that in corresponding adjacent normal tissues (Fig. 1A). Meanwhile, we also determined that the expression of mir-181b in human gastric adenocarcinoma cells, MGC-803, BGC-823, and SGC-7901 cells was

3 630 CHEN ET AL. Figure 1. Deregulation of mir-181b in gastric adenocarcinoma tissue and suppression of mir-181b in gastric adenocarcinoma cell growth. (A) Differential expression of mir-181b in 10 pairs of human gastric adenocarcinomas and corresponding adjacent normal tissues. U6 snrna was used as an endogenous normalizer. (B) The relative expression level of mir-181b in MGC-803, BGC-823, SGC-7901, and GES-1 cells are shown. (C) The relative level of mir-181b in BGC-823 cells after transfection with pcdna3/primir-181b (pri-mir-181b), ASO-miR-181b, mir-181b mimics or mir-181b-mut, and the expression level of mature mir-181b was detected by quantitative RT-PCR. (D F) MGC-803 (D), BGC-823 (E), and SGC-7901 (F) cells were transfected with pri-mir-181b or ASO-miR-181b. Cell viability was determined at 24, 48, and 72 H post-transfection by the MTT assay. (G) MGC-803, BGC- 823, and SGC-7901 cells were transfected with pri-mir-181b or control vector. The cell growth capacity in vitro was assessed by the colony formation assay. (H) MGC-803, BGC-823, and SGC-7901 cells were transfected with ASO-miR-181b or control vector (*P \0.05, **P \0.005). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] lower than that in GES-1 human gastric epithelial cells using qrt-pcr (Fig. 1B). Overexpression of mir-181b Suppresses the Proliferation of Gastric Adenocarcinoma Cells To determine the effects of mir-181b on gastric adenocarcinoma cell proliferation, a mir-181b overexpression vector, a synthesized anti-mir inhibitor (ASO-miR-181b), synthesized mir-181b mimics and mir-181b-mut were used to alter the expression levels of mir-181b in gastric adenocarcinoma cells. First, we used quantitative RT-PCR to validate the expression of mir-181b in BGC-823 cells after transfection with pri-mir- 181b, ASO-miR-181b, mir-181b mimics or mir-181b-mut (Fig. 1C). To test the effects of mir-181b on cell viability and growth of gastric adenocarcinoma cells, we transfected the primir-181b or ASO-miR-181b into BGC-823 cells. The MTT assay showed that the enhanced level of mir-181b reduced the viability of MGC-803 cells by approximately 20% (Fig. 1D). In contrast, ASO-miR-181b increased the viability of MGC-803 cells by approximately 30% (Fig. 1D). The colony formation assay indicated that the colony formation rate of MGC-803 cells

4 MIR-181B TARGETS CREB1 IN GASTRIC CANCER 631 Figure 2. CREB1 is a direct target of mir-181b. (A) The predicted binding sites of mir-181b on the CREB1 mrna are shown. The mutated CREB1 3 UTR and mutated mir-181b are also shown. (B D) BGC-823 cells were transfected with the wild type pcdna3/egfp-creb1 3 UTR reporter vector along with pri-mir-181b or ASO-miR-181b. The EGFP expression was measured by fluorescence intensity (B), quantitative RT-PCR (C) and Western blot (D). (E G) BGC-823 cells were transfected with the mutated pcdna3/egfp-creb1-3 UTR (pcdna3/egfp-creb1-3 UTR-mut) reporter vector along with pri-mir-181b or ASOmiR-181b. H-J: BGC-823 cells were transfected with the wild type pcdna3/egfp-creb1 3 UTR reporter vector along with mir- 181b mimics or mir-181b mutant (mir-181b-mut) (*P < 0.05).

5 632 CHEN ET AL. Figure 3. mir-181b negatively regulates CREB1 at the post-transcriptional level. (A) BGC-823 cells were transfected with primir-181b or ASO-miR-181b, and the CREB1 protein expression level was evaluated by western blot. The GAPDH protein was used as an endogenous normalizer. (B) BGC-823 cells were transfected with pri-mir-181b or ASO-miR-181b, and expression of CREB1 mrna was measured by qrt-pcr. b-actin mrna was used as an endogenous normalizer. (C) The expression levels of CREB1 mrna in the 10 pairs of gastric adenocarcinoma tissues (Ca) and the matched normal tissues (N) were assessed by qrt- PCR. b-actin mrna was used as an endogenous normalizer (*P \ 0.05, ** P \0.005). transfected with pri-mir-181b was 30 40% lower than the control group (Fig. 1G), whereas the addition of ASO-miR-181b enhanced colony formation rates by approximately 30% in MGC-803 cells (Fig. 1H). Similar results were found in BGC- 823 and SGC-7901 cells (Figs. 1E 1H). These results demonstrate that mir-181b can suppress cell growth in gastric adenocarcinoma cells. mir-181b Binds Directly to the CREB1 3 UTR in Gastric Adenocarcinoma Cells To identify the target genes mediating the function of mir- 181b, bioinformatic methods were used to predict potential target genes. We found that the 3 UTR of the CREB1 mrna contains a mir-181b-complementary binding site (Fig. 2A). To validate that CREB1 can be directly targeted by mir-181b, we performed an EGFP reporter assay using engineered EGFP reporter vectors that had either the wild type 3 UTR of CREB1 or the mutant 3 UTR with a 4-base mutation in the complementary seed sequence. Also, mir-181b mimics and mir-181b mutant bearing four mutated bases are also synthesized (Fig. 2A). Overexpression of mir-181b by addition of pri-mir-181b significantly repressed EGFP expression compared to the control vector (Figs. 2B 2D), whereas downregulation of mir-181b with ASO-miR-181b significantly increased the intensity of EGFP fluorescence compared to the control group (Figs. 2B 2D). In contrast, with the mutated CREB1 3 UTR reporter vector, EGFP expression levels were not influenced by overexpression or downregulation of mir-181b (Figs. 2E 2G). To further confirm the direct binding between mir-181b and CREB1 3 UTR, we also mutated the mir-181b seed sequence. We found that mir-181b mimics could suppress EGFP expression from pcdna3/egfp-creb1 3 UTR, whereas mutated mir- 181b could not (Figs. 2H 2J). All the results indicated that mir-181b could bind to the seed sequence present in the CREB1 mrna 3 UTR and negatively regulate the expression of the CREB1 gene. mir-181b Negatively Regulates CREB1 at the Post Transcriptional Level To determine if mir-181b decreases endogenous CREB1 expression, we transfected BGC-823 cells with pri-mir-181b or ASO-miR-181b and examined the protein expression of CREB1 by western blot analysis. The results indicated that overexpression of mir-181b reduced the expression level of the CREB1 protein by 30% (Fig. 3A) and qrt-pcr indicated a decrease in endogenous CREB1 mrna (Fig. 3B). Conversely, in BGC-823 cells transfected with ASO-miR-181b, the protein level of endogenous CREB1 was significantly increased (Fig. 3A), and the

6 MIR-181B TARGETS CREB1 IN GASTRIC CANCER 633 Figure 4. Knockdown of CREB1 inhibits cell growth in gastric adenocarcinoma cell lines. (A) Transfection with the psilencer/sir- CREB1 reduced CREB1 protein levels in BGC-823 cells compared with the control group. (B D) MTT assay analysis showed that transfection with the pcdna3/sir-creb1 plasmid suppressed cell viability in MGC-803 cells (B), BGC-823 cells (C), and SGC cells (D). (E) Colony formation assay rates in MGC-803, BGC823, and SGC-7901 cells transfected with pcdna3/sir- CREB1 plasmid were lower compared with the control vector (*P \ 0.05, ** P \0.005). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] CREB1 mrna level was also elevated (Fig. 3B). In addition, we found that the expression level of CREB1 mrna in gastric adenocarcinoma tissues was significantly higher than in matching adjacent normal tissues in the 10 pairs of gastric tissues (Fig. 3C). Both the mrna and protein levels of CREB1 are responsive to the mir-181b level, indicating that mir-181b negatively regulates the expression of CREB1 mainly by the mrna degradation mechanism. Knockdown of CREB1 Inhibits Cell Growth in Gastric Adenocarcinoma Cells Previous studies have shown that CREB1 plays an important role in promoting tumor cell proliferation and functions as an oncogene in acute myeloid leukemia (12). To determine if CREB1 affects BGC-823 cell growth, we constructed the psilencer/sir-creb1 plasmid to knockdown the expression of CREB1. Western blot analysis indicated that the psilencer/sir- CREB1 plasmid could effectively reduce the CREB1 protein expression level in BGC-823 cells by approximately 20% (Fig. 4A). The MTT and colony formation assays indicated that CREB1 knockdown resulted in a reduction of cell viability and growth activity compared to the control group in MGC-803, BGC-823, and SGC-7901 cells (Figs. 4B 4E). Ectopic Expression of CREB1 Counteracts the Suppression of Cell Growth Caused by mir-181b in Gastric Cancer Cells To further confirm that mir-181b suppresses the growth of gastric cancer cells through downregulation of CREB1, we constructed the expression vector pcdna3/creb1 without the 3 UTR to avoid the mirna interference. Western blot analysis was used to validate the CREB1 expression in transfected BGC-823 cells (Fig. 5A). Ectopic expression of CREB1 was able to rescue the growth inhibition caused by mir-181b in

7 634 CHEN ET AL. Figure 5. Ectopic expression of CREB1 rescues the suppression of cell growth caused by mir-181b in gastric adenocarcinoma cell lines. (A) BGC-823 cells were transfected with pri-mir-181b and control vector, pri-mir-181b and either pcdna3/creb1 or pcdna3/creb1 and control vector. CREB1 protein levels were detected by western blot. The GAPDH protein was used as an endogenous normalizer. (B D) MGC-803 cells (B), BGC-823 cells (C), and SGC-7901 cells (D) were transfected with pcdna3/ CREB1 and pcdna3 with or without pri-mir-181b, and cell viability was determined using the MTT assay at 24, 48, and 72 H post-transfection. (E G) CREB1 ectopic expression reversed the cell growth suppression caused by mir-181b in the colony formation assay. MGC-803 cells (E), BGC-823 cells (F), and SGC-7901 cells (G) were transfected with pcdna3/creb1 and pcdna3 with or without pri-mir-181b and then seeded in 12-well plates. The colony formation rate is shown (*P \ 0.05, **P \0.005). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] MGC-803, BGC-823, and SGC-7901 cells (Figs. 5B 5D) as measured by the MTT and colony formation assays (Figs. 5E 5G). DISCUSSION mirnas are involved in a variety of cellular activities by negatively regulating specific target genes, and approximately one-third of human genes are believed to be mirna targets (13). Previous studies have shown that hsa-mir-181b functions as tumor suppressor that triggers growth inhibition, induces apoptosis, and inhibits invasion in glioma cells (14). It was also reported that mir-181b may contribute to multidrug resistance in human gastric and lung cancer cell lines by modulation of apoptosis via BCL2 targeting (15). As mir-181b was downregulated in gastric adenocarcinoma tissues (8), we here focused on the functions of mir-181b in gastric cancer cells and the regulation of its target genes. Initially, we used qrt-pcr to discover that mir-181b was significantly downregulated in human gastric adenocarcinoma tissue samples compared to corresponding adjacent normal tissues (Fig. 1A). The results suggested that mir-181b could play an important role in the development of gastric adenocarcinoma. Because of the low expression levels of mir-181b in cancer tissues, we speculated that mir-181b could be an inhibitory factor in gastric adenocarcinoma cells. To test this hypothesis, we used the MTT assay and colony formation assay to investigate the relationship between mir-181b and the growth capacity of gastric adenocarcinoma cell lines. The viability and the colony formation activity of MGC-803, BGC-823, and SGC-7901 cells transfected with the mir-181b overexpression vector (pri-mir-181b) were significantly decreased when compared to their control groups (Figs. 1D 1G). Moreover, ASO-miR-181b increased cell viability and colony formation activity when compared to ASO-NC (Figs. 1D 1F and 1H). Thus, we concluded that mir-181b functions as a tumor suppressor gene in gastric adenocarcinoma. Typically, mirnas affect cell processes by regulating target gene expression (2). To identify the target gene responsible for

8 MIR-181B TARGETS CREB1 IN GASTRIC CANCER 635 effects of mir-181b in gastric adenocarcinoma cells, we used bioinformatic analyses to predict a mir-181b binding site on the CREB1 transcript (Fig. 2A). Next, we used the fluorescence reporter assay to determine that CREB1 was indeed a target of mir-181b. The ability of mir-181b to regulate CREB1 expression was likely direct, because it bound the region of the CREB1 3 UTR that was complementary to the mir-181b seed region. The EGFP fluorescence intensity of EGFP-CREB1-3 UTR was specifically responsive to mir-181b alteration. However, mutation of either the mir-181b binding site on CREB1 3 UTR or the mir-181b itself could abolish the effect of mir-181b on the regulation of EGFP fluorescence intensity. Additionally, endogenous CREB1 expression was decreased at both the mrna and protein level in BGC-823 cells transfected with pri-mir-181b but increased in BGC-823 cells transfected with ASO-miR-181b (Figs. 3A and B). In addition, we observed an inverse correlation between the expression of mir-181b and CREB1 in tumor tissues and corresponding normal gastric tissues by qrt-pcr (Fig. 3C). These results indicate that mir- 181b regulates CREB1 expression by binding its 3 UTR directly. Cyclic AMP responsive element binding protein (CREB) family members are transcription factors which play critical roles in regulating numerous biological processes such as development, cancer, and memory formation (16, 17). CREB1 is one of the CREB family proteins that has been shown to be an oncogene that promotes tumor cell growth and proliferation. It can activate gene expression in a phosphorylation-dependent manner and bind as a dimer to a cyclic AMP-responsive element in the promoter region of target genes (18). Our findings uncover the relationship between mirnas and CREB1 in gastric adenocarcinoma cells. Knockdown of CREB1 suppresses the growth of three gastric cancer cell lines, which is consistent with the results of mir-181b overexpression. Ectopic expression of CREB1 rescues the suppression of cell growth caused by mir-181b. Thus, the low expression of mir-181b in gastric adenocarcinoma cells leads to an abnormally high expression level of the CREB1 oncogene resulting in cell proliferation. In summary, our results show that mir-181b functions as a tumor suppressor gene and inhibits cell proliferation in gastric adenocarcinoma cell lines. The low expression of mir-181b in gastric adenocarcinoma tissues leads to an abnormally high expression level of the CREB1 oncogene and results in the activation of cell proliferation. The elucidation of the effect of mir-181b on CREB1 regulation helps us to further understand the mechanism of gastric adenocarcinoma initiation and progression. ACKNOWLEDGEMENTS The authors like to thank the Tumor Bank Facility of Tianjin Medical University Cancer Institute and Hospital and National Foundation of Cancer Research (TBF of TMUCIH & NFCR) for providing human gastric tissue samples. They also thank the College of Public Health, Tianjin Medical University, for technical assistance with fluorescence detection. This work was supported by the National Natural Science Foundation of China (No , No , and No ) and the Natural Science Foundation of Tianjin (No. 08JCZDJC23300 and No. 09JCZDJC17500). REFERENCES 1. Parkin, D. M., Pisani, P., and Ferlay, J. (1999) Estimates of the worldwide incidence of 25 major cancers in Int. J. Cancer 80, Bartel, D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, Lee, Y., Jeon, K., Lee, J. T., Kim, S., and Kim, V. N. (2002) Micro- RNA maturation: stepwise processing and subcellular localization. EMBO J. 21, Esquela-Kerscher, A., and Slack, F. J. 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