Tumor Biol. DOI 10.1007/s13277-016-5426-y

ORIGINAL ARTICLE

The long non-coding RNA maternally expressed gene 3 activates p53 and is downregulated in esophageal squamous cell cancer Desheng Lv 1 & Run Sun 2 & Qian Yu 1 & Xuefei Zhang 1

Received: 16 May 2016 / Accepted: 23 September 2016 # International Society of Oncology and BioMarkers (ISOBM) 2016

Abstract Esophageal squamous cell cancer (ESCC) is an aggressive malignancy with poor survival. Long non-coding RNAs (lncRNAs) play important roles in tumorigenesis and cancer progression; hence, lncRNAs are also involved in the development and progression of ESCC. In this study, we used quantitative real-time polymerase chain reaction (qRT-PCR) to investigate expression of lncRNA, maternally expressed gene 3 (MEG3) in ESCC. Ectopic expression of MEG3 was performed in ESCC cell lines. Proliferation and apoptosis of ESCC cell lines were analyzed after ectopic expression of MEG3. We found MEG3 was significantly downregulated in ESCC tissues compared with normal tissues by qRT-PCR. Low expression of MEG3 was correlated with lymph node metastasis and advanced TNM stages of ESCC patients and indicated shorter survival (HR = 0.471, 95 % CI 0.234–0.950, P = 0.035), which was confirmed by The Cancer Genome Atlas (TCGA ) esophageal cancer dataset. D NAdemethylating agent (5-aza-2-deoxy-cytidine (5-aza-CdR)) treatment significantly increased MEG3 expression level in ESCC cells, and TCGA esophageal cancer dataset also showed that DNA methylation of MEG3 predicted survival. Ectopic expression of MEG3 in ESCC cells inhibited cell proliferation, promoted apoptosis, and suppressed metastasis. Further investigation showed enforced expression of MEG3 activated p53 and its target genes by downregulation of mouse

* Desheng Lv [email protected]

1

Department of Thoracic Surgery, The Second Affiliated Hospital of Dalian Medical University, 467 Zhongshan Road, Dalian 116023, China

2

Department of VIP Ward, The Second Affiliated Hospital of Dalian Medical University, Dalian 116023, China

double minute 2 homolog (MDM2). Overall, our study indicated that MEG3 expression loss is common in ESCC and MEG3 could activate p53 and predict prognosis in ESCC. Keywords ESCC . MEG3 . Prognosis . p53

Background Esophageal cancer is one of the most common types of cancer worldwide [1]. Esophageal cancer ranks as the sixth most frequent cause of cancer-related death in the world [2]. Although the occurrence rates vary greatly by geographic location, about half nearly diagnosed esophageal cancer cases occur in China each year [3]. There are two major histological types of esophageal cancer: adenocarcinoma and squamous cell carcinoma (esophageal squamous cell cancer (ESCC)). In China, more than 90 % cases of esophageal cancer are ESCC [4]. ESCC is highly aggressive and the prognosis is very poor with the overall 5-year survival rate less than 10 %. It is urgent to discover the genetic and molecular disorders of ESCC to early diagnosis and improved survival of ESCC patients. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides without protein-coding capacity [5, 6]. LncRNA was once considered as Btranscription noise^ and Bdark matter,^ since more than 98 % of human transcriptome is non-coding transcripts. LncRNA has attracted increasing interest and is found involved in diverse biological processes in recent years [7–9]. Numerous lncRNAs have been identified, but their biological functions remain largely unknown [9]. Many lncRNAs are aberrantly expressed in solid cancers, such as well-known lncRNA MALAT1 [10], HOTAIR [11], TUG1 [12], and PVT1 [13]. For ESCC, the long intergenic non-coding RNA linc-POU3F3 is highly expressed in ESCC

Tumor Biol.

tissues and inhibits the expression of its neighbor gene POU3F3 by mediated hypermethylation of CpG islands in POU3F3 [14]. Maternally expressed gene 3 (MEG3) is an lncRNA widely expressed in many human tissues but MEG3 expression is lost in many cancers due to hypermethylation at promoter or the intergenic region [15–17]. Low expression of MEG3 has been found in non-small cell lung cancer, bladder cancer, and many other cancer tissues [17–19]. However, the expression and functional role of MEG3 in the development of ESCC is little known. In this study, we demonstrated that MEG3 expression was significantly downregulated in ESCC tissues and MEG3 low expression was correlated with lymph node metastasis and advanced TNM stages. Ectopic expression of MEG3 inhibited ESCC cell proliferation, promoted apoptosis, and suppressed metastasis. Further investigation revealed that MEG3 could activate p53 by downregulation of mouse double minute 2 homolog (MDM2).

Methods

RNA extraction and qRT-PCR analysis Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Five hundred nanograms of total RNA was reverse transcribed in a final volume of 10 μl using random primers under standard conditions using the PrimeScript RT Master Mix (TaKaRa, Dalian, China). The PrimeScript RT reagent Kit and SYBR Premix Ex Taq (TaKaRa, Dalian, China) were used for quantitative real-time polymerase chain reaction (qRTPCR) according to the manufacturer’s instructions. GAPDH was measured as an internal control, as its expression showed minimal variation in different cell lines and cancer specimens. 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′. The PCR reaction was conducted at 95 °C for 30 s and followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s in the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The qPCR results were analyzed and expressed as relative mRNA expression of CT (threshold cycle) value, which was then converted to fold changes.

Patients and tissue samples The study was approved by the Ethics Committee of the Second Affiliated Hospital of Dalian Medical University. The written informed consent was obtained from every patient. A total of 96 pairs of primary ESCC tissues and adjacent normal tissues were collected from patients who underwent surgery at the Department of Thoracic Surgery, the Second Affiliated Hospital of Dalian Medical University between 2007 and 2011. After removal from the resected esophagus, the tumor samples were collected immediately and then frozen stored at −80 °C till RNA extraction. All tumor specimens and paired normal tissues were confirmed by experienced pathologists. The clinical and pathological characteristics of each patient were also collected.

Treatment of 5-aza-2-deoxy-cytidine TE-13 and ECA-109 cells (2.5 * 105) were seeded into a sixwell culture plate on day 0 and exposed to 0, 2, or 5 μM 5-aza2-deoxy-cytidine (5-aza-CdR) (Sigma-Aldrich, USA) from day 1 to day 3. The cells treated with 5-aza-CdR were harvested on day 3 and used for the detection of MEG3 expression. Plasmid construction The sequence of MEG3 was synthesized and subcloned into pcDNA3.1 (Invitrogen, Shanghai, China). Ectopic expression of MEG3 was achieved by using the pcDNA-MEG3 transfection, and empty pCDNA vector (empty) was used as control. The expression level of MEG3 was detected by qPCR.

Cell lines and culture conditions Transfection of NCSCL cells All cell lines were obtained from Shanghai Institutes for Biological Science, China. KYSE-410 and human normal esophageal epithelial cell line (HEEC) were grown in RPMI 1640 medium (GIBICO-BRL), supplemented with penicillinstreptomycin (Invitrogen, Shanghai, China) and 10 % FBS (Life Technologies, Australia). ECA-109, TE-1, and TE-13 were grown in DMEM containing high glucose (GIBICOBRL). All cells were grown in an incubator at 37 °C with 5 % CO2.

All plasmid vectors (pCDNA-MEG3 and empty vector) for transfection were extracted by DNA Midiprep or Midiprep kit (Qiagen, Hilden, Germany). TE-13 and ECA-109 cells cultured on a six-well plate were transfected with the pCDNAMEG3 or empty vector 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.

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Cell counting kit-8 assay

Cell invasion assay

Cell proliferation was monitored by the cell counting kit-8 (CCK8) assay (Promega) every 24 h following the manufacturer’s protocol. In brief, the transfected cells were plated in 96-well plates (2000 cells/well), and then 10 μl of CCK8 solution was added and incubated for 2 h. Each solution was measured spectrophotometrically at 450 nm.

For invasion assay, transfected cells (5 * 105) were plated in the top chamber with a matrigel-coated membrane (BD Biosciences) in 500 μl serum-free medium. Also, the bottom chambers were filled with culture medium supplemented with 10 % FBS. After 48 h of incubation, the cells on the filter surface were fixed with methanol, stained with crystal violet,

Fig. 1 MEG3 expression was profiled in 96 pairs of ESCC and normal tissues (a). MEG3 expression level was significantly correlated with differentiation (b), lymph node metastasis (c), and TNM stages (d).

Kaplan-Meier and Cox regression showed that low MEG3 expression indicated poor survival (e) and the poor prognosis was confirmed by TCGA dataset (f). *P < 0.05, **P < 0.01

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and photographed. Migration was assessed by counting the number of stained cell nuclei from five random fields per filter in each group. Flow cytometric analysis of apoptosis Cells transfected with pcDNA-MEG3 and empty vector were harvested 48 h after transfection by trypsinization. Following double staining with FITC-Annexin V and propidium iodide (PI), the cells were analyzed using flow cytometry (FACScan®; BD Biosciences) equipped with a CellQuest software (BD Biosciences). Cells were discriminated into viable cells, dead cells, early apoptotic cells, and apoptotic cells. The percentage of early apoptotic cells were compared to control groups from each experiment. All of the samples assayed were in triplicates. Western blotting assay Cells were lysed using mammalian protein extraction reagent RIPA (Beyotime, China) supplemented with protease inhibitor cocktail (Roche, Switzerland) and PMSF (Roche, Switzerland). Protein concentration was measured with the Bio-Rad protein assay kit. Fifty-microgram protein extractions were separated by 12 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to 0.22 μm nitrocellulose membranes (Sigma-Aldrich. USA) and incubated with specific antibodies. ECL chromogenic substrate was used to visualize the bands, and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad). GAPDH was used as control. GAPDH antibody was purchased from Sigma-Aldrich (USA), P53 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and P21 and BAX antibodies were purchased from Cell Signaling Technology (MA, USA). Statistical analysis Student’s t test (two-tailed), one-way ANOVA, Kaplan-Meier curve, and Cox regression were performed to analyze the data using SPSS 16.0 software. P values less than 0.05 were considered statistically significant. The Cancer Genome Atlas dataset was assessed and analyzed using the online tool, Cancer Browser (https://genome-cancer.ucsc. edu/proj/site/hgHeatmap/).

Results MEG3 is downregulated in ESCC The expression of MEG3 in ESCC was firstly profiled by qRT-PCR in 96 pairs of ESCC and non-tumor tissues. The

expression of MEG3 was significantly downregulated in ESCC tissues compared with paired non-tumor tissues (Fig. 1a), with an average downregulation fold of −15.46. Correlation between MEG3 expression level and clinicopathologic characteristics was further analyzed (Table 1). Low MEG3 expression was significantly associated with high differentiation (Fig. 1b), lymph node metastasis (Fig. 1c), and advanced TNM stages (Fig. 1d). However, MEG3 expression level was not significantly correlated with tumor location, smoking history, age, or gender. Then Kaplan-Meier survival analysis and Cox regression were performed to analyze the relationship between MEG3 expression level and survival. The 96 patients were classified into two groups according to MEG3 expression level. The survival analyses showed that patients with high MEG3 expression level had significantly longer survival times than those with high MEG3 expression level (Fig. 1e, HR = 0.471, 95 % CI 0.234–0.950, P = 0.035). In addition, The Cancer Genome Atlas (TCGA) project proved the largest cohort of esophageal cancer patients, including both clinical data and comprehensive data of gene expression, DNA methylation, and diverse high-throughput data. MEG3 expression Table 1 Correlation between TINCR expression and clinicopathologic characteristics Characteristics

Age (years) <60 >60 Sex Male Female Smoking Yes No Cancer location Upper Middle Lower Differentiation

Number of patients

Percentage (%)

Fold change

34

35.42

−14.01

62

64.58

−16.38

77 19

80.21 19.79

−14.54 −19.59

43 53

44.79 55.21

−15.37 −15.68

14 52 30

14.58 54.17 31.25

−14.93 −17.46 −12.50

P values

0.64

0.58

0.85

0.18

High 12 Middle 53 Low 31 Lymph node metastasis No 41 Yes 55 TNM stage III–IV 49 I–II 47

0.02* 12.50 55.20 32.30

−28.45 −15.61 −10.77

42.71 59.14

−7.36 −21.64

51.04 48.96

−21.22 −9.62

<0.01**

<0.01**

Statistical significance, *P < 0.05, **P < 0.01

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and survival of esophageal cancer patients were analyzed using TCGA data, and the results also demonstrated that esophageal cancer patients with low MEG3 level had shorter overall survival times than those with high MEG3 level (Fig. 1f). These evidences prove that decreased MEG3 may play a key role in ESCC development and progression. DNA methylation of MEG3 We then examined MEG3 expression in four human ESCC cell lines (TE-13, TE3, ECA109, and KYSE-410) and a normal esophageal epithelium cell line (HEEC). Compared with the normal esophageal epithelium cell line, MEG3 was downregulated in all four ESCC cell lines and the expression was lowest in TE-13 and ECA-109 (Fig. 2a). Lu K showed that hypermethylation of MEG3 promoter was associated with downregulation of MEG3 in lung cancer

Fig. 2 Compared with human normal esophageal epithelial cell line (HEEC), MEG3 expression level was lower in ESCC cell lines (a). After treatment of 5-aza-CdR, MEG3 expression level significantly

[20]. We then tested this hypothesis in ESCC cell lines by treatment with DNA-demethylating agent (5-aza-CdR), and MEG3 expression level was significantly increased (Fig. 2b). Methylation of MEG3 was also analyzed in the TCGA esophageal cancer cohort. Kaplan-Meier survival curve showed patients with high level of methylation had shortest overall survival (Fig. 2c), which was in consistence with the fact that low expression of MEG3 indicated poor survival. Effect of MEG3 on ESCC tumor behavior Since MEG3 expression level was lowest in TE-13 and ECA109 cells, we restored MEG3 expression by transfection of TE-13 and ECA-109 cell with pcDNA-MEG3, and qRTPCR demonstrated MEG3 expression was dramatically increased (Fig. 3a).

increased in TE-13 and ECA-109 cells (b). TCGA dataset of esophageal cancer showed that higher DNA methylation level of MEG3 indicated poor survival (c). *P < 0.05, **P < 0.01

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ƒFig. 3

Compared with empty vector (EV), MEG3 expression was restored after transfection with pcDNA-MEG3 (a). CCK8 assay showed that transfection of pcDNA-MEG3 significantly inhibited proliferation of TE-13 (b, upper panel) and ECA-109 (b, lower panel). Enforced expression of MEG3 induced apoptosis in TE-13 (c) and ECA-109 (d) cells. Metastasis ability of TE-13 (e) and ECA-109 (f) cells was also suppressed by MEG3

To evaluate the biological role of MEG3 in ESCC, CCK8 assay was performed in TE-13 and ECA-109 cells and cell proliferation was significantly inhibited after overexpression of MEG3 (Fig. 3b). Then, we further analyzed whether apoptosis was affected by MEG3 overexpression by PI staining followed by flow cytometric analysis. After transfection of pcDNA-MEG3, the percentage of apoptotic cells was significantly increased both in TE-13 (Fig. 3c) and ECA-109 (Fig. 3d) cells. Matrigel assay was also conducted, and the results showed that overexpression of

Fig. 4 Expression of p53-related genes was detected by qRT-PCR (a) and Western blot (b). MDM2 was downregulated whereas p53 and its target genes p21 and BAX were upregulated after ectopic expression of MEG3. Negative correlation between MEG3 and MDM2 was confirmed by qRTPCR in ESCC tissues (c). *P < 0.05, **P < 0.01

MEG3 significantly inhibited invasion ability of ESCC cells (Fig. 3e, f).

MEG3 exerts function by activating p53 Recent studies have indicated that lncRNAs may play an important role in the regulation of cell growth by modulating p53 pathway [26]. It has been reported that MEG3 could activate p53 [21, 22]. qRT-PCR (Fig. 4a) and Western blot (Fig. 4b) were performed to determine the changes of p53 and its target genes after pcDNA-MEG3 transfection. The results showed that MDM2 was downregulated and expression of p53 and its target genes, P21 and BAX, was significantly upregulated in TE-13 cells after transfection with pcDNA-MEG3. In addition, the reverse correlation between MEG3 and MDM2 was confirmed by qRT-PCR in a cohort of ESCC tissues (r = −0.86, P < 0.01). These data confirm that MEG3

Tumor Biol.

functions as a tumor suppressor gene by regulating p53 activation in ESCC.

Discussion With the advance of high-throughput technology, researchers have encoded human genome and found ~98 % of human genome are non-coding genes and they are actively transcribed, yielding huge number of non-coding transcripts [23, 24]. LncRNA accounts for the major part of non-coding transcripts. According to GENCODE project, the number of human lncRNA is more than 13,000 [25] but only small part of lncRNAs has been well characterized [26]. Although few lncRNAs have been fully characterized, published evidence proves that lncRNAs are involved in almost every aspect of cell biology. In cancer research, some well-known lncRNAs have been well characterized, like HOTAIR [27] and MALAT1 [28]. But few studies have characterized ESCCassociated lncRNAs. Previous studies have reported that MEG3 is downregulated in many kinds of solid tumors, like gastric cancer [29], hepatocellular cancers [30], and cervical cancer [31]. In the present study, we found lncRNA MEG3 is significantly downregulated in human ESCC tissues compared with paired normal tissues, and low expression of MEG3 is significantly correlated with high level differentiation, lymph node metastasis, and advanced TNM stages. Additionally, both our own survival data and TCGA esophageal cancer dataset supported that low expression level of MEG3 correlated shorter survival of esophageal cancer patients. Our results showed that DNA methylation may contribute to the loss of MEG3 expression in ESCC, and TCGA esophageal cancer dataset also proved that DNA methylation of MEG3 predicted survival. Thus, these lines of evidence show that MEG3 is a prognostic biomarker of ESCC and downregulation of MEG3 is mediated by DNA methylation. To characterize the impact of downregulated expression and function of MEG3, we constructed expression vector and successfully restore MEG3 expression. In two ESCC cell lines, ectopic expression of MEG3 significantly inhibited proliferation, promoted apoptosis, and inhibited metastasis in vivo. These findings were consistent with previous reports, which showed that MEG3 functions as a tumor suppressor and inhibited tumor proliferation and invasion [32, 33]. Previous evidence has suggested that MEG3 could induce the activation of p53 [22]. P53 is a well-known tumor suppressor, which regulates expression of many target genes. The protein level of p53 is regulated by MDM2, an E3 ubiquitin ligase. We found MDM2 RNA and protein level decreased significantly and observed increased p53 level after ectopic expression of MEG3, indicating that MEG3 induces p53 activation may be due to MDM2 downregulation. As expected,

we also observed upregulation of p21 and BAX, well-known p53 target genes. These findings demonstrate that MEG3 could activate p53 and its target genes in ESCC. In a word, we find lncRNA MEG3 downregulation is a common event in ESCC and low expression of MEG3 is a negative prognostic factor of ESCC patients. Ectopic expression of MEG3 could inhibit ESCC cell proliferation and promoted apoptosis in vivo by activating p53. Acknowledgments None Compliance with ethical standards The study was approved by the Ethics Committee of the Second Affiliated Hospital of Dalian Medical University. The written informed consent was obtained from every patient. Conflicts of interest None

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