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MicroRNA-195 and microRNA-378 mediate tumor growth suppression by epigenetical regulation in gastric cancer
Gene 518 (2013) 351–359
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Gene journal homepage: www.elsevier.com/locate/gene
MicroRNA-195 and microRNA-378 mediate tumor growth suppression by epigenetical regulation in gastric cancer Hongxia Deng a, Yanan Guo a, Haojun Song a, Bingxiu Xiao a, Weiliang Sun b, Zhong Liu c, Xiuchong Yu a, Tian Xia a, Long Cui a, Junming Guo a,⁎ a b c
Department of Biochemistry and Molecular Biology, and Zhejiang Provincial Key Laboratory of Pathophysiology, Ningbo University School of Medicine, Ningbo, 315211, China Ningbo Yinzhou People's Hospital and the Affiliated Hospital, Ningbo University School of Medicine, Ningbo 315040, China The Affiliated Hospital, Ningbo University School of Medicine, Ningbo 315010, China
a r t i c l e
i n f o
Article history: Accepted 24 December 2012 Available online 17 January 2013 Keywords: MicroRNA Gastric cancer Tumor suppressor Cell cycle Epigenetic
a b s t r a c t The epigenetic regulation of microRNAs is one of several mechanisms underlying carcinogenesis. We found that microRNA-195 (miR-195) and microRNA-378 (miR-378) were significantly down-regulated in gastric cancer tissues and gastric cancer cell lines. The expression of miR-195 and miR-378 in gastric cancer cells was significantly restored by 5-aza-dC, a demethylation reagent. The low expression of miR-195 and miR-378 was closely related to the presence of promoter CpG island methylation. Treatment with miR-195/miR-378 mimics strikingly suppressed the growth of gastric cancer cells whereas promoted the growth of normal gastric epithelial cells. In contrast, administration of miR-195/miR-378 inhibitors significantly prevented the growth of normal gastric epithelial cells. Expression of cyclin-dependent kinase 6 and vascular endothelial growth factor was downregulated by exogenous miR-195 and miR-378, respectively. In conclusion, miR-195 and miR-378 are abnormally expressed and epigenetically regulated in gastric cancer cell lines and tissues via the suppression of CDK6 and VEGF signaling, suggesting that miR-195 and miR-378 have tumor suppressor properties in gastric cancer. © 2013 Elsevier B.V. All rights reserved.
1. Introduction It is increasingly recognized that epigenetic modifications play an important role in the process of tumorigenesis through the regulation of gene transcription (Bird, 2002). DNA methylation is one of the most important types of epigenetic modifications (Stein, 2011). This modification occurs primarily on cytosine residues of CpG dinucleotides, which are frequently clustered into CpG islands at the regulatory sites of gene promoters (Li et al., 2011a). DNA methylation is dynamically mediated by at least five independent DNA methyltransferases (DNMTs) (Brooks et al., 2010). Given the critical roles of DNMTs in the regulation of cancer cell growth, DNMT inhibitors such as 5-aza-2′-deoxycytidine (5-aza-dC, decitabine) have been widely used in demethylation studies and in clinical practice (Gore, 2010). Abbreviations: DNMT, DNA methyltransferase; 5-aza-dC, 5-aza-2′-deoxycytidine; TSG, tumor suppressor gene; miRNA, microRNA; MSP, methylation specific PCR; TNM, tumor-node-metastasis; FBS, fetal bovine serum; NC, negative control; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; RTCA, real-time cell analyzer; CI, cell index; NCIti, normalized cell index; PBS, phosphate buffered saline; PI, propidium iodide; RT, reverse transcription; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; Ct, threshold cycle; RIPA, radioimmunoprecipitation; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel; PVDF, polyvinylidene fluoride; CDK, cyclin-dependent kinase; VEGF, vascular endothelial growth factor; SPSS, Statistical Program for Social Sciences; miR-195, microRNA-195; miR-378, microRNA-378; 3′-UTR, 3′-untranslated region. ⁎ Corresponding author. Tel.: +86 574 87600758; fax: +86 574 87608638. E-mail address: [email protected] (J. Guo). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.12.103
Hypermethylation of human tumor suppressor genes (TSGs) may lead to their transcriptional inactivation and contribute to carcinogenesis (Laird, 2003). MicroRNAs (miRNAs) are a class of short, highly conserved noncoding RNAs (Zhou et al., 2010). miRNAs play essential roles in cell growth, differentiation and apoptosis, as well as carcinogenesis by regulating the expression of target mRNAs. Thus, in the context of over-expression or aberrant repression, miRNAs may function as either oncogenes or TSGs, respectively (Jiang et al., 2010; Zhou et al., 2010). Interestingly, emerging studies report that the expression of many miRNA genes is associated with promoter methylation (Fabbri et al., 2007; Lujambio et al., 2007). Radpour et al. (2011) revealed that miR-21 and miR-155 were down-regulated or over-expressed in breast cancer cell lines treated with 5-aza-dC, respectively. A study by Hiroki et al. (2012) also showed that miR-34b expression was restored by 5-aza-dC treatment in endometrial serous adenocarcinoma cell lines. Gastric cancer is the most common malignant tumor of the digestive system (Li et al., 2012), however the mechanisms underlying its pathogenesis remain incompletely understood. Dysregulation of miRNA expression caused by genetic and epigenetic alterations is considered to play important roles in gastric cancer development (Guo et al., 2009; Jiang et al., 2010). Previously, we identified seven miRNAs downregulated in gastric cancer compared with normal tissues (Guo et al., 2009). In this work, we investigate the role and relevant mechanisms of these miRNAs in gastric cancer progression. The integrated approach involving bioinformatics, enzyme inhibitors and mimics of miRNAs was
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designed and adopted in several gastric cancer cell lines and normal gastric epithelial cells (Fig. S1). 2. Materials and methods 2.1. Patients and specimens miRNA expression levels were analyzed in surgical tissue specimens obtained from 44 patients with gastric cancer treated at the Affiliated Hospital, Ningbo University School of Medicine or Ningbo Yinzhou People's Hospital (Table S1) after obtaining informed, written consent. Non-cancerous tissues from 17 randomly selected patients were used as controls. All non-cancerous tissue specimens were located 5 cm from the tumor and no cancer cells were observed, as evaluated by an experienced pathologist. Tumors were staged according to the tumor-node-metastasis (TNM) staging system. Histological grade was assessed according to the National Comprehensive Cancer Network clinical practice guideline of oncology (V.2.2010). No chemotherapy or other treatments were employed at the time tissue samples were obtained. The Human Research Ethics Committee from Ningbo University approved all aspects of this study. 2.2. Cell culture The human gastric cancer cell lines MGC-803, SGC-7901 and AGS were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The human, normal gastric epithelial cell line, GES-1, was purchased from the Cancer Institute and Hospital, Chinese Academy of Medical Sciences (Beijing, China). Cells were cultured in flasks at 37 °C in a humidified atmosphere of 5% CO2 with RPMI 1640 Medium (Invitrogen, Grand Island, NY) containing 10% fetal bovine serum (FBS) with 50 U/ml penicillin and 50 U/ml streptomycin. Log phase cells were used for experiments. Cells were counted using a hemocytometer. 2.3. 5-Aza-dC treatment Cells were seeded at 2 × 10 5 cells/well in 6-well plates. After 24 h, 5-aza-dC (Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 1 μM or 2 μM. Following 24 or 48 h treatment, cells were collected for cell cycle analysis, reverse transcription polymerase chain reaction (RT-PCR), or western blot.
Science, Mannheim, Germany), as described by the manufacturer. Briefly, the background signal of the culture medium was first determined by incubating E-Plates with 100 μl of RPMI 1640 with 10% FBS at room temperature for 30 min. A total of 8 × 103 cells were seeded per well in E-Plates 96 in a final volume of 100 μl RPMI 1640 and incubated at room temperature for 30 min. Cells were subsequently placed in the RTCA and incubated at 37 °C in 5% CO2. After 24 h, cells were transfected with miRNA mimic or inhibitor. Impedance was measured at 15 min intervals commencing immediately after cell seeding for 72 h. Impedance is represented by the cell index (CI), calculated as follows: CI= (Zi − Z0) / 15 Ω, where Zi is the impedance at an individual time-point; and Z0 is the background impedance. The average CI per time-point and experiment was calculated from a minimum of three wells. Raw CI values were normalized to the time-point of cell adherence. The normalized cell index (NCIti) was calculated as the cell index CIti at a given time-point divided by the cell index CInml_time at the normalized time-point (nml_time), that is NCIti = CIti / CInml_time. 2.7. Cell cycle analysis Gastric cancer cells treated with 5-aza-dC or transfected with miRNA mimic/inhibitor were collected and washed twice with phosphate buffered saline (PBS). After fixation in 75% ice-cold ethanol overnight at − 20 °C, cell pellets were washed twice with ice-cold PBS, and stained with a propidium iodide (PI) solution. Stained cells were maintained on ice in the dark, and analyzed on a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA). Data were analyzed using Cell Quest Pro software (BD Biosciences). 2.8. RNA extraction and reverse transcription Total RNA was extracted from fresh cultured cells or human tissues using Trizol reagent (Invitrogen, Karlsruhe, Germany) following the manufacturer's protocol. The concentration and purity of RNA samples were measured using the SmartSpec Plus spectrophotometer (Bio-Rad, Hercules, CA). The ratio of A260/A280 was used to indicate the purity of total RNA. cDNA was generated using the miScript Reverse Transcription (RT) Kit (Qiagen GmbH, Hilden, Germany) by combining 6 μl total RNA, 1 μl miScript Reverse Transcriptase Mix, and 4 μl miScript RT buffer and incubating for 60 min at 37 °C, followed by 5 min at 95 °C, in accordance with the manufacturer's protocol (Cheng et al., 2012). cDNA was then diluted with 80 μl of autoclaved-distilled water. 2.9. Real-time PCR
2.4. Transient transfection Cells were seeded at 2 × 10 5 cells/well in 6-well plates. When cells reached 40–60% confluence, 0, 50, 100, and 150 nM miRNA mimic/ inhibitor, or 150 nM negative control (NC) was transfected using Lipofectamine 2000 reagent (Invitrogen, Karlsruhe, Germany) following the manufacturer's protocol. The specific 2′-methoxy-modified RNA oligonucleotides (Shanghai GenePharma Co., Shanghai, China), were used as miRNA mimic and inhibitor (Jiang et al., 2010) (Table S2). 2.5. MTT assay The effect of 5-aza-dC or miRNA mimic/inhibitor treatment on gastric cancer and normal gastric epithelial cell growth was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay as previously described (Jiang et al., 2010).
Real-time PCR was performed using the miScript SYBR Green PCR Kit (Qiagen). PCR was performed in a 20 μl reaction volume containing 6 μl cDNA product, 10 μl 2× QuantiTect SYBR Green PCR Master Mix, 1 μl 10× miScript Universal Primer, 1 μl 10× miScript Primer Assay (for miRNAs, Qiagen) and 2 μl autoclaved-distilled water. Amplification was performed using an MX3005P QPCR System (Stratagene, La Jolla, CA) with the following cycling conditions; 95 °C for 15 min, followed by 40 cycles of 94 °C for 15 s, 60 °C for 30 s, and 70 °C for 30 s. Levels of U6 small RNA were determined with the Hs_RNU6B_2 miScript Primer Assay (Qiagen) and used for normalizing the levels of miRNAs (Jiang et al., 2010). The ΔCt method was used to calculate miRNA expression levels (Jiang et al., 2010). All experiments were repeated in biological duplicate. The sequences of primers are listed in Table S3. 2.10. Western blot analysis
2.6. Real-time analysis of cell proliferation Proliferation assays were performed in E-Plates 96 (Roche Applied Science, Mannheim, Germany) using a Roche DP real-time cell analyzer (RTCA), an impedance-based xCELLigence System (Roche Applied
Cells treated with 5-aza-dC or miRNA mimic/inhibitor, were lysed with radioimmunoprecipitation (RIPA) buffer. Lysates were clarified by centrifugation at 16,000 rpm for 20 min at 4 °C, boiled, separated by a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and
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transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Bedford, MA). Membranes were blocked with 5% skim milk powder for 1 h and incubated with rabbit anti-human cyclindependent kinase (CDK)1, CDK2, CDK6, cyclin A, cyclin D1 or vascular endothelial growth factor (VEGF) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight and subsequently incubated with alkaline phosphatase conjugated goat anti-rabbit antibody (Boster, Wuhan, China) at room temperature for 1 h. β-Actin (Sigma-Aldrich, Saint Louis, MO) was used as a normalization control for protein loading.
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line, and AGS and MGC-803, low differential gastric cancer cell lines) compared with normal gastric epithelial cells (GES-1), and in a larger panel of primary gastric cancer tissues compared with non-cancerous tissues by real-time PCR. As shown in Fig. 1B, the level of miR-195 and miR-378 was significantly lower in all gastric cancer cells and gastric cancer tissues compared with normal gastric epithelial cells (GES-1) and non-cancerous tissues, respectively. We also found that the level of miR-195 was decreased in low differential gastric cancer cell lines, AGS and MGC-803, compared with SGC-7901, the middle differential gastric cancer cell lines. The level of miR-378 in MGC-803 cells was also lower compared with SGC-7901 cells (Fig. 1B).
2.11. 3′-UTR reporter assay To study the targets of these miRNAs, we investigated the predicted binding site of miRNAs and the mRNA 3′-untranslated region (UTR) using the two most widely used target prediction databases: PicTar (http://www.mdc-berlin.de/en/research/research_teams/systems_ biology_of_gene_regulatory_elements/projects/pictar/) and microRNA targets (http://www.microrna.org/microrna/home.do). A fragment of the wild-type (wt) mRNA 3′-UTR containing the predicted miRNA binding site was amplified by RT-PCR. The mutation-type (mt) mRNA 3′-UTR was created by site-directed mutagenesis using primers listed in Table S4. The target mRNA 3′-UTR was cloned into the Psicheck 2 dual luciferase reporter vector (Promega). Human embryonic kidney (HEK) 293T or MGC-803 cells were co-transfected with reporter constructs and miRNA mimic/NC. Cells were lysed 48 h post after transfection and the ratio of Renilla to firefly luciferase activity was measured using the dual luciferase assay kit (Promega). Normalized Renilla to firefly luciferase activity ratios were determined in the presence or absence of miRNA inhibition. 2.12. Statistical analysis Statistical analysis was performed using the Statistical Program for Social Sciences (SPSS) software 17.0 (SPSS Inc., Chicago, IL). All data represent as the mean± standard deviation (SD). Differences between experimental groups and control groups were analyzed using the two-tailed Student's t-test. Statistical significance was accepted at P b 0.05. 3. Results 3.1. Presence of CpG islands in the promoters of miR-378 and miR-195 Previously, we identified seven miRNAs (miR-768-3p, miR-139-5p, miR-378, miR-31, miR-195, miR-497 and miR-133b) down-regulated in gastric cancer tissues compared with non-tumor tissues (Guo et al., 2009). Down-regulation of miRNAs in tumors is often due to aberrant DNA hypermethylation of gene promoters (Brooks et al., 2010; Li et al., 2011a), therefore in this study, we focused on investigating the epigenetic regulation of candidate miRNA genes. To identify the existence of CpG islands in the promoters of miRNAs down-regulated in gastric cancer, we analyzed the sequence 5 kb upstream of these seven miRNA genes using CpGplot software (http://www.ebi.ac.uk/ emboss/cpgplot/) (Previti et al., 2009). As shown in Fig. 1A, we identified one and two CpG island(s) in the promoters of miR-378 and miR-195, respectively. 3.2. Expression of miR-195 and miR-378 is significantly down-regulated in gastric cancer cells and gastric cancer tissues In our previous study, we showed that miR-195 and miR-378 were down-regulated in gastric cancer tissues by microarray analysis (Guo et al., 2009). To further investigate the expression of miR-195 and miR-378 in gastric cancer, we assessed their levels in three human gastric cancer cell lines (SGC-7901, a middle differential gastric cancer cell
3.3. Expression of miR-195 and miR-378 is restored in gastric cancer cells treated with DNMT inhibitor To investigate the role of DNA methylation in the regulation of miR-195 and miR-378, we assessed the expression of these miRNAs in MGC-803 cells following treatment with 5-aza-dC (1 or 2 μM). We observed a dose-dependent up-regulation of miR-195 and miR-378 expression in 5-aza-dC treated cells compared with controls (Fig. 1C). Previously, we identified seven miRNAs (including miR-195 and miR-378) down-regulated in gastric cancer tissues (Guo et al., 2009), therefore we next investigated whether the expression of these other miRNAs was induced by treatment with DNMT inhibitor. miR-768-3p was not studied since this miRNA has been removed from the miRNA database (http://www.mirbase.org/cgi-bin/query.pl?terms=miR-768). In contrast to miR-195 and miR-378, however, the expression of the four remaining miRNAs (miR-139-5p, miR-133b, miR-31, and miR-497), which do not contain promoter CpG islands, was not restored by 5-aza-dC (Fig. 1D). 3.4. Treatment with low doses of 5-aza-dC is not toxic to gastric cancer cells Since the expression of miR-195 and miR-378 was shown to be upregulated in gastric cancer cells treated with 5-aza-dC (Fig. 1C), we next examined the growth of MGC-803 gastric cancer cells following DNMT inhibitor treatment. MGC-803 cell growth was not significantly inhibited following exposure to 5-aza-dC for 24, 48 and 72 h (Fig. 2A). These results indicate that at low concentrations (1 or 2 μM), 5-aza-dC has no toxic effect on gastric cancer cells. Next, we monitored the changes in cell cycle distribution by flow cytometry. Treatment of MGC-803 and SGC-7901 gastric cancer cells with 5-aza-dC for 24 h or 48 h led to cell cycle arrest at the G2/M phase or S phase, respectively (Table 1). Changes in the expression of cell cycle regulators such as cyclins and CDKs, were consistent with these changes in cell cycle distribution. As shown in Figs. 2B and C, treatment with 5-aza-dC led to a significant increase in CDK1, CDK2 and cyclin A protein levels. 3.5. Growth of gastric cancer cells is affected by modulation of miR-195/miR-378 As miR-195 and miR-378 were shown to be down-regulated in gastric cancer tissues (Guo et al., 2009, Fig. 1B) and gastric cancer cells (Fig. 1B), we next examined their possible tumor suppressor activities using two independent methods, RTCA and MTT assay. We first examined the effect of increasing the cellular level of miR-195 and miR-378 in gastric cancer cells (MGC-803, SGC-7901 and AGS) and normal gastric epithelial GES-1 cells, by transfection of mimics. The effects on cell growth were detected by RTCA, a novel real-time cell growth analyzer. As shown in Fig. S2, gastric cancer cell growth was significantly inhibited following introduction of miR-195 or miR-378 mimics, while the growth of normal gastric epithelial cells was promoted by miR-195 mimic. To confirm the effects of miR-195 and miR-378 mimics on gastric cancer cell growth, we next assessed cell growth by MTT assay. Following transfection with miR-195 or miR-378 mimics for 24, 48 and 72 h,
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the growth of gastric cancer MGC-803 (Figs. 3A and D) and AGS (Figs. 3B and E) cells was significantly inhibited in a dose-dependent manner. In contrast, the growth of GES-1 cells was promoted (Figs. 3C and F). To confirm successful transfection of miR-195 and miR-378 mimics into cells, we measured the level of miR-195 and miR-378 in MGC-803 cells by real time PCR. We found that their levels were elevated compared with negative control (NC) treated cells (Fig. S3). We next examined the effect of inhibiting miR-195 and miR-378 on both gastric cancer and normal gastric epithelial cell growth. Gastric cancer cells (AGS and SGC-7901) and normal gastric epithelial cells (GES-1) were transfected with miR-195 or miR-378 inhibitors or NC miRNA inhibitor, and the effect on cell growth was assessed by RTCA.
As shown in Fig. S4, the growth of normal gastric epithelial GES-1 cells was significantly impaired by miR-195 or miR-378 inhibitors. As expected, the growth of both SGC-7901 and AGS cells, in which the basal levels of miR-195 and miR-378 were very low (Fig. 1B), was not significantly affected by treatment with miR-195 or miR-378 inhibitors (Fig. S4). 3.6. Effect of miR-195 or miR-378 mimics on cell cycle distribution of gastric cancer cells To further investigate the growth suppressive mechanisms of miR-195 or miR-378 mimics on gastric cancer cells, we monitored changes in the cell cycle distribution by flow cytometry. Treatment
Fig. 1. Expression and methylation analysis of miRNAs in gastric cancer. (A) Bioinformatic analysis identified CpG islands in the promoters of miR-195 and miR-378. (B) miR-195 and miR-378 expression was down-regulated in gastric cancer tissues and cells. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to detect miRNA expression in tissues, normal gastric epithelial cells (GES-1) and gastric cancer cell lines (MGC-803, SGC-7901 and AGS). Comparison with non-cancerous tissues or GES-1 cells, ⁎P b 0.001; comparison with SGC-7901, #P b 0.001. Data represent the mean of three independent experiments. 5-Aza-dC, a DNMT inhibitor, increased the expression of miR-195 and miR-378 in MGC-803 gastric cancer cells (C), but did not affect the expression of miRNAs lacking CpG islands in their gene promoters (D). Cells were treated with 0, 1 or 2 μM 5-aza-dC for 48 h. The expression levels of miR-195 and miR-378 were detected by RT-PCR. Comparison with control (0 μM), ⁎P b 0.05.
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4. Discussion
Fig. 1 (continued).
of MGC-803 and AGS gastric cancer cells with miR-195 and miR-378 mimics led to an arrest at the G0/G1 and G2/M phases, respectively (Table 2). These results were generally consistent with those following treatment with 5-aza-dC (Table 1), with minor differences. This may be owing to differences in the increase in miR-195 and miR-378 levels induced by 5-aza-dC treatment and transfection with miRNA mimics. For example, transfection of cells with 100 nM miR-195/miR-378 mimics led to a >300-fold increase in miR-195 and miR-378 levels (Fig. S3). In contrast, treatment of cells with 2 μM 5-aza-dC, led to an increase in miR-195 and miR-378 levels of less than 80-fold (Fig. 1C). Nevertheless, the effects of 5-aza-dC on gastric cancer were not limited to the gene expression of miRNAs.
3.7. Expression of cancer-related miR-195 or miR-378 targets is suppressed by miR-195 or miR-378 mimics In order to confirm the putative tumor suppressor activities of miR-195 and miR-378 in gastric cancer, we studied the expression changes of their targets. Recently, CDK6 and VEGF were identified as targets of miR-195 (Wang et al., 2012) and miR-378 (Hua et al., 2006), respectively, in other cancer types. As a result, we focused on these targets in this study. As shown in Fig. 4, miR-195 and miR-378 mimics suppressed the expression of CDK6 and VEGF, respectively.
3.8. miR-195 is a negative regulator of CDK6 expression To understand the mechanism by which miR-195 regulates CDK6 expression, we searched for potential targeted sequences in 3′-UTR of CDK6 mRNA using two software programs, PicTar and miRanda. The result showed that two binding sites of miR-195 were found in 3′-UTR of CDK6 mRNA (Fig. S5). To further validate this bioinformatic finding, we assessed the expression of 3′-UTR of CDK6 mRNA in luciferase reporter assays. Sequencing results confirmed that the vectors of 3′-UTR reporter assay were successfully constructed (Fig. S6). The expression of the CDK6 reporter was significantly reduced to ≤50% in miR-195 mimic-transfected MGC-803 cells, however this effect was abolished using the CDK6 mutation-type reporter (Fig. 5).
In recent years, a number of miRNAs were shown to be expressed at low levels in human tumors, and many of these miRNAs may function as tumor suppressors (Nie et al., 2012; Ugras et al., 2011). DNA methylation in promoter regions of these miRNA genes represents one possible mechanism for their loss of tumor suppressor function in cancer cells (Wong et al., 2011). Indeed, several down-regulated miRNAs that act as tumor suppressors have been confirmed to be associated with DNA methylation (Lopez-Serra and Esteller, 2012; Yu et al., 2012). DNA methylation primarily occurs on cytosine residues of CpG dinucleotides, especially on CpG islands in the gene promoter regions (Deaton and Bird, 2011; Fabbri and Calin, 2010). 5-aza-dC, a DNMT inhibitor, has been widely used to study DNA methylation (Kulis and Esteller, 2010). Recently, miR-195 expression was shown to be down-regulated in various cancers, including bladder cancer (Han et al., 2011), colorectal cancer (Wang et al., 2012), hepatocellular cancer (Xu et al., 2009) and breast cancer (Li et al., 2011b). miR-378 was shown to be downregulated in colorectal cancer (Mosakhani et al., 2012) and oral cancer (Scapoli et al., 2010). Previously, we reported seven miRNAs, including miR-195 and miR-378, to be down-regulated in gastric cancer (Guo et al., 2009). In this study, we extended this analysis to show that the promoter regions of miR-195 and miR-378 contained CpG islands (Fig. 1A). Furthermore, we confirmed that miR-195 and miR-378 were significantly down-regulated not only in gastric cancer tissues (Guo et al., 2009), but also in human gastric cancer cell lines (Fig. 1B). Although we and others, have previously shown that miR-195 and miR-378 are expressed at low levels in gastric cancer (Guo et al., 2009; Liu et al., 2012; Wu et al., 2011; Yao et al., 2009), the mechanism underlying their down-regulation and their roles in carcinogenesis remain unclear. Our data suggest that the expression of both miR-195 and miR-378 is regulated by DNA methylation in their upstream promoters (Fig. 1). Recent studies showed that the promoters of several miRNAs, including miR-34a (Lodygin et al., 2008), miR-34c (Wong et al., 2011), miR-200c (Neves et al., 2010) and miR-1 (Datta et al., 2008), contain CpG islands, which are methylated. To understand whether CpG island methylation is responsible for the silencing of miR-195 and miR-378 in gastric cancer, we treated gastric cancer cells with 5-aza-dC, a demethylation reagent, and found that it restored the expression of miR-195 and miR-378 (Fig. 1C). However, the expression of other miRNAs lacking promoter CpG islands, was not restored by 5-aza-dC treatment (Fig. 1C). This implies that CpG island methylation of the miR-195 and miR-378 gene promoters plays a central role in the reduction of their expression in gastric cancer. The regulation of gene expression is very complicated, however, and those miRNAs lacking promoter CpG islands may be regulated by other mechanisms, which also warrant further study. A previous study showed that treatment of breast cancer MDAMB-435S cells with 5 μM of 5-aza-dC inhibited cell growth (Zhang et al., 2007). In order to prevent the toxicity of 5-aza-dC on gastric cells and demonstrate its inhibitory activity on methyltransferase, we treated cells with low doses (1 μM or 2 μM), and observed no toxic effects at these concentrations (Fig. 2A). Our data results demonstrate that low doses of 5-aza-dC arrest gastric cancer cells at the G2/M or S phase (Table 1). Furthermore, low doses of 5-aza-dC enhanced the expression of certain cell cycle-related proteins (Figs. 2B and C). Taken together, this suggests that the effects of low dose treatment of gastric cancer cells with 5-aza-dC may occur through the elevation of specific miRNAs, which may function as TSGs. Cell cycle progression is a highly regulated process and is controlled by the sequential activation and inactivation of families of the CDKs and cyclins. CDK1 combined with cyclin A or cyclin B, plays an essential role in S and G2/M phase transitions of the eukaryotic cell cycle (Dorée and Hunt, 2002). CDK2 with cyclin A or cyclin E, restricts cells at the G1/S phase, and is essential for G1 phase to S phase transition (Chen et al.,
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Fig. 2. The effect of 5-aza-dC on gastric cancer cell growth. (A) Cell growth was measured by MTT assay. Cells were treated with 1 or 2 μM of 5-aza-dC for 24, 48 and 72 h, respectively. Three independent experiments were performed. Western blot analysis of cell cycle-related proteins in MGC-803 (B) and SGC-7901 (C). Cells were treated with 1 or 2 μM of 5-aza-dC for 48 h. Western blot analysis was performed with antibodies recognizing human CDK1, CDK2, cyclin A and cyclin D1. β-Actin was used as a loading control. A representative result (left) and results from three independent experiments (right) are shown. Comparison with control, *P b 0.05.
Table 1 Analysis of cell cycle distribution in gastric cancer cells treated with 5-aza-dC (%). Treatment time
Cell line
5-Aza-dC (μM)
G0/G1
S
G2/M
24 h
MGC-803
0 1 2 0 1 2 0 1 2 0 1 2
36.16 ± 1.36 32.14 ± 0.18 32.20 ± 2.52 54.33 ± 0.18 48.68 ± 0.49⁎ 46.56 ± 0.26⁎
38.91 ± 3.16 36.60 ± 1.24 31.14 ± 1.87 30.63 ± 0.69 31.15 ± 1.70 29.69 ± 1.39 32.45 ± 0.37 47.65 ± 0.25⁎ 47.89 ± 1.79⁎ 32.6 ± 0.25 50.26 ± 0.12⁎ 57.25 ± 0.09⁎
24.93 ± 1.81 31.26 ± 1.06⁎ 36.67 ± 0.65⁎ 15.06 ± 0.51 20.19 ± 2.18 23.75 ± 1.13⁎
SGC-7901
48 h
MGC-803
SGC-7901
35.76 ± 0.40 20.79 ± 0.49⁎ 19.25 ± 0.01# 60.37 ± 1.32 49.74 ± 0.43 40.38 ± 0.05
Comparison with control treated cells (0 μM), ⁎P b 0.05, #P b 0.01.
31.79 ± 0.77 31.57 ± 0.29 32.86 ± 1.78 7.03 ± 1.82 0.00 ± 0.25 2.37 ± 0.19
2003). Cyclin D1 also regulates cell cycle checkpoints by binding its catalytic partners, CDK4 or CDK6 (Vodermaier, 2004). Cell proliferation and cell cycle progression are closely related to these proteins. To clarify the mechanism(s) by which miR-195 and miR-378 suppress gastric cancer cell growth, we studied the expression of several cell cycle associated proteins including CDK1, CDK2, cyclin A and cyclin D1 (Fig. 2). In these experiments, we found that the ectopic expression of miR-195 and miR-378 was capable of inhibiting the proliferation of gastric cancer cells, while promoting the proliferation of normal gastric epithelial cells (Figs. 3 and S2). In keeping with this finding, treatment with miR-195 and miR-378 inhibitors suppressed the growth of normal gastric epithelial cells, but had a minimal effect on the growth of gastric cancer cells (Fig. S4). This is likely owing to the low basal expression of miR-195 and miR-378 in gastric cancer cells (Fig. 1B). Since the expression of these miRNAs was already low, the growth of gastric cancer cells was not significantly altered following treatment with miR-195 and miR-378 inhibitors. In order to investigate the mechanisms underlying the growth inhibitory effects of miR-195 and miR-378 on gastric cancer
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Fig. 3. The effect of miR-195 or miR-378 mimics on the growth of gastric cancer and normal gastric epithelial cells. The effect of miR-195 mimic on MGC-803 (A), AGS (B) or GES-1 (C). The effect of miR-378 mimic on MGC-803 (D), AGS (E) or GES-1 (F). Cells were treated with 50 nM, 100 nM or 150 nM of miR-195 or miR-378 mimics or 150 nM negative control (NC) for 24, 48 or 72 h, respectively. Cell growth was measured by MTT assay. Data represent the mean of three independent experiments. Comparison with NC, *P b 0.05.
cells, we monitored the cell cycle distributions in gastric cancer cells transfected with miR-195 and miR-378 mimics. We observed that cancer cells were arrested in G0/G1 or G2/M phase following treatment
Table 2 Cell cycle analysis of gastric cancer cells transfected with miRNA mimics (%). Mimic
Cell line
Concentration G0/G1 (nM)
miR-195 MGC-803 NC 50 150 AGS NC 50 150 miR-378 MGC-803 NC 50 150 AGS NC 50 150
S
39.70 ± 0.50 47.53 ± 1.12⁎ 45.63 ± 0.41⁎ 43.09 ± 0.63 50.51 ± 0.93⁎
28.78 ± 0.50 27.33 ± 1.15 26.45 ± 0.88 48.30 ± 1.56 33.64 ± 1.87⁎ 53.28 ± 0.43# 29.51 ± 0.55# 39.70 ± 0.50 28.78 ± 0.50 39.12 ± 0.08 27.03 ± 0.35 27.03 ± 0.25⁎ 33.75 ± 1.33 43.09 ± 0.63 48.30 ± 1.56 47.34 ± 0.32⁎ 33.87 ± 1.06⁎ 42.11 ± 2.18 39.14 ± 0.74⁎
Comparison with negative control (NC), ⁎P b 0.05, #P b 0.01.
G2/M 31.53 ± 0.99 25.15 ± 0.02 27.93 ± 0.47 8.61 ± 2.18 15.86 ± 0.95 17.22 ± 0.12 31.53 ± 0.99 33.86 ± 0.26 39.23 ± 1.58⁎ 8.61 ± 2.18 18.80 ± 0.75⁎ 18.75 ± 1.44⁎
with miR-195 or miR-378 mimics, respectively (Table 2). Based on these data, we postulate that miR-195 and miR-378 have possible tumor suppressor roles in gastric cancer. Furthermore, we confirmed that CDK6 and VEGF are regulated by miR-195 and miR-378, respectively (Fig. 4). CDK6 is a key element of D-type cyclin holoenzymes, and is involved in regulation of the G1/S phase transition of the cell cycle (Liu et al., 2011). CDK6 has been shown to be over-expressed in gastric cancer and many other cancers (Zhang et al., 2009). Recently, CDK6 was found to be a target of several miRNAs including miR-129 (Wu et al., 2010), miR-22 (Xu et al., 2011) and miR-195 (Wang et al., 2012). One study showed that a decrease in CDK6 blocks gastric cancer cells in the G0/G1 phase (Feng et al., 2012). In our study, we found that over-expression of a miR-195 mimic inhibited the expression of CDK6 in gastric cancer cells (Fig. 4A), and arrested cells in the G0/G1 phase (Table 2). These results are in accordance with the tumor suppressor function of miR-195 (Fig. 3). We suspect that the growth inhibitory mechanism of miR-195 in gastric cancer cells is associated with the down-regulation of CDK6, and cell cycle arrest. Indeed, we confirmed CDK6 as a target of miR-195 using luciferase reporter assays (Fig. 5). VEGF plays a role in various processes, including angiogenesis, promoting cell migration and inhibiting
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Fig. 4. Western blot analysis of the expression level changes of miR-195 or miR-378 mimics on their targets. Various concentrations of miR-195 mimic (50, 100 and 150 nm) or 150 nM negative control (NC) on the expression of CDK6 in MGC-803 cells (A). Various concentrations of miR-378 mimic (50, 100 and 150 nm) or 150 nM negative control (NC) on the expression of VEGF in MGC-803 (B) and AGS (C) cells. β-Actin was used as loading control. A representative result (up) and results from three independent experiments (down) were shown. Comparison with NC, *P b 0.05.
apoptosis. Recently, VEGF was shown to be a target of miR-206 (Zhang et al., 2011) and miR-378 (Hua et al., 2006) in laryngeal and nasopharyngeal cancers. However, the relationship between miRNAs and VEGF has not been investigated in gastric cancer. Here, we present the first report that VEGF is regulated by miR-378 in gastric cancer (Figs. 4B and C). In summary, using a combination of several gastric cancer and normal gastric epithelial cell lines as models, and by modulating the levels of cellular miR-195 and miR-378 levels, we demonstrate that the down-regulation of miR-195 and miR-378 in gastric cancer is a result of promoter CpG island methylation. We also show that miR-195 and miR-378 may act as tumor suppressors in gastric cancer by negatively regulating the expression of CDK6 and VEGF, respectively. Taken
together, these data indicate that miR-195 and miR-378 may be potential therapeutic targets in gastric cancer therapy. Acknowledgments This work was supported by the Excellent Dissertation Fund of Ningbo University (No. PY201014 and PY20110020); College Students' Science–Technology Innovation Program of Zhejiang Province (No. 200959); the Scientific Innovation Team Project of Ningbo (No. 2011B82014); Ningbo Natural Science Foundation (No. 2010A610044 and No. 2012A610207); Zhejiang Provincial Research Project (No. 2010C33112 and No. 2012C23127); National Natural Science Foundation
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Fig. 5. miR-195 directly targets CDK6. miR-195 suppressed the expression of a luciferase vector containing the 3′-UTR of CDK6 mRNA. A luciferase expression vector with the 3′-UTR of CDK6 mRNA and miR-195 mimic or NC (blank) was co-transfected into MGC-803 cells. Cells were harvested after 48 h and assayed for luciferase activity using the dual luciferase assay kit (Promega). Firefly luciferase was used for normalization. Data represent the mean of three independent experiments. Comparison with blank, *Pb 0.05. luc-wt1, the vector CDK6-wild type (wt)-1 contained the binding site of miR-195 (120–126: TGCTGCT); luc-mt1, the mutant (mt) of luc-wt1 (Fig. S6). luc-wt2, the vector CDK6-wt-2 contained the binding site of miR-195 (117–123: TGCTGCT); luc-mt2, the mutant (mt) of luc-wt2 (Fig. S6).
of China (No. 81171660); the Medical Science–Technology Project of Ningbo (No. 2010B06); and KC Wong Magna Fund of Ningbo University. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.gene.2012.12.103. References Bird, A., 2002. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21. Brooks, W.H., Le Dantec, C., Pers, J.O., Youinou, P., Renaudineau, Y., 2010. Epigenetics and autoimmunity. J. Autoimmun. 34, J207–J219. Chen, J.H., Tseng, T.H., Ho, Y.C., Lin, H.H., Lin, W.L., Wang, C.J., 2003. Gaseous nitrogen oxides stimulate cell cycle progression by retinoblastoma phosphorylation via activation of cyclins/Cdks (correction). Toxicol. Sci. 76, 83–90. Cheng, J., et al., 2012. piR-823, a novel non-coding small RNA, demonstrates in vitro and in vivo tumor suppressive activity in human gastric cancer cells. Cancer Lett. 315, 12–17. Datta, J., et al., 2008. Methylation mediated silencing of MicroRNA-1 gene and its role in hepatocellular carcinogenesis. Cancer Res. 68, 5049–5058. Deaton, A.M., Bird, A., 2011. CpG islands and the regulation of transcription. Genes Dev. 25, 1010–1022. Dorée, M., Hunt, T., 2002. From Cdc2 to Cdk1: when did the cell cycle kinase join its cyclin partner? J. Cell Sci. 115, 2461–2464. Fabbri, M., Calin, G.A., 2010. Epigenetics and miRNAs in human cancer. Adv. Genet. 70, 87–99. Fabbri, M., Ivan, M., Cimmino, A., Negrini, M., Calin, G.A., 2007. Regulatory mechanisms of microRNAs involvement in cancer. Expert Opin. Biol. Ther. 7, 1009–1019. Feng, L., Xie, Y., Zhang, H., Wu, Y., 2012. miR-107 targets cyclin-dependent kinase 6 expression, induces cell cycle G1 arrest and inhibits invasion in gastric cancer cells. Med. Oncol. 29, 856–863. Gore, S.D., 2010. Combination therapy with DNA methyltransferase inhibitors in hematologic malignancies. Nat. Clin. Pract. Oncol. 2, S30–S35. Guo, J., et al., 2009. Differential expression of microRNA species in human gastric cancer versus non-tumorous tissues. J. Gastroenterol. Hepatol. 24, 652–657. Han, Y., et al., 2011. MicroRNA expression signatures of bladder cancer revealed by deep sequencing. PLoS One 6, e18286. Hiroki, E., et al., 2012. MicroRNA-34b functions as a potential tumor suppressor in endometrial serous adenocarcinoma. Int. J. Cancer 131, E395–E404.
359
Hua, Z., et al., 2006. MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia. PLoS One 1, e116. Jiang, Z., et al., 2010. Increased expression of miR-421 in human gastric carcinoma and its clinical association. J. Gastroenterol. 45, 17–23. Kulis, M., Esteller, M., 2010. DNA methylation and cancer. Adv. Genet. 70, 27–56. Laird, P.W., 2003. The power and the promise of DNA methylation markers. Nat. Rev. Cancer 3, 253–266. Li, Y., Daniel, M., Tollefsbol, T.O., 2011a. Epigenetic regulation of caloric restriction in aging. BMC Med. 9, 98. Li, D., et al., 2011b. Analysis of MiR-195 and MiR-497 expression, regulation and role in breast cancer. Clin. Cancer Res. 17, 1722–1730. Li, L.Z., et al., 2012. Growth inhibitory effect of 4-phenyl butyric acid on human gastric cancer cells is associated with cell cycle arrest. World J. Gastroenterol. 18, 79–83. Liu, Y.F., Zan, L.S., Cui, W.T., Xin, Y.P., Jiao, Y., Li, K., 2011. Molecular cloning, characterization and association analysis of the promoter region of the bovine CDK6 gene. Genet. Mol. Res. 10, 1777–1786. Liu, H., et al., 2012. Genome-wide microRNA profiles identify miR-378 as a serum biomarker for early detection of gastric cancer. Cancer Lett. 316, 196–203. Lodygin, D., et al., 2008. Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer. Cell Cycle 7, 2591–2600. Lopez-Serra, P., Esteller, M., 2012. DNA methylation-associated silencing of tumorsuppressor microRNAs in cancer. Oncogene 31, 1609–1622. Lujambio, A., et al., 2007. Genetic unmasking of an epigenetically silenced microRNA in human cancer cells. Cancer Res. 67, 1424–1429. Mosakhani, N., et al., 2012. MicroRNA profiling differentiates colorectal cancer according to KRAS status. Genes Chromosomes Cancer 51, 1–9. Neves, R., et al., 2010. Role of DNA methylation in miR-200c/141 cluster silencing in invasive breast cancer cells. BMC Res. Notes 3, 219. Nie, J., et al., 2012. MicroRNA-365, down-regulated in colon cancer, inhibits cell cycle progression and promotes apoptosis of colon cancer cells by probably targeting Cyclin D1 and Bcl-2. Carcinogenesis 33, 220–225. Previti, C., Harari, O., Zwir, I., del Val, C., 2009. Profile analysis and prediction of tissuespecific CpG island methylation classes. BMC Bioinformatics 10, 116. Radpour, R., et al., 2011. Integrated epigenetics of human breast cancer: synoptic investigation of targeted genes, microRNAs and proteins upon demethylation treatment. PLoS One 6, e27355. Scapoli, L., et al., 2010. MicroRNA expression profiling of oral carcinoma identifies new markers of tumor progression. Int. J. Immunopathol. Pharmacol. 23, 1229–1234. Stein, R.A., 2011. DNA methylation profiling: a promising tool and a long road ahead for clinical applications. Int. J. Clin. Pract. 65, 1212–1213. Ugras, S., et al., 2011. Small RNA sequencing and functional characterization reveals MicroRNA-143 tumor suppressor activity in liposarcoma. Cancer Res. 71, 5659–5669. Vodermaier, H.C., 2004. APC/C and SCF: controlling each other and the cell cycle. Curr. Biol. 14, R787–R796. Wang, X., Wang, J., Ma, H., Zhang, J., Zhou, X., 2012. Downregulation of miR-195 correlates with lymph node metastasis and poor prognosis in colorectal cancer. Med. Oncol. 29, 919–927. Wong, K.Y., Yu, L., Chim, C.S., 2011. DNA methylation of tumor suppressor miRNA genes: a lesson from the miR-34 family. Epigenomics 3, 83–92. Wu, J., et al., 2010. miR-129 regulates cell proliferation by downregulating Cdk6 expression. Cell Cycle 9, 1809–1818. Wu, W.Y., et al., 2011. Potentially predictive microRNAs of gastric cancer with metastasis to lymph node. World J. Gastroenterol. 17, 3645–3651. Xu, T., Zhu, Y., Xiong, Y., Ge, Y.Y., Yun, J.P., Zhuang, S.M., 2009. MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells. Hepatology 50, 113–121. Xu, D., et al., 2011. miR-22 represses cancer progression by inducing cellular senescence. J. Cell Biol. 193, 409–424. Yao, Y., et al., 2009. MicroRNA profiling of human gastric cancer. Mol. Med. Rep. 2, 963–970. Yu, F., et al., 2012. MicroRNA 34c gene down-regulation via DNA methylation promotes self-renewal and epithelial–mesenchymal transition in breast tumor-initiating cells. J. Biol. Chem. 287, 465–473. Zhang, B., Huang, T., Liu, K., Chen, J., Wang, G., 2007. Effects of 5-Aza-CdR on cell proliferation of breast cancer cell line MDA-MB-435S and expression of maspin gene. J. Huazhong Univ. Sci. Technol. Med. Sci. 27, 543–546. Zhang, S., et al., 2009. RhoA regulates G1-S progression of gastric cancer cells by modulation of multiple INK4 family tumor suppressors. Mol. Cancer Res. 7, 570–580. Zhang, T., Liu, M., Wang, C., Lin, C., Sun, Y., Jin, D., 2011. Down-regulation of miR-206 promotes proliferation and invasion of laryngeal cancer by regulating VEGF expression. Anticancer Res. 31, 3859–3863. Zhou, H., et al., 2010. Detection of circulating tumor cells in peripheral blood from patients with gastric cancer using microRNA as a marker. J. Mol. Med. 88, 709–717.
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Gene journal homepage: www.elsevier.com/locate/gene
MicroRNA-195 and microRNA-378 mediate tumor growth suppression by epigenetical regulation in gastric cancer Hongxia Deng a, Yanan Guo a, Haojun Song a, Bingxiu Xiao a, Weiliang Sun b, Zhong Liu c, Xiuchong Yu a, Tian Xia a, Long Cui a, Junming Guo a,⁎ a b c
Department of Biochemistry and Molecular Biology, and Zhejiang Provincial Key Laboratory of Pathophysiology, Ningbo University School of Medicine, Ningbo, 315211, China Ningbo Yinzhou People's Hospital and the Affiliated Hospital, Ningbo University School of Medicine, Ningbo 315040, China The Affiliated Hospital, Ningbo University School of Medicine, Ningbo 315010, China
a r t i c l e
i n f o
Article history: Accepted 24 December 2012 Available online 17 January 2013 Keywords: MicroRNA Gastric cancer Tumor suppressor Cell cycle Epigenetic
a b s t r a c t The epigenetic regulation of microRNAs is one of several mechanisms underlying carcinogenesis. We found that microRNA-195 (miR-195) and microRNA-378 (miR-378) were significantly down-regulated in gastric cancer tissues and gastric cancer cell lines. The expression of miR-195 and miR-378 in gastric cancer cells was significantly restored by 5-aza-dC, a demethylation reagent. The low expression of miR-195 and miR-378 was closely related to the presence of promoter CpG island methylation. Treatment with miR-195/miR-378 mimics strikingly suppressed the growth of gastric cancer cells whereas promoted the growth of normal gastric epithelial cells. In contrast, administration of miR-195/miR-378 inhibitors significantly prevented the growth of normal gastric epithelial cells. Expression of cyclin-dependent kinase 6 and vascular endothelial growth factor was downregulated by exogenous miR-195 and miR-378, respectively. In conclusion, miR-195 and miR-378 are abnormally expressed and epigenetically regulated in gastric cancer cell lines and tissues via the suppression of CDK6 and VEGF signaling, suggesting that miR-195 and miR-378 have tumor suppressor properties in gastric cancer. © 2013 Elsevier B.V. All rights reserved.
1. Introduction It is increasingly recognized that epigenetic modifications play an important role in the process of tumorigenesis through the regulation of gene transcription (Bird, 2002). DNA methylation is one of the most important types of epigenetic modifications (Stein, 2011). This modification occurs primarily on cytosine residues of CpG dinucleotides, which are frequently clustered into CpG islands at the regulatory sites of gene promoters (Li et al., 2011a). DNA methylation is dynamically mediated by at least five independent DNA methyltransferases (DNMTs) (Brooks et al., 2010). Given the critical roles of DNMTs in the regulation of cancer cell growth, DNMT inhibitors such as 5-aza-2′-deoxycytidine (5-aza-dC, decitabine) have been widely used in demethylation studies and in clinical practice (Gore, 2010). Abbreviations: DNMT, DNA methyltransferase; 5-aza-dC, 5-aza-2′-deoxycytidine; TSG, tumor suppressor gene; miRNA, microRNA; MSP, methylation specific PCR; TNM, tumor-node-metastasis; FBS, fetal bovine serum; NC, negative control; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; RTCA, real-time cell analyzer; CI, cell index; NCIti, normalized cell index; PBS, phosphate buffered saline; PI, propidium iodide; RT, reverse transcription; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; Ct, threshold cycle; RIPA, radioimmunoprecipitation; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel; PVDF, polyvinylidene fluoride; CDK, cyclin-dependent kinase; VEGF, vascular endothelial growth factor; SPSS, Statistical Program for Social Sciences; miR-195, microRNA-195; miR-378, microRNA-378; 3′-UTR, 3′-untranslated region. ⁎ Corresponding author. Tel.: +86 574 87600758; fax: +86 574 87608638. E-mail address: [email protected] (J. Guo). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.12.103
Hypermethylation of human tumor suppressor genes (TSGs) may lead to their transcriptional inactivation and contribute to carcinogenesis (Laird, 2003). MicroRNAs (miRNAs) are a class of short, highly conserved noncoding RNAs (Zhou et al., 2010). miRNAs play essential roles in cell growth, differentiation and apoptosis, as well as carcinogenesis by regulating the expression of target mRNAs. Thus, in the context of over-expression or aberrant repression, miRNAs may function as either oncogenes or TSGs, respectively (Jiang et al., 2010; Zhou et al., 2010). Interestingly, emerging studies report that the expression of many miRNA genes is associated with promoter methylation (Fabbri et al., 2007; Lujambio et al., 2007). Radpour et al. (2011) revealed that miR-21 and miR-155 were down-regulated or over-expressed in breast cancer cell lines treated with 5-aza-dC, respectively. A study by Hiroki et al. (2012) also showed that miR-34b expression was restored by 5-aza-dC treatment in endometrial serous adenocarcinoma cell lines. Gastric cancer is the most common malignant tumor of the digestive system (Li et al., 2012), however the mechanisms underlying its pathogenesis remain incompletely understood. Dysregulation of miRNA expression caused by genetic and epigenetic alterations is considered to play important roles in gastric cancer development (Guo et al., 2009; Jiang et al., 2010). Previously, we identified seven miRNAs downregulated in gastric cancer compared with normal tissues (Guo et al., 2009). In this work, we investigate the role and relevant mechanisms of these miRNAs in gastric cancer progression. The integrated approach involving bioinformatics, enzyme inhibitors and mimics of miRNAs was
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designed and adopted in several gastric cancer cell lines and normal gastric epithelial cells (Fig. S1). 2. Materials and methods 2.1. Patients and specimens miRNA expression levels were analyzed in surgical tissue specimens obtained from 44 patients with gastric cancer treated at the Affiliated Hospital, Ningbo University School of Medicine or Ningbo Yinzhou People's Hospital (Table S1) after obtaining informed, written consent. Non-cancerous tissues from 17 randomly selected patients were used as controls. All non-cancerous tissue specimens were located 5 cm from the tumor and no cancer cells were observed, as evaluated by an experienced pathologist. Tumors were staged according to the tumor-node-metastasis (TNM) staging system. Histological grade was assessed according to the National Comprehensive Cancer Network clinical practice guideline of oncology (V.2.2010). No chemotherapy or other treatments were employed at the time tissue samples were obtained. The Human Research Ethics Committee from Ningbo University approved all aspects of this study. 2.2. Cell culture The human gastric cancer cell lines MGC-803, SGC-7901 and AGS were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The human, normal gastric epithelial cell line, GES-1, was purchased from the Cancer Institute and Hospital, Chinese Academy of Medical Sciences (Beijing, China). Cells were cultured in flasks at 37 °C in a humidified atmosphere of 5% CO2 with RPMI 1640 Medium (Invitrogen, Grand Island, NY) containing 10% fetal bovine serum (FBS) with 50 U/ml penicillin and 50 U/ml streptomycin. Log phase cells were used for experiments. Cells were counted using a hemocytometer. 2.3. 5-Aza-dC treatment Cells were seeded at 2 × 10 5 cells/well in 6-well plates. After 24 h, 5-aza-dC (Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 1 μM or 2 μM. Following 24 or 48 h treatment, cells were collected for cell cycle analysis, reverse transcription polymerase chain reaction (RT-PCR), or western blot.
Science, Mannheim, Germany), as described by the manufacturer. Briefly, the background signal of the culture medium was first determined by incubating E-Plates with 100 μl of RPMI 1640 with 10% FBS at room temperature for 30 min. A total of 8 × 103 cells were seeded per well in E-Plates 96 in a final volume of 100 μl RPMI 1640 and incubated at room temperature for 30 min. Cells were subsequently placed in the RTCA and incubated at 37 °C in 5% CO2. After 24 h, cells were transfected with miRNA mimic or inhibitor. Impedance was measured at 15 min intervals commencing immediately after cell seeding for 72 h. Impedance is represented by the cell index (CI), calculated as follows: CI= (Zi − Z0) / 15 Ω, where Zi is the impedance at an individual time-point; and Z0 is the background impedance. The average CI per time-point and experiment was calculated from a minimum of three wells. Raw CI values were normalized to the time-point of cell adherence. The normalized cell index (NCIti) was calculated as the cell index CIti at a given time-point divided by the cell index CInml_time at the normalized time-point (nml_time), that is NCIti = CIti / CInml_time. 2.7. Cell cycle analysis Gastric cancer cells treated with 5-aza-dC or transfected with miRNA mimic/inhibitor were collected and washed twice with phosphate buffered saline (PBS). After fixation in 75% ice-cold ethanol overnight at − 20 °C, cell pellets were washed twice with ice-cold PBS, and stained with a propidium iodide (PI) solution. Stained cells were maintained on ice in the dark, and analyzed on a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA). Data were analyzed using Cell Quest Pro software (BD Biosciences). 2.8. RNA extraction and reverse transcription Total RNA was extracted from fresh cultured cells or human tissues using Trizol reagent (Invitrogen, Karlsruhe, Germany) following the manufacturer's protocol. The concentration and purity of RNA samples were measured using the SmartSpec Plus spectrophotometer (Bio-Rad, Hercules, CA). The ratio of A260/A280 was used to indicate the purity of total RNA. cDNA was generated using the miScript Reverse Transcription (RT) Kit (Qiagen GmbH, Hilden, Germany) by combining 6 μl total RNA, 1 μl miScript Reverse Transcriptase Mix, and 4 μl miScript RT buffer and incubating for 60 min at 37 °C, followed by 5 min at 95 °C, in accordance with the manufacturer's protocol (Cheng et al., 2012). cDNA was then diluted with 80 μl of autoclaved-distilled water. 2.9. Real-time PCR
2.4. Transient transfection Cells were seeded at 2 × 10 5 cells/well in 6-well plates. When cells reached 40–60% confluence, 0, 50, 100, and 150 nM miRNA mimic/ inhibitor, or 150 nM negative control (NC) was transfected using Lipofectamine 2000 reagent (Invitrogen, Karlsruhe, Germany) following the manufacturer's protocol. The specific 2′-methoxy-modified RNA oligonucleotides (Shanghai GenePharma Co., Shanghai, China), were used as miRNA mimic and inhibitor (Jiang et al., 2010) (Table S2). 2.5. MTT assay The effect of 5-aza-dC or miRNA mimic/inhibitor treatment on gastric cancer and normal gastric epithelial cell growth was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay as previously described (Jiang et al., 2010).
Real-time PCR was performed using the miScript SYBR Green PCR Kit (Qiagen). PCR was performed in a 20 μl reaction volume containing 6 μl cDNA product, 10 μl 2× QuantiTect SYBR Green PCR Master Mix, 1 μl 10× miScript Universal Primer, 1 μl 10× miScript Primer Assay (for miRNAs, Qiagen) and 2 μl autoclaved-distilled water. Amplification was performed using an MX3005P QPCR System (Stratagene, La Jolla, CA) with the following cycling conditions; 95 °C for 15 min, followed by 40 cycles of 94 °C for 15 s, 60 °C for 30 s, and 70 °C for 30 s. Levels of U6 small RNA were determined with the Hs_RNU6B_2 miScript Primer Assay (Qiagen) and used for normalizing the levels of miRNAs (Jiang et al., 2010). The ΔCt method was used to calculate miRNA expression levels (Jiang et al., 2010). All experiments were repeated in biological duplicate. The sequences of primers are listed in Table S3. 2.10. Western blot analysis
2.6. Real-time analysis of cell proliferation Proliferation assays were performed in E-Plates 96 (Roche Applied Science, Mannheim, Germany) using a Roche DP real-time cell analyzer (RTCA), an impedance-based xCELLigence System (Roche Applied
Cells treated with 5-aza-dC or miRNA mimic/inhibitor, were lysed with radioimmunoprecipitation (RIPA) buffer. Lysates were clarified by centrifugation at 16,000 rpm for 20 min at 4 °C, boiled, separated by a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and
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transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Bedford, MA). Membranes were blocked with 5% skim milk powder for 1 h and incubated with rabbit anti-human cyclindependent kinase (CDK)1, CDK2, CDK6, cyclin A, cyclin D1 or vascular endothelial growth factor (VEGF) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight and subsequently incubated with alkaline phosphatase conjugated goat anti-rabbit antibody (Boster, Wuhan, China) at room temperature for 1 h. β-Actin (Sigma-Aldrich, Saint Louis, MO) was used as a normalization control for protein loading.
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line, and AGS and MGC-803, low differential gastric cancer cell lines) compared with normal gastric epithelial cells (GES-1), and in a larger panel of primary gastric cancer tissues compared with non-cancerous tissues by real-time PCR. As shown in Fig. 1B, the level of miR-195 and miR-378 was significantly lower in all gastric cancer cells and gastric cancer tissues compared with normal gastric epithelial cells (GES-1) and non-cancerous tissues, respectively. We also found that the level of miR-195 was decreased in low differential gastric cancer cell lines, AGS and MGC-803, compared with SGC-7901, the middle differential gastric cancer cell lines. The level of miR-378 in MGC-803 cells was also lower compared with SGC-7901 cells (Fig. 1B).
2.11. 3′-UTR reporter assay To study the targets of these miRNAs, we investigated the predicted binding site of miRNAs and the mRNA 3′-untranslated region (UTR) using the two most widely used target prediction databases: PicTar (http://www.mdc-berlin.de/en/research/research_teams/systems_ biology_of_gene_regulatory_elements/projects/pictar/) and microRNA targets (http://www.microrna.org/microrna/home.do). A fragment of the wild-type (wt) mRNA 3′-UTR containing the predicted miRNA binding site was amplified by RT-PCR. The mutation-type (mt) mRNA 3′-UTR was created by site-directed mutagenesis using primers listed in Table S4. The target mRNA 3′-UTR was cloned into the Psicheck 2 dual luciferase reporter vector (Promega). Human embryonic kidney (HEK) 293T or MGC-803 cells were co-transfected with reporter constructs and miRNA mimic/NC. Cells were lysed 48 h post after transfection and the ratio of Renilla to firefly luciferase activity was measured using the dual luciferase assay kit (Promega). Normalized Renilla to firefly luciferase activity ratios were determined in the presence or absence of miRNA inhibition. 2.12. Statistical analysis Statistical analysis was performed using the Statistical Program for Social Sciences (SPSS) software 17.0 (SPSS Inc., Chicago, IL). All data represent as the mean± standard deviation (SD). Differences between experimental groups and control groups were analyzed using the two-tailed Student's t-test. Statistical significance was accepted at P b 0.05. 3. Results 3.1. Presence of CpG islands in the promoters of miR-378 and miR-195 Previously, we identified seven miRNAs (miR-768-3p, miR-139-5p, miR-378, miR-31, miR-195, miR-497 and miR-133b) down-regulated in gastric cancer tissues compared with non-tumor tissues (Guo et al., 2009). Down-regulation of miRNAs in tumors is often due to aberrant DNA hypermethylation of gene promoters (Brooks et al., 2010; Li et al., 2011a), therefore in this study, we focused on investigating the epigenetic regulation of candidate miRNA genes. To identify the existence of CpG islands in the promoters of miRNAs down-regulated in gastric cancer, we analyzed the sequence 5 kb upstream of these seven miRNA genes using CpGplot software (http://www.ebi.ac.uk/ emboss/cpgplot/) (Previti et al., 2009). As shown in Fig. 1A, we identified one and two CpG island(s) in the promoters of miR-378 and miR-195, respectively. 3.2. Expression of miR-195 and miR-378 is significantly down-regulated in gastric cancer cells and gastric cancer tissues In our previous study, we showed that miR-195 and miR-378 were down-regulated in gastric cancer tissues by microarray analysis (Guo et al., 2009). To further investigate the expression of miR-195 and miR-378 in gastric cancer, we assessed their levels in three human gastric cancer cell lines (SGC-7901, a middle differential gastric cancer cell
3.3. Expression of miR-195 and miR-378 is restored in gastric cancer cells treated with DNMT inhibitor To investigate the role of DNA methylation in the regulation of miR-195 and miR-378, we assessed the expression of these miRNAs in MGC-803 cells following treatment with 5-aza-dC (1 or 2 μM). We observed a dose-dependent up-regulation of miR-195 and miR-378 expression in 5-aza-dC treated cells compared with controls (Fig. 1C). Previously, we identified seven miRNAs (including miR-195 and miR-378) down-regulated in gastric cancer tissues (Guo et al., 2009), therefore we next investigated whether the expression of these other miRNAs was induced by treatment with DNMT inhibitor. miR-768-3p was not studied since this miRNA has been removed from the miRNA database (http://www.mirbase.org/cgi-bin/query.pl?terms=miR-768). In contrast to miR-195 and miR-378, however, the expression of the four remaining miRNAs (miR-139-5p, miR-133b, miR-31, and miR-497), which do not contain promoter CpG islands, was not restored by 5-aza-dC (Fig. 1D). 3.4. Treatment with low doses of 5-aza-dC is not toxic to gastric cancer cells Since the expression of miR-195 and miR-378 was shown to be upregulated in gastric cancer cells treated with 5-aza-dC (Fig. 1C), we next examined the growth of MGC-803 gastric cancer cells following DNMT inhibitor treatment. MGC-803 cell growth was not significantly inhibited following exposure to 5-aza-dC for 24, 48 and 72 h (Fig. 2A). These results indicate that at low concentrations (1 or 2 μM), 5-aza-dC has no toxic effect on gastric cancer cells. Next, we monitored the changes in cell cycle distribution by flow cytometry. Treatment of MGC-803 and SGC-7901 gastric cancer cells with 5-aza-dC for 24 h or 48 h led to cell cycle arrest at the G2/M phase or S phase, respectively (Table 1). Changes in the expression of cell cycle regulators such as cyclins and CDKs, were consistent with these changes in cell cycle distribution. As shown in Figs. 2B and C, treatment with 5-aza-dC led to a significant increase in CDK1, CDK2 and cyclin A protein levels. 3.5. Growth of gastric cancer cells is affected by modulation of miR-195/miR-378 As miR-195 and miR-378 were shown to be down-regulated in gastric cancer tissues (Guo et al., 2009, Fig. 1B) and gastric cancer cells (Fig. 1B), we next examined their possible tumor suppressor activities using two independent methods, RTCA and MTT assay. We first examined the effect of increasing the cellular level of miR-195 and miR-378 in gastric cancer cells (MGC-803, SGC-7901 and AGS) and normal gastric epithelial GES-1 cells, by transfection of mimics. The effects on cell growth were detected by RTCA, a novel real-time cell growth analyzer. As shown in Fig. S2, gastric cancer cell growth was significantly inhibited following introduction of miR-195 or miR-378 mimics, while the growth of normal gastric epithelial cells was promoted by miR-195 mimic. To confirm the effects of miR-195 and miR-378 mimics on gastric cancer cell growth, we next assessed cell growth by MTT assay. Following transfection with miR-195 or miR-378 mimics for 24, 48 and 72 h,
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the growth of gastric cancer MGC-803 (Figs. 3A and D) and AGS (Figs. 3B and E) cells was significantly inhibited in a dose-dependent manner. In contrast, the growth of GES-1 cells was promoted (Figs. 3C and F). To confirm successful transfection of miR-195 and miR-378 mimics into cells, we measured the level of miR-195 and miR-378 in MGC-803 cells by real time PCR. We found that their levels were elevated compared with negative control (NC) treated cells (Fig. S3). We next examined the effect of inhibiting miR-195 and miR-378 on both gastric cancer and normal gastric epithelial cell growth. Gastric cancer cells (AGS and SGC-7901) and normal gastric epithelial cells (GES-1) were transfected with miR-195 or miR-378 inhibitors or NC miRNA inhibitor, and the effect on cell growth was assessed by RTCA.
As shown in Fig. S4, the growth of normal gastric epithelial GES-1 cells was significantly impaired by miR-195 or miR-378 inhibitors. As expected, the growth of both SGC-7901 and AGS cells, in which the basal levels of miR-195 and miR-378 were very low (Fig. 1B), was not significantly affected by treatment with miR-195 or miR-378 inhibitors (Fig. S4). 3.6. Effect of miR-195 or miR-378 mimics on cell cycle distribution of gastric cancer cells To further investigate the growth suppressive mechanisms of miR-195 or miR-378 mimics on gastric cancer cells, we monitored changes in the cell cycle distribution by flow cytometry. Treatment
Fig. 1. Expression and methylation analysis of miRNAs in gastric cancer. (A) Bioinformatic analysis identified CpG islands in the promoters of miR-195 and miR-378. (B) miR-195 and miR-378 expression was down-regulated in gastric cancer tissues and cells. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to detect miRNA expression in tissues, normal gastric epithelial cells (GES-1) and gastric cancer cell lines (MGC-803, SGC-7901 and AGS). Comparison with non-cancerous tissues or GES-1 cells, ⁎P b 0.001; comparison with SGC-7901, #P b 0.001. Data represent the mean of three independent experiments. 5-Aza-dC, a DNMT inhibitor, increased the expression of miR-195 and miR-378 in MGC-803 gastric cancer cells (C), but did not affect the expression of miRNAs lacking CpG islands in their gene promoters (D). Cells were treated with 0, 1 or 2 μM 5-aza-dC for 48 h. The expression levels of miR-195 and miR-378 were detected by RT-PCR. Comparison with control (0 μM), ⁎P b 0.05.
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4. Discussion
Fig. 1 (continued).
of MGC-803 and AGS gastric cancer cells with miR-195 and miR-378 mimics led to an arrest at the G0/G1 and G2/M phases, respectively (Table 2). These results were generally consistent with those following treatment with 5-aza-dC (Table 1), with minor differences. This may be owing to differences in the increase in miR-195 and miR-378 levels induced by 5-aza-dC treatment and transfection with miRNA mimics. For example, transfection of cells with 100 nM miR-195/miR-378 mimics led to a >300-fold increase in miR-195 and miR-378 levels (Fig. S3). In contrast, treatment of cells with 2 μM 5-aza-dC, led to an increase in miR-195 and miR-378 levels of less than 80-fold (Fig. 1C). Nevertheless, the effects of 5-aza-dC on gastric cancer were not limited to the gene expression of miRNAs.
3.7. Expression of cancer-related miR-195 or miR-378 targets is suppressed by miR-195 or miR-378 mimics In order to confirm the putative tumor suppressor activities of miR-195 and miR-378 in gastric cancer, we studied the expression changes of their targets. Recently, CDK6 and VEGF were identified as targets of miR-195 (Wang et al., 2012) and miR-378 (Hua et al., 2006), respectively, in other cancer types. As a result, we focused on these targets in this study. As shown in Fig. 4, miR-195 and miR-378 mimics suppressed the expression of CDK6 and VEGF, respectively.
3.8. miR-195 is a negative regulator of CDK6 expression To understand the mechanism by which miR-195 regulates CDK6 expression, we searched for potential targeted sequences in 3′-UTR of CDK6 mRNA using two software programs, PicTar and miRanda. The result showed that two binding sites of miR-195 were found in 3′-UTR of CDK6 mRNA (Fig. S5). To further validate this bioinformatic finding, we assessed the expression of 3′-UTR of CDK6 mRNA in luciferase reporter assays. Sequencing results confirmed that the vectors of 3′-UTR reporter assay were successfully constructed (Fig. S6). The expression of the CDK6 reporter was significantly reduced to ≤50% in miR-195 mimic-transfected MGC-803 cells, however this effect was abolished using the CDK6 mutation-type reporter (Fig. 5).
In recent years, a number of miRNAs were shown to be expressed at low levels in human tumors, and many of these miRNAs may function as tumor suppressors (Nie et al., 2012; Ugras et al., 2011). DNA methylation in promoter regions of these miRNA genes represents one possible mechanism for their loss of tumor suppressor function in cancer cells (Wong et al., 2011). Indeed, several down-regulated miRNAs that act as tumor suppressors have been confirmed to be associated with DNA methylation (Lopez-Serra and Esteller, 2012; Yu et al., 2012). DNA methylation primarily occurs on cytosine residues of CpG dinucleotides, especially on CpG islands in the gene promoter regions (Deaton and Bird, 2011; Fabbri and Calin, 2010). 5-aza-dC, a DNMT inhibitor, has been widely used to study DNA methylation (Kulis and Esteller, 2010). Recently, miR-195 expression was shown to be down-regulated in various cancers, including bladder cancer (Han et al., 2011), colorectal cancer (Wang et al., 2012), hepatocellular cancer (Xu et al., 2009) and breast cancer (Li et al., 2011b). miR-378 was shown to be downregulated in colorectal cancer (Mosakhani et al., 2012) and oral cancer (Scapoli et al., 2010). Previously, we reported seven miRNAs, including miR-195 and miR-378, to be down-regulated in gastric cancer (Guo et al., 2009). In this study, we extended this analysis to show that the promoter regions of miR-195 and miR-378 contained CpG islands (Fig. 1A). Furthermore, we confirmed that miR-195 and miR-378 were significantly down-regulated not only in gastric cancer tissues (Guo et al., 2009), but also in human gastric cancer cell lines (Fig. 1B). Although we and others, have previously shown that miR-195 and miR-378 are expressed at low levels in gastric cancer (Guo et al., 2009; Liu et al., 2012; Wu et al., 2011; Yao et al., 2009), the mechanism underlying their down-regulation and their roles in carcinogenesis remain unclear. Our data suggest that the expression of both miR-195 and miR-378 is regulated by DNA methylation in their upstream promoters (Fig. 1). Recent studies showed that the promoters of several miRNAs, including miR-34a (Lodygin et al., 2008), miR-34c (Wong et al., 2011), miR-200c (Neves et al., 2010) and miR-1 (Datta et al., 2008), contain CpG islands, which are methylated. To understand whether CpG island methylation is responsible for the silencing of miR-195 and miR-378 in gastric cancer, we treated gastric cancer cells with 5-aza-dC, a demethylation reagent, and found that it restored the expression of miR-195 and miR-378 (Fig. 1C). However, the expression of other miRNAs lacking promoter CpG islands, was not restored by 5-aza-dC treatment (Fig. 1C). This implies that CpG island methylation of the miR-195 and miR-378 gene promoters plays a central role in the reduction of their expression in gastric cancer. The regulation of gene expression is very complicated, however, and those miRNAs lacking promoter CpG islands may be regulated by other mechanisms, which also warrant further study. A previous study showed that treatment of breast cancer MDAMB-435S cells with 5 μM of 5-aza-dC inhibited cell growth (Zhang et al., 2007). In order to prevent the toxicity of 5-aza-dC on gastric cells and demonstrate its inhibitory activity on methyltransferase, we treated cells with low doses (1 μM or 2 μM), and observed no toxic effects at these concentrations (Fig. 2A). Our data results demonstrate that low doses of 5-aza-dC arrest gastric cancer cells at the G2/M or S phase (Table 1). Furthermore, low doses of 5-aza-dC enhanced the expression of certain cell cycle-related proteins (Figs. 2B and C). Taken together, this suggests that the effects of low dose treatment of gastric cancer cells with 5-aza-dC may occur through the elevation of specific miRNAs, which may function as TSGs. Cell cycle progression is a highly regulated process and is controlled by the sequential activation and inactivation of families of the CDKs and cyclins. CDK1 combined with cyclin A or cyclin B, plays an essential role in S and G2/M phase transitions of the eukaryotic cell cycle (Dorée and Hunt, 2002). CDK2 with cyclin A or cyclin E, restricts cells at the G1/S phase, and is essential for G1 phase to S phase transition (Chen et al.,
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Fig. 2. The effect of 5-aza-dC on gastric cancer cell growth. (A) Cell growth was measured by MTT assay. Cells were treated with 1 or 2 μM of 5-aza-dC for 24, 48 and 72 h, respectively. Three independent experiments were performed. Western blot analysis of cell cycle-related proteins in MGC-803 (B) and SGC-7901 (C). Cells were treated with 1 or 2 μM of 5-aza-dC for 48 h. Western blot analysis was performed with antibodies recognizing human CDK1, CDK2, cyclin A and cyclin D1. β-Actin was used as a loading control. A representative result (left) and results from three independent experiments (right) are shown. Comparison with control, *P b 0.05.
Table 1 Analysis of cell cycle distribution in gastric cancer cells treated with 5-aza-dC (%). Treatment time
Cell line
5-Aza-dC (μM)
G0/G1
S
G2/M
24 h
MGC-803
0 1 2 0 1 2 0 1 2 0 1 2
36.16 ± 1.36 32.14 ± 0.18 32.20 ± 2.52 54.33 ± 0.18 48.68 ± 0.49⁎ 46.56 ± 0.26⁎
38.91 ± 3.16 36.60 ± 1.24 31.14 ± 1.87 30.63 ± 0.69 31.15 ± 1.70 29.69 ± 1.39 32.45 ± 0.37 47.65 ± 0.25⁎ 47.89 ± 1.79⁎ 32.6 ± 0.25 50.26 ± 0.12⁎ 57.25 ± 0.09⁎
24.93 ± 1.81 31.26 ± 1.06⁎ 36.67 ± 0.65⁎ 15.06 ± 0.51 20.19 ± 2.18 23.75 ± 1.13⁎
SGC-7901
48 h
MGC-803
SGC-7901
35.76 ± 0.40 20.79 ± 0.49⁎ 19.25 ± 0.01# 60.37 ± 1.32 49.74 ± 0.43 40.38 ± 0.05
Comparison with control treated cells (0 μM), ⁎P b 0.05, #P b 0.01.
31.79 ± 0.77 31.57 ± 0.29 32.86 ± 1.78 7.03 ± 1.82 0.00 ± 0.25 2.37 ± 0.19
2003). Cyclin D1 also regulates cell cycle checkpoints by binding its catalytic partners, CDK4 or CDK6 (Vodermaier, 2004). Cell proliferation and cell cycle progression are closely related to these proteins. To clarify the mechanism(s) by which miR-195 and miR-378 suppress gastric cancer cell growth, we studied the expression of several cell cycle associated proteins including CDK1, CDK2, cyclin A and cyclin D1 (Fig. 2). In these experiments, we found that the ectopic expression of miR-195 and miR-378 was capable of inhibiting the proliferation of gastric cancer cells, while promoting the proliferation of normal gastric epithelial cells (Figs. 3 and S2). In keeping with this finding, treatment with miR-195 and miR-378 inhibitors suppressed the growth of normal gastric epithelial cells, but had a minimal effect on the growth of gastric cancer cells (Fig. S4). This is likely owing to the low basal expression of miR-195 and miR-378 in gastric cancer cells (Fig. 1B). Since the expression of these miRNAs was already low, the growth of gastric cancer cells was not significantly altered following treatment with miR-195 and miR-378 inhibitors. In order to investigate the mechanisms underlying the growth inhibitory effects of miR-195 and miR-378 on gastric cancer
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Fig. 3. The effect of miR-195 or miR-378 mimics on the growth of gastric cancer and normal gastric epithelial cells. The effect of miR-195 mimic on MGC-803 (A), AGS (B) or GES-1 (C). The effect of miR-378 mimic on MGC-803 (D), AGS (E) or GES-1 (F). Cells were treated with 50 nM, 100 nM or 150 nM of miR-195 or miR-378 mimics or 150 nM negative control (NC) for 24, 48 or 72 h, respectively. Cell growth was measured by MTT assay. Data represent the mean of three independent experiments. Comparison with NC, *P b 0.05.
cells, we monitored the cell cycle distributions in gastric cancer cells transfected with miR-195 and miR-378 mimics. We observed that cancer cells were arrested in G0/G1 or G2/M phase following treatment
Table 2 Cell cycle analysis of gastric cancer cells transfected with miRNA mimics (%). Mimic
Cell line
Concentration G0/G1 (nM)
miR-195 MGC-803 NC 50 150 AGS NC 50 150 miR-378 MGC-803 NC 50 150 AGS NC 50 150
S
39.70 ± 0.50 47.53 ± 1.12⁎ 45.63 ± 0.41⁎ 43.09 ± 0.63 50.51 ± 0.93⁎
28.78 ± 0.50 27.33 ± 1.15 26.45 ± 0.88 48.30 ± 1.56 33.64 ± 1.87⁎ 53.28 ± 0.43# 29.51 ± 0.55# 39.70 ± 0.50 28.78 ± 0.50 39.12 ± 0.08 27.03 ± 0.35 27.03 ± 0.25⁎ 33.75 ± 1.33 43.09 ± 0.63 48.30 ± 1.56 47.34 ± 0.32⁎ 33.87 ± 1.06⁎ 42.11 ± 2.18 39.14 ± 0.74⁎
Comparison with negative control (NC), ⁎P b 0.05, #P b 0.01.
G2/M 31.53 ± 0.99 25.15 ± 0.02 27.93 ± 0.47 8.61 ± 2.18 15.86 ± 0.95 17.22 ± 0.12 31.53 ± 0.99 33.86 ± 0.26 39.23 ± 1.58⁎ 8.61 ± 2.18 18.80 ± 0.75⁎ 18.75 ± 1.44⁎
with miR-195 or miR-378 mimics, respectively (Table 2). Based on these data, we postulate that miR-195 and miR-378 have possible tumor suppressor roles in gastric cancer. Furthermore, we confirmed that CDK6 and VEGF are regulated by miR-195 and miR-378, respectively (Fig. 4). CDK6 is a key element of D-type cyclin holoenzymes, and is involved in regulation of the G1/S phase transition of the cell cycle (Liu et al., 2011). CDK6 has been shown to be over-expressed in gastric cancer and many other cancers (Zhang et al., 2009). Recently, CDK6 was found to be a target of several miRNAs including miR-129 (Wu et al., 2010), miR-22 (Xu et al., 2011) and miR-195 (Wang et al., 2012). One study showed that a decrease in CDK6 blocks gastric cancer cells in the G0/G1 phase (Feng et al., 2012). In our study, we found that over-expression of a miR-195 mimic inhibited the expression of CDK6 in gastric cancer cells (Fig. 4A), and arrested cells in the G0/G1 phase (Table 2). These results are in accordance with the tumor suppressor function of miR-195 (Fig. 3). We suspect that the growth inhibitory mechanism of miR-195 in gastric cancer cells is associated with the down-regulation of CDK6, and cell cycle arrest. Indeed, we confirmed CDK6 as a target of miR-195 using luciferase reporter assays (Fig. 5). VEGF plays a role in various processes, including angiogenesis, promoting cell migration and inhibiting
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Fig. 4. Western blot analysis of the expression level changes of miR-195 or miR-378 mimics on their targets. Various concentrations of miR-195 mimic (50, 100 and 150 nm) or 150 nM negative control (NC) on the expression of CDK6 in MGC-803 cells (A). Various concentrations of miR-378 mimic (50, 100 and 150 nm) or 150 nM negative control (NC) on the expression of VEGF in MGC-803 (B) and AGS (C) cells. β-Actin was used as loading control. A representative result (up) and results from three independent experiments (down) were shown. Comparison with NC, *P b 0.05.
apoptosis. Recently, VEGF was shown to be a target of miR-206 (Zhang et al., 2011) and miR-378 (Hua et al., 2006) in laryngeal and nasopharyngeal cancers. However, the relationship between miRNAs and VEGF has not been investigated in gastric cancer. Here, we present the first report that VEGF is regulated by miR-378 in gastric cancer (Figs. 4B and C). In summary, using a combination of several gastric cancer and normal gastric epithelial cell lines as models, and by modulating the levels of cellular miR-195 and miR-378 levels, we demonstrate that the down-regulation of miR-195 and miR-378 in gastric cancer is a result of promoter CpG island methylation. We also show that miR-195 and miR-378 may act as tumor suppressors in gastric cancer by negatively regulating the expression of CDK6 and VEGF, respectively. Taken
together, these data indicate that miR-195 and miR-378 may be potential therapeutic targets in gastric cancer therapy. Acknowledgments This work was supported by the Excellent Dissertation Fund of Ningbo University (No. PY201014 and PY20110020); College Students' Science–Technology Innovation Program of Zhejiang Province (No. 200959); the Scientific Innovation Team Project of Ningbo (No. 2011B82014); Ningbo Natural Science Foundation (No. 2010A610044 and No. 2012A610207); Zhejiang Provincial Research Project (No. 2010C33112 and No. 2012C23127); National Natural Science Foundation
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Fig. 5. miR-195 directly targets CDK6. miR-195 suppressed the expression of a luciferase vector containing the 3′-UTR of CDK6 mRNA. A luciferase expression vector with the 3′-UTR of CDK6 mRNA and miR-195 mimic or NC (blank) was co-transfected into MGC-803 cells. Cells were harvested after 48 h and assayed for luciferase activity using the dual luciferase assay kit (Promega). Firefly luciferase was used for normalization. Data represent the mean of three independent experiments. Comparison with blank, *Pb 0.05. luc-wt1, the vector CDK6-wild type (wt)-1 contained the binding site of miR-195 (120–126: TGCTGCT); luc-mt1, the mutant (mt) of luc-wt1 (Fig. S6). luc-wt2, the vector CDK6-wt-2 contained the binding site of miR-195 (117–123: TGCTGCT); luc-mt2, the mutant (mt) of luc-wt2 (Fig. S6).
of China (No. 81171660); the Medical Science–Technology Project of Ningbo (No. 2010B06); and KC Wong Magna Fund of Ningbo University. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.gene.2012.12.103. References Bird, A., 2002. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21. Brooks, W.H., Le Dantec, C., Pers, J.O., Youinou, P., Renaudineau, Y., 2010. Epigenetics and autoimmunity. J. Autoimmun. 34, J207–J219. Chen, J.H., Tseng, T.H., Ho, Y.C., Lin, H.H., Lin, W.L., Wang, C.J., 2003. Gaseous nitrogen oxides stimulate cell cycle progression by retinoblastoma phosphorylation via activation of cyclins/Cdks (correction). Toxicol. Sci. 76, 83–90. Cheng, J., et al., 2012. piR-823, a novel non-coding small RNA, demonstrates in vitro and in vivo tumor suppressive activity in human gastric cancer cells. Cancer Lett. 315, 12–17. Datta, J., et al., 2008. Methylation mediated silencing of MicroRNA-1 gene and its role in hepatocellular carcinogenesis. Cancer Res. 68, 5049–5058. Deaton, A.M., Bird, A., 2011. CpG islands and the regulation of transcription. Genes Dev. 25, 1010–1022. Dorée, M., Hunt, T., 2002. From Cdc2 to Cdk1: when did the cell cycle kinase join its cyclin partner? J. Cell Sci. 115, 2461–2464. Fabbri, M., Calin, G.A., 2010. Epigenetics and miRNAs in human cancer. Adv. Genet. 70, 87–99. Fabbri, M., Ivan, M., Cimmino, A., Negrini, M., Calin, G.A., 2007. Regulatory mechanisms of microRNAs involvement in cancer. Expert Opin. Biol. Ther. 7, 1009–1019. Feng, L., Xie, Y., Zhang, H., Wu, Y., 2012. miR-107 targets cyclin-dependent kinase 6 expression, induces cell cycle G1 arrest and inhibits invasion in gastric cancer cells. Med. Oncol. 29, 856–863. Gore, S.D., 2010. Combination therapy with DNA methyltransferase inhibitors in hematologic malignancies. Nat. Clin. Pract. Oncol. 2, S30–S35. Guo, J., et al., 2009. Differential expression of microRNA species in human gastric cancer versus non-tumorous tissues. J. Gastroenterol. Hepatol. 24, 652–657. Han, Y., et al., 2011. MicroRNA expression signatures of bladder cancer revealed by deep sequencing. PLoS One 6, e18286. Hiroki, E., et al., 2012. MicroRNA-34b functions as a potential tumor suppressor in endometrial serous adenocarcinoma. Int. J. Cancer 131, E395–E404.
359
Hua, Z., et al., 2006. MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia. PLoS One 1, e116. Jiang, Z., et al., 2010. Increased expression of miR-421 in human gastric carcinoma and its clinical association. J. Gastroenterol. 45, 17–23. Kulis, M., Esteller, M., 2010. DNA methylation and cancer. Adv. Genet. 70, 27–56. Laird, P.W., 2003. The power and the promise of DNA methylation markers. Nat. Rev. Cancer 3, 253–266. Li, Y., Daniel, M., Tollefsbol, T.O., 2011a. Epigenetic regulation of caloric restriction in aging. BMC Med. 9, 98. Li, D., et al., 2011b. Analysis of MiR-195 and MiR-497 expression, regulation and role in breast cancer. Clin. Cancer Res. 17, 1722–1730. Li, L.Z., et al., 2012. Growth inhibitory effect of 4-phenyl butyric acid on human gastric cancer cells is associated with cell cycle arrest. World J. Gastroenterol. 18, 79–83. Liu, Y.F., Zan, L.S., Cui, W.T., Xin, Y.P., Jiao, Y., Li, K., 2011. Molecular cloning, characterization and association analysis of the promoter region of the bovine CDK6 gene. Genet. Mol. Res. 10, 1777–1786. Liu, H., et al., 2012. Genome-wide microRNA profiles identify miR-378 as a serum biomarker for early detection of gastric cancer. Cancer Lett. 316, 196–203. Lodygin, D., et al., 2008. Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer. Cell Cycle 7, 2591–2600. Lopez-Serra, P., Esteller, M., 2012. DNA methylation-associated silencing of tumorsuppressor microRNAs in cancer. Oncogene 31, 1609–1622. Lujambio, A., et al., 2007. Genetic unmasking of an epigenetically silenced microRNA in human cancer cells. Cancer Res. 67, 1424–1429. Mosakhani, N., et al., 2012. MicroRNA profiling differentiates colorectal cancer according to KRAS status. Genes Chromosomes Cancer 51, 1–9. Neves, R., et al., 2010. Role of DNA methylation in miR-200c/141 cluster silencing in invasive breast cancer cells. BMC Res. Notes 3, 219. Nie, J., et al., 2012. MicroRNA-365, down-regulated in colon cancer, inhibits cell cycle progression and promotes apoptosis of colon cancer cells by probably targeting Cyclin D1 and Bcl-2. Carcinogenesis 33, 220–225. Previti, C., Harari, O., Zwir, I., del Val, C., 2009. Profile analysis and prediction of tissuespecific CpG island methylation classes. BMC Bioinformatics 10, 116. Radpour, R., et al., 2011. Integrated epigenetics of human breast cancer: synoptic investigation of targeted genes, microRNAs and proteins upon demethylation treatment. PLoS One 6, e27355. Scapoli, L., et al., 2010. MicroRNA expression profiling of oral carcinoma identifies new markers of tumor progression. Int. J. Immunopathol. Pharmacol. 23, 1229–1234. Stein, R.A., 2011. DNA methylation profiling: a promising tool and a long road ahead for clinical applications. Int. J. Clin. Pract. 65, 1212–1213. Ugras, S., et al., 2011. Small RNA sequencing and functional characterization reveals MicroRNA-143 tumor suppressor activity in liposarcoma. Cancer Res. 71, 5659–5669. Vodermaier, H.C., 2004. APC/C and SCF: controlling each other and the cell cycle. Curr. Biol. 14, R787–R796. Wang, X., Wang, J., Ma, H., Zhang, J., Zhou, X., 2012. Downregulation of miR-195 correlates with lymph node metastasis and poor prognosis in colorectal cancer. Med. Oncol. 29, 919–927. Wong, K.Y., Yu, L., Chim, C.S., 2011. DNA methylation of tumor suppressor miRNA genes: a lesson from the miR-34 family. Epigenomics 3, 83–92. Wu, J., et al., 2010. miR-129 regulates cell proliferation by downregulating Cdk6 expression. Cell Cycle 9, 1809–1818. Wu, W.Y., et al., 2011. Potentially predictive microRNAs of gastric cancer with metastasis to lymph node. World J. Gastroenterol. 17, 3645–3651. Xu, T., Zhu, Y., Xiong, Y., Ge, Y.Y., Yun, J.P., Zhuang, S.M., 2009. MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells. Hepatology 50, 113–121. Xu, D., et al., 2011. miR-22 represses cancer progression by inducing cellular senescence. J. Cell Biol. 193, 409–424. Yao, Y., et al., 2009. MicroRNA profiling of human gastric cancer. Mol. Med. Rep. 2, 963–970. Yu, F., et al., 2012. MicroRNA 34c gene down-regulation via DNA methylation promotes self-renewal and epithelial–mesenchymal transition in breast tumor-initiating cells. J. Biol. Chem. 287, 465–473. Zhang, B., Huang, T., Liu, K., Chen, J., Wang, G., 2007. Effects of 5-Aza-CdR on cell proliferation of breast cancer cell line MDA-MB-435S and expression of maspin gene. J. Huazhong Univ. Sci. Technol. Med. Sci. 27, 543–546. Zhang, S., et al., 2009. RhoA regulates G1-S progression of gastric cancer cells by modulation of multiple INK4 family tumor suppressors. Mol. Cancer Res. 7, 570–580. Zhang, T., Liu, M., Wang, C., Lin, C., Sun, Y., Jin, D., 2011. Down-regulation of miR-206 promotes proliferation and invasion of laryngeal cancer by regulating VEGF expression. Anticancer Res. 31, 3859–3863. Zhou, H., et al., 2010. Detection of circulating tumor cells in peripheral blood from patients with gastric cancer using microRNA as a marker. J. Mol. Med. 88, 709–717.