Tumor Biol. (2014) 35:1065–1073 DOI 10.1007/s13277-013-1142-z

RESEARCH ARTICLE

Downregulated long noncoding RNA MEG3 is associated with poor prognosis and promotes cell proliferation in gastric cancer Ming Sun & Rui Xia & Feiyan Jin & Tongpeng Xu & Zhijun Liu & Wei De & Xianghua Liu

Received: 25 July 2013 / Accepted: 23 August 2013 / Published online: 5 September 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract Long noncoding RNAs (lncRNAs) have emerged recently as major players in governing fundamental biological processes, and many of which are altered in expression and likely to have a functional role in tumorigenesis. Maternally expressed gene 3 (MEG3) is an imprinted gene located at 14q32 that encodes a lncRNA associated with various human cancers. However, its biological role and clinical significance in gastric cancer development and progression are unknown. In this study, to investigate the lncRNA MEG3 expression in gastric cancer, quantitative reverse-transcription polymerase chain reaction was conducted. We found that MEG3 levels were markedly decreased in gastric cancer tissues compared with adjacent normal tissues. Its expression level was significantly correlated with TNM stages, depth of invasion, and tumor size. Moreover, patients with low levels of MEG3 expression had a relatively poor prognosis. Furthermore, knockdown of MEG3 expression by siRNA could promote cell proliferation, while ectopic expression of MEG3 inhibited cell proliferation, promoted cell apoptosis, and modulated p53 expression in gastric cancer cell lines. By 5-aza-CdR treatment, we also observed that MEG3 expression can be modulated by DNA methylation. Our findings present that MEG3 downexpression can be identified as a poor prognostic biomarker in gastric cancer and regulate cell proliferation and apoptosis in vitro. Ming Sun and Rui Xia contributed equally to this work and should be regarded as joint first authors. M. Sun : R. Xia : F. Jin : Z. Liu : W. De : X. Liu (*) Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing 210029, Jiangsu, People’s Republic of China e-mail: [email protected] T. Xu Department of Oncology, First Affiliated Hospital, Nanjing Medical University, Nanjing, People’s Republic of China

Keywords Gastric cancer . Long noncoding RNA . MEG3 . Poor prognosis . Proliferation

Introduction Gastric cancer is the second leading cause of cancer death and is the most common gastrointestinal malignancy in East Asia, Eastern Europe, and parts of Central and South America [1]. In most patients, gastric cancer is diagnosed at advanced stage accompanied by malignant proliferation, extensive invasion, and lymphatic metastasis. Gastrectomy remains the mainstay treatment of gastric cancer, but the prognosis for advancedstage patients is still very poor and the mortality is high [2]. Therefore, better understanding of the pathogenesis and identification of the molecular alterations is essential for the development of diagnostic markers that aid novel effective therapies for gastric cancer [3–5]. Recent improvements in genome-wide surveys and highthroughput transcriptome analysis have revealed that human genome contains only ~20,000 protein-coding genes, representing <2 % of the total genome while a substantial fraction of the human genome can be transcripted into many short or long noncoding RNAs (lncRNAs) [6–9]. Up to date, over 3,000 lncRNAs have been identified and well characterized to participate in a large range of biological processes, such as modulation of apoptosis and invasion, marker of cell fate, reprogramming of stem cell pluripotency, and parental imprinting, indicating that they may play a major role in the regulation of eukaryotic genome [10–13]. Moreover, multiple lines of evidences link dysregulation of these lncRNAs to diverse human diseases, especially cancers [14–16]. Therefore, identification of cancer-associated lncRNAs and investigation of their molecular and biological functions are important to understand the molecular biology of tumor and its progression.

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Maternally expressed gene 3 (MEG3) represents a tumorsuppressor gene located in chromosome 14q32 that encodes a noncoding RNA (ncRNA) associated with tumorigenesis [17, 18]. MEG3 RNA is expressed in many normal tissues, while it was lost in an expanding list of primary human tumors including glioma, hepatocellular cancers, meningiomas, and bladder cancer [19–22]. Hypermethylation of promoter or the differentially methylated regions (DMRs) upstream of the MEG3 gene has been found to contribute to the loss of MEG3 expression in tumors [17, 20]. Overexpression of MEG3 could induce cell growth arrest and promote cell apoptosis in human glioma cell lines [19]. However, very little is known about MEG3 expression level and biological role in gastric cancer pathogenesis. In this study, we demonstrated that decreased MEG3 expression is a characteristic molecular change in gastric cancer and investigated the effect of altered MEG3 levels on the phenotypes of gastric cancer cells in vitro. The effect of DNA methylation on MEG3 expression was also investigated. Our findings suggest that lncRNA MEG3 may represent a new marker of poor prognosis and is a potential therapeutic target for gastric cancer intervention.

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RNA extraction and quantitative reverse-transcription polymerase chain reaction analyses Total RNA was extracted from tissues or cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA). For quantitative reverse-transcription polymerase chain reaction (qRT-PCR), RNA was reverse-transcribed to cDNA by using a reverse transcription kit (Takara, Dalian, China). Real-time PCR analyses were performed with Power SYBR Green (Takara, Dalian China). Results were normalized to the expression of GAPDH. The PCR primers for MEG3 or GAPDH were as follows: MEG3 sense, 5′-CTGCCCATCTACACCTCACG-3′ and reverse, 5′-CTCTCCGCCGTCTGCGCTAGGGGCT-3′; GAPDH sense, 5′-GTCAACGGATTTGGTCTGTATT-3′ and reverse, 5′-AGTCTTCTGGGTGGCAGTGAT-3′. qRT-PCR and data collection were performed on ABI 7500.The relative expression of MEG3 was calculated and normalized using the 2−ΔΔCt method relative to GAPDH . Treatment of AGS and MGC803 cells with 5-aza-CdR

Materials and methods

AGS and MGC803 cells (2.5×105) were seeded into a sixwell culture plate on day 0 and exposed to 0, 5, or 10 μM 5aza-CdR (Sigma-Aldrich, USA) for 3 days. The cells treated with 5-aza-CdR were harvested and used for detection of MEG3 expression.

Tissue collection

Plasmid constructs

Seventy-two gastric cancer samples were obtained from patients who had underwent surgery at Jiangsu Province Hospital between 2006 and 2008 and were diagnosed with gastric cancer (stages II, III, and IV; seventh edition of the AJCC Cancer Staging Manual) based on histopathological evaluation. Clinical pathology information was available for all samples (Table 1). No local or systemic treatment was conducted in these patients before the operation. All specimens were immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction. The study was approved by the Research Ethics Committee of Nanjing Medical University, China. Informed consents were obtained from all patients.

The sequence of MEG3 was synthesized and subcloned into pCDNA3.1 (Invitrogen, Shanghai, China) vector. Ectopic expression of MEG3 was achieved by using the pCDNAMEG3 transfection and empty pCDNA vector (empty) was used as a control. The expression level of MEG3 was detected by qPCR.

Cell lines and culture conditions Five gastric cancer cell lines (SGC7901, AGS, MGC803, MKN45, and BGC823) and a normal gastric epithelium cell line (GES-1) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (GIBCOBRL) medium supplemented with 10 % fetal bovine serum (10 % FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen) in humidified air at 37 °C with 5 % CO2.

Transfection of gastric cancer cells All plasmid vectors (pCDNA-MEG3 and empty vector) for transfection were extracted by DNA Midiprep or Midiprep Kit (QIAGEN, Hilden, Germany). Gastric cells cultured on a sixwell plate were transfected with the pCDNA-MEG3, empty vector, si-MEG3 , or si-NC using Lipofectamine 2000 (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. Cells were harvested after 48 h for qRT-PCR and western blot analyses. Target sequence for MEG3 siRNA was as follows: 5′-UUAGGUAAGAGGGACAGCUGGCUGG-3′. Cell proliferation assays A cell proliferation assay was performed with an MTT kit (Sigma, St. Louis, MO) according to the manufacturer’s instruction. Cells were placed into a six-well plate and

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Table 1 Correlation between MEG3 expression and clinicopathological characteristics in gastric cancer Clinical parameter

MEG3 Low-MEG3 group, no. of cases

Chi-squared test P value

Table 1 (continued) Clinical parameter

Low-MEG3 group, no. of cases

High-MEG3 group, no. of cases

Age (years)

0.916

MEG3

Chi-squared test P value High-MEG3 group, no. of cases

Lymphatic metastasis

0.071

<60

18

14

Yes

26

14

>60

22

18

No

14

18

Gender

0.873

Male

23

19

Female

17

13

Location

0.506

Regional lymph nodes PN0

14

18

PN1

6

8

PN2

10

3

10

3

Distal

15

15

PN3

Middle

16

13

Proximal

9

4

Distant metastasis

Size

0.006

<5 cm

11

19

>5 cm

29

13

Histological differentiation

0.972

Well

2

2

Moderately

17

8

Poorly

17

19

Undifferentiated 4

0.037

T1

6

8

T2

8

14

T3

18

6

T4

8

4

TNM stages

0.022

I

3

8

II

12

15

III

20

8

IV

5

1

0.775

Yes

3

3

No

37

29

maintained in media containing 10 % FBS for 2 weeks. Colonies were fixed with methanol and stained with 0.1 % crystal violet (Sigma, St. Louis, MO). Visible colonies were manually counted. Hoechst staining assay

3

Invasion depth

0.057

AGS and MGC803 cells transiently transfected with pCDNAMEG3 or empty vector were cultured in six-well cell culture plates, and Hoechst 33342 (Sigma, St. Louis, MO) was added to the culture medium; changes in nuclear morphology were detected by fluorescence microscopy using a filter for Hoechst 33342 (365 nm). For quantification of Hoechst 33342 staining, the percentage of Hoechst-positive nuclei per optical field (at least 50 fields) was counted. Western blot assay and antibodies Cells protein lysates were separated by 10 % SDS–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22-μm NC membranes (Sigma), and incubated with specific antibodies. ECL chromogenic substrate was used and signals were quantified by densitometry (Quantity One software, BioRad). GAPDH antibody was used as a control; anti-P53 (1:1,000) was purchased from Cell Signaling Technology, Inc. (CST).

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Statistical analysis Statistical analysis was performed using the SPSS software package (version 20.0, SPSS Inc.). Statistical significance was tested by a Student’s t test or a chi-squared test as appropriate. Survival analysis was performed using the Kaplan–Meier method, and the log-rank test was used to compare the differences between patient groups. P values less than 0.05 were considered significant.

Results Expression of MEG3 is downregulated and correlated with poor prognosis of gastric cancer

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frequently decreased in gastric cancer tissues and cells. Hypermethylation of the MEG3 regulatory region, known as MEG3 -DMRs, has been reported to contribute to MEG3 transcriptional inactivation. To examine the role of aberrant methylation in deregulation of MEG3 in gastric cancer cells, we evaluated the effect of a DNA demethylating agent (5-azaCdR) on MEG3 expression. Following treatment of AGS and MGC803 cells with 5-aza-CdR, we found that MEG3 expression was significantly increased by 5.51- or 5.86-fold in 5-azaCdR-treated cells compared with control (Fig. 2b). The results indicate that downregulation of MEG3 observed in gastric cancer cells might have been partly due to hypermethylation of MEG3-DMRs. MEG3 inhibits gastric cancer cell proliferation in vitro

The level of MEG3 was detected in 72 paired gastric cancer samples and adjacent histological normal tissues by qRT-PCR and normalized to GAPDH. In cancerous tissues, MEG3 expression was at a level lower than the average level of normal specimens, with an average expression level of 0.377 compared with normal tissue (Fig. 1a). Examination of the correlation between MEG3 expression and clinical pathological features showed that decreased MEG3 expression was correlated with larger tumor size, advanced pathological stage, and deeper depth of invasion (Fig. 1b, c, Table 1). However, we did not find any association between MEG3 expression levels and other clinical pathological features including patients’ age, gender, lymph node metastasis, etc. (Table 1). Kaplan–Meier survival analysis and log-rank tests using patient postoperative survival were performed to further investigate the correlation between MEG3 expression and gastric cancer patient prognosis. According to the mean ratio of relative MEG3 expression (mean ration of 0.377-fold) in tumor tissues, the 72 gastric cancer patients were classified into two groups: relatively high-MEG3 group (n =36, MEG3 expression ratio≥mean ratio) and relatively low-MEG3 group (n =36, MEG3 expression ratio≤median ratio). With regard to overall survival, patients with lower MEG3 expression had a significantly poorer prognosis than those with higher MEG3 expression (P <0.001, log-rank test) (Fig. 1d). Thus, it was concluded that downregulation of MEG3 might have important roles in gastric cancer development and progression.

The significant decrease of MEG3 expression in gastric cancer samples and cells prompted us to explore the possible biological significance of MEG3 in tumorigenesis. In order to manipulate MEG3 levels in gastric cancer cells, pCDNAMEG3 vector was transfected into AGS and MGC803 cells. qRT-PCR analysis of MEG3 levels was performed 48 h posttransfection and revealed that MEG3 expression was increased 294- and 367-fold in AGS and MGC-803 cells, compared with respective control cells (Fig. 2c). Furthermore, MTT assay revealed that cell growth was significantly impaired in AGS and MGC803 cells transfected with pCDNAMEG3 compared with respective controls (Fig. 3a. b). Similarly, the results of colony formation assay also showed that clonogenic survival was decreased following overexpression of MEG3 in AGS and MGC803 cells (Fig. 3c, d).

MEG3 expression is modulated by DNA methylation

Inhibition of MEG3 promotes gastric cancer cell proliferation

We next performed qRT-PCR analysis to examine the expression levels of MEG3 in five gastric cancer cell lines (SGC7901, AGS, MGC803, MKN45, and MKN28). The results showed that MEG3 expression was significantly downregulated in gastric cancer cells when compared with the normal gastric epithelium cell line GES-1 (Fig. 2a). Taken together, our data suggested that MEG3 expression may be

To determine whether inhibition of MEG3 could promote gastric cancer cell proliferation, we performed targeted knockdown of MEG3 expression using RNAi in SGC7901 cells. The expression levels of MEG3 in SGC7901/si-MEG3 cells were significantly decreased compared with si-NC-transfected cells (Fig. 4a). MTT and colony formation assays revealed that inhibition of MEG3 promoted cell proliferation (Fig. 4b, c).

MEG3 promotes gastric cancer cell apoptosis in vitro To determine whether apoptosis was a contributing factor to cell growth inhibition, we performed Hoechst staining analysis of pCDNA-MEG3-transfected AGS and MGC803 cells. The results showed that the number of cells with condensed and fragmented nuclei indicating the fraction of early apoptotic cells was significantly different in pCDNA-MEG3transfected cells compared with respective controls (Fig. 3e). This indicates that upregulation of MEG3 can induce cell gastric cancer apoptosis in vitro.

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Fig. 1 Relative MEG3 expression in gastric cancer tissues and its clinical significance. a Relative expression of MEG3 in gastric cancer tissues (n =72) in comparison with the corresponding nontumor normal tissues (n =72). MEG3 expression was examined by qRTPCR and normalized to GAPDH expression. Data were presented as fold change in tumor tissues relative to normal tissues. b MEG3 expression was significantly lower in patients with big tumors than in patients with small tumors. c MEG3 expression was significantly lower in patients with higher pathological stage than in those with lower pathological stage. d Kaplan–Meier overall survival curves according to MEG3 expression level. The overall survival of the high-MEG3 group (n =36: MEG3 expression ratio≥ median ratio) was significantly higher than that of the low-MEG3 group (n =36: MEG3 expression ratio≤median ratio). P <0.001, log-rank test. *P <0.05, **P < 0.01

These data indicate that downregulation of MEG3 expression promotes gastric cancer cell proliferation.

MEG3 induces activation of p53 protein Recently, there is evidence that numerous lncRNAs may play an important role in the regulation of cell growth by modulating the p53 pathway [23]. Previous studies have reported that reexpression of MEG3 could lead to accumulation of p53 (TP53) protein and its target gene expression that contribute to cell growth inhibition [24]. To further investigate how MEG3 induces gastric cancer cell growth arrest and apoptosis, we examined the level of p53 after transfection of pCDNAMEG3 in p53-wild AGS cells. The results of western blot analysis indicated that the expression level of p53 was significantly increased in AGS cells transfected with pCDNAMEG3 compared to those with empty vector (Fig. 4c). These data suggested that MEG3 may function as a tumorsuppressor gene partly via the activation of p53 in gastric cancer.

Discussion Recently, many lncRNAs have been identified, and the participation of lncRNAs in a wide repertoire of biological processes has been a topic of intense contemporary research, as virtually every step in the life cycle of genes from transcription to mRNA splicing, RNA decay, and translation can be influenced by these molecules [25–27]. Moreover, dysregulation of these lncRNAs may also affect epigenetic information and provide a cellular growth advantage, resulting in progressive and uncontrolled tumor growth [28, 29]. Therefore, lncRNAs may provide the missing piece of the wellknown oncogenic and tumor-suppressor network puzzle. Recent studies are beginning to unravel their importance in tumorigenesis. A famous long ncRNA involved in tumor pathogenesis is known as HOTAIR, which has been consistently upregulated and identified as a strong prognosis marker of patient outcomes such as metastasis and patient survival in diverse human cancers [15, 29–31]. The studies also revealed that HOTAIR binds the polycomb complex PRC2, which methylates histone H3 on K27 to promote gene repression

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Fig. 2 The level of MEG3 expression in gastric cancer cells. a Results from qRT-PCR demonstrating MEG3 expression levels of gastric cancer cell lines (SGC7901, AGS, MGC803, MKN45, and MKN28) compared with a normal human gastric epithelial cell line (GES-1). b qRT-PCR

analyses of MEG3 expression level following treatment of AGS and MGC803 cells with 5 μM 5-aza-CdR. c qRT-PCR analyses of MEG3 expression level following treatment of AGS and MGC803 cells with pCDNA-MEG3 or empty vector. **P <0.01

[15, 31]. In liver cancer, lncRNA HULC is highly upregulated and plays an important role in tumorigenesis. In particular, HULC may function as competing endogenous RNAs to sponge miR-372, thereby modulating the derepression of miRNA targets and imposing an additional level of posttranscriptional regulation [16]. Khaitan et al. [32] reported that lncRNA SPRY4-IT1 may have an important role in the molecular etiology of human melanoma, and SPRY4-IT1 RNAi knockdown resulted in defects in cell growth, differentiation, and higher rates of apoptosis in melanoma cell lines. However, the overall pathophysiological contributions of lncRNAs to gastric cancer remain largely unknown. In this study, we found that the expression of lncRNA MEG3 was decreased in gastric cancer tissues when compared to normal tissues. Specifically, MEG3 expression was found to be significantly lower that had undergone later stages of tumor development in gastric cancer patients. Simultaneously, the overall survival time of patients with lower

MEG3 expression levels was significantly shorter than that of patients with higher MEG3 expression levels. Moreover, significant reduction or loss of MEG3 expression has also been found in various human primary tumors [19–22]. Furthermore, we found that MEG3 expression was lost in multiple gastric cancer cell lines, similar to studies in many cancer cell lines including those derived from brain, bladder, bone marrow, breast, cervix, colon, liver, lung, meninges, and prostate [18]. We also showed that DNA methylation may contribute to the lost expression of MEG3 in gastric cancer cells. This suggests that the decreased expression of MEG3 may be useful in the development of novel prognostic or progression markers for gastric cancer. In order to highlight the impact of altered expression and function of MEG3, we show the biological role of MEG3 in gastric carcinoma cells by applying gain-of-function and lossof-function approaches. Ectopic expression of MEG3 inhibited the gastric cancer cell growth and led to the

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Fig. 3 The effect of MEG3 on gastric cancer cell proliferation and apoptosis in vitro. AGS and MGC803 cells were transfected with pCDNA-MEG3 vector (or empty vector). a, b MTT assay was performed to determine the proliferation of pCDNA-MEG3-transfected AGS and MGC803 cells. Data represent the mean±SD from three independent

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experiments. c, d Colony-forming growth assay was performed to determine the proliferation of pCDNA-MEG3-transfected AGS and MGC803 cells. The colonies were counted and captured. e Hoechst staining assay for cell apoptosis; the percentage of Hoechst-positive nuclei per optical field (at least 50 fields) was counted. *P <0.05, **P <0.01

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Fig. 4 Inhibition of MEG3 promotes gastric cancer cell proliferation. a Results from qRT-PCR demonstrating MEG3 expression levels of SGC7901 cells transfected with si-MEG3. b MTT assay was performed to determine the proliferation of si-MEG3-transfected SGC7901 cells. c Colony-forming growth assay was performed to determine the

proliferation of si-MEG3-transfected SGC7901 cells. d Western blot analysis of p53 after pCDNA-MEG3 or empty vector transfected into p53-wild AGS cells. Results shown are from three independent experiments. GAPDH protein was used as an internal control. *P <0.05, **P < 0.01

promotion of cell apoptosis in vitro, while downregulation of MEG3 could promote gastric cancer cell proliferation. To further investigate the underlying mechanisms by which MEG3 induced growth arrest and apoptosis, we examined whether MEG3 can affect p53 protein expression because the tumor suppressor p53 potently inhibits cell growth by inducing block of proliferation or by activating cell death programs. We found that reexpression of MEG3 could significantly stimulate the level of p53 protein in gastric cell lines AGS p53+/+, similar to studies in human colon cancer cells

and glioma cell lines [19, 24]. As an important transcription factor, p53 is able to regulate many target gene expression, leading to the suppression of tumor growth and development, and it is lost or mutated in most human cancers [33]. These findings indicated that lncRNA MEG3 may function as a tumor suppressor and its deficiency or decreased expression could contribute to gastric cancer development. In summary, we demonstrate that the decreased lncRNA MEG3 expression is a common event underlying gastric cancer, indicating that MEG3 may play a key functional role

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as an indicator of poor survival rate and a negative prognostic factor for gastric cancer patients. We also show that MEG3 expression level could be affected by DNA methylation and regulate gastric cancer cell proliferation and apoptosis in vitro. These data suggest an important role of MEG3 in the molecular etiology of gastric cancer and facilitate the development of miRNA-directed prognosis and therapeutics against this deadly disease. Acknowledgments This work was supported by the National Natural Scientific Foundation of China (no. 81301824, no. 81070620). Conflicts of interest None

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