MOLECULAR CARCINOGENESIS

Long Non-Coding RNA 91H Contributes to the Occurrence and Progression of Esophageal Squamous Cell Carcinoma by Inhibiting IGF2 Expression Tianyi Gao,1 Bangshun He,1 Yuqin Pan,1 Yeqiong Xu,1, 2 Rui Li,1, 2 Qiwen Deng,1 Huilin Sun,1, 2 and Shukui Wang1* 1 2

Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China Department of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu, China

Long non-coding RNAs (lncRNAs) have been recently recognized as a major class of regulators in mammalian systems. 91H, a novel long noncoding antisense transcripts located on the position of the H19/IGF2 locus has been suggested to play a potential tumor-suppressor role in tumor development. However, little study has proved the mechanism in esophageal squamous cell carcinoma (ESCC). We carried out this study to explore the role of lncRNA 91H in the regulation of H19 imprinting control regions (ICR) and IGF2 expression and the association between 91H and ESCC progression. The cell line TE-1, Eca-109, and 232 ESCC patients' matched sets of paraffin-embedded adjacent normal and tumor samples were obtained in this study. The results showed that 91H expression was significantly lower in patients with higher depth of invasion, neoplastic grading and TNM which usually leads to the overexpression of IGF2 in tumor progression. The expression of 91H usually decreased in TE-1 and Eca-109 when treated with demethylation agent. Further analysis revealed that, in 91H knockdown cell lines, IGF2 expression was also significantly higher than negative controls. Therefore, the results demonstrated that the lncRNA 91H was associated with H19 ICR methylation and inhibited IGF2 expression of ESCC patients which may optimize the mechanism of IGF2 regulation in tumor development. Patients with higher depth of invasion, neoplastic grading and TNM usually demonstrated lower 91H expression potentially represent a novel clinically relevant event to identify individuals at increased risk for the occurrence, progression and prognosis of ESCC. © 2014 Wiley Periodicals, Inc.

Key words: lncRNA; 91H; IGF2; expression; H19 ICR; methylation; ESCC

INTRODUCTION Over the past decades, esophageal cancer (EC) is one of the most common cancers in the world with an extremely poor prognosis due to late tumor presentation and rapid progression, causing more than 40,000 deaths each year [1]. The most prevalent type of EC is esophageal squamous cell carcinoma (ESCC), and China is among the highest risk areas [1,2]. Epigenetic, genetic, or posttranscriptional factors may be involved in ESCC development and the identification of such factors may improve the prevention of this malignancy and reduce mortality [3]. Genomic imprinting plays a critical role in modulating gene expression during embryogenesis and normal development [4]. It consists of an epigenetic modification that results in the silencing of a specific allele, depending on its parental origin. Most of the 80 mammalian imprinted genes identified thus far are organized into clusters [5]. It has been suggested that the clustering of genes enables sharing of cis-acting elements located several kilobase pairs away [6]. Differentially methylated regions associated with the imprinted clusters play a crucial role in the imprinted expression patterns, which are hence called imprinting control regions (ICRs) [7,8]. The H19/IGF2 locus is a well-characterized cluster. Both genes share ß 2014 WILEY PERIODICALS, INC.

a common set of enhancers located downstream from the H19 gene. The ICR, located 2 kb upstream of the H19 promoter, controls the monoallelic expression of the H19 and IGF2 genes by insulating communication between the 30 enhancers and the IGF2 promoter. In the past years, the enhancer competition model which has been constructed in many studies is mostly used to illustrate the relationship between H19 ICR and IGF2 expression [9,10]. A more recently identified characteristic of imprinted genes is their association, in some cases, with noncoding antisense transcripts (ncRNAs), which

Abbreviations: EC, esophageal cancer; ESCC, esophageal squamous cell carcinoma; ICRs, called imprinting control regions; ncRNAs, noncoding antisense transcripts; lncRNA, long noncoding antisense transcripts; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; IF, immunofluorescence; IOD, optical densities. Tianyi Gao and Bangshun He contributed equally to this work. *Correspondence to: Shukui Wang, Central Laboratory, Nanjing First Hospital, Nanjing Medical University, 68 Changle Road, Nanjing 210029, Jiangsu, China. Received 31 July 2013; Revised 14 October 2013; Accepted 23 October 2013 DOI 10.1002/mc.22106 Published online in Wiley Online Library (wileyonlinelibrary.com).

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have been suggested to constitute a new epigenetic regulatory system in carcinogenesis and cancer metastasis [11–13]. It has been proposed that antisense transcripts serve to regulate overlapping genes by promoter or transcript occlusion or by competing with these loci for regulatory elements such as transcription factors or enhancers [14–17]. 91H, a novel 119.392-kb long noncoding antisense transcripts (lncRNA), located on the position of the H19/IGF2 locus (Accession number NC_000011.9). It has been proved to be consistently overexpressed in breast cancer and increases IGF2 expression which demonstrated that another mechanism between H19 ICR and IGF2 expression may exist [18]. However, little study has proved the mechanism and the association with tumor progression also remains unclear. Therefore, we perform this study to explore the role of lncRNA 91H in the regulation of H19 ICR and IGF2 expression and the association between 91H and ESCC progression. MATERIALS AND METHODS Tumor Tissues Matched sets of paraffin-embedded adjacent histologically normal esophageal tissues and ESCC tumor samples obtained from 232 patients at Nanjing First Hospital Affiliated to Nanjing Medical University. The patients’ age varied from 43 to 81 yr old, with a mean age of 63.13  9.67. The study was conducted according to our institutional guidelines.under the supervision of the Institutional Review Board of Nanjing First Hospital Affiliated to Nanjing Medical University, and written informed consent was obtained from all participants. Characteristics of patients and their pathology were recorded, including age, sex, depth of invasion (T1: The cancer has grown through the muscularis mucosa and extends into the submucosa; T2: The cancer has grown through the submucosa and extends into the muscularis propria (thick outer muscle layer); T3: The cancer has grown through the muscularis propria and into the outermost layers of the colon or rectum but not through them. It has not reached any nearby organs or tissues; or T4: The cancer has grown through the serosa or through the wall of the colon or rectum and attached to or invades into nearby tissues or organs), lymph nodes involvement, neoplastic grading (Grade) of all the patients which were based on the guidelines of the American Joint Commission on Cancer, and the TNM Classification of Malignant Tumors (T: tumor size, N: nearby lymph nodes, M: distant metastasis) determined by the guidelines of the International Union Against Cancer (IUAC). Cell Lines The human esophageal squamous cell lines TE-1 and Eca-109 were obtained from the American Type Culture Collection (ATCC). All the cell lines were Molecular Carcinogenesis

maintained in RPMI 1640 with 10% FBS (Gibco, Grand Island, NY) and cultured at 378C with 5% CO2. The cell lines authenticity were frequently confirmed by analyzing various genetic and epigenetic markers every 6–8 mo. RNA Interference RNA interference was carried out by using synthetic siRNA duplexes, as described by earlier studies [18,19]. Two synthetic siRNA duplexes (si91H 1 and si91H 2) corresponding to the 91H RNA sequences 50 -GGCGUCAUUCUGAUGGGACTT-30 and 50 -UUCAGGAGCUUAAGAUGCUTT-30 were used and 50 -CGUGGGUGGAUGCAUGGAUTT-30 were used as a negative control. The cells (5  102) were grown on coverslips in six-well plates and transfected with 400 pmol of siRNAs. After transfection for 24 h, followed by lysed for total RNA isolation. 5-aza-CdR Treatment The demethylating agent 5-aza-CdR (Sigma, MO, USA) was freshly prepared in DMSO at 2.5, 5, 10 mM and small aliquots were kept frozen at 208C. Twentyfour hours after seeding, ESCC cells (5  102) seeded in six-well plates were treated with 5-aza-CdR. The medium was removed and replaced with fresh medium containing 5-aza-CdR every 24 h for a period of 48 h. Extraction of Nucleic Acids Genomic DNA and total RNA were extracted from paraffin-embedded tissues by using E.Z.N.A.FFPE DNA Kit and E.Z.N.A.FFPE RNA Kit (Omega) following the manufacturers’ protocol. To avoid the possible DNA contamination, RNA was digested with RNasefree DNaseI and cleaned with the RNA easy MinElute Cleanup Kit (Qiagen, Mainz, Germany). Genomic DNA and total RNA of cell lines were extracted with E.Z.N.A.Total DNA Kit and E.Z.N.A.Total RNA Kit (Omega). Reverse Transcription Total reverse transcriptions were performed as follows: 1 mg RNA, 4 ml of buffer containing random hexamers and reverse transcriptase (RT; Takara, Shiga, Japan) were incubated for 15 min at 378C, for 5 s at 858C, and stored at 48C in a final volume of 20 ml. Real-Time RT-PCR Real-time PCR amplifications were performed by using a QuantiTect Sybr green PCR kit (Qiagen) with 2 ml of cDNA and 500 nM concentrations of the primers. The primers used for the 91H were Fw 50 GCTTGTCAGTAGAGTGCGCC-30 and Rev 50 -CATCCAGTTGACCGAGCTTG-30 . The primers used for IGF2 were Fw: 50 -CCTTGGACTTTGAGTCAAATT-30 and IGF2 Rev: 50 -GGTCGTGCCAATTACATTTCA-30 . We selected the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene, a housekeeping gene as an

lncRNA 91H CONTRIBUTES TO THE OCCURRENCE AND PROGRESSION OF ESCC

internal control: GAPDH Fw: 50 -GTCAACGGATTTGGTCGTATT-30 and Rev: 50 -AGTCTTCTGGGTGGCAGTGAT-30 . Relative quantification of gene expression changes was recorded after normalizing for GAPDH expression, computed by using the 2DDCt method (user manual 2, ABI Prism 7700 SDS). Immunofluorescence (IF) Cells were plated onto sterile coverslips (WHB-24CS, diameter: 14 mm) at a density of 5  105 cells in fully supplemented keratinocyte media and allowed to adhere for approximately 4 h. Cells were then washed twice with PBS and fixed with 10% neutralbuffered formalin for an hour at room temperature. Coverslips with fixed cells were incubated overnight at 418C with antibodies specific for the IGF2 (1:50; R&D Systems Inc., Minneapolis, MN). FITC and Rhodamine B-conjugated anti-rabbit secondary antibody (1:100; Sigma, Oakville, ON, Canada) was used for detection and cells were counterstained with propidium iodide. We used integrated optical densities (IOD) of Image-Pro Plus6.0 to score the expression of IGF2 in each sections, a software from http://www. mediacy.com/index.aspx?page¼IP_Premier&gclid¼ CIy68orZnbMCFchU4godYEEAEg. Methylation Analysis of H19 ICR Genomic DNA from tumor tissues and cell lines were treated with bisulfite (EpiTect Plus DNA Bisulfite Kit 59124) to convert unmethylated cytosines to uracils, whereas methylated cytosines are unaffected. Bisulfite-treated DNA was subsequently amplified using the forward primer 50 -TGGGTATTTTTGGAGGTTTTTTT-30 and reverse primer 50 -ATAAATATCCTATTCCCAAATAA-30 . These primers allowed the amplification of both the methylated and the unmethylated alleles by spanning a region with 18 differentially methylated CpGs. The region corresponded to GenBank nucleotides 7881–8100 (accession no. AF125183). The PCR condition was as follows: denatured at 958C for 15 min, followed by 958C for 30 s, 578C for 30 s and 728C for 20 s respectively for 50 cycles, and enlongated at 728C for 5 min. Each PCR product (5 ml) was analyzed on 2% agarose gel and the remaining 20 ml were purified using QIAquick purification kit (Qiagen) to be sequenced using the forward primer, and the sequencing results were analyzed using CpG viewer, a software tool for DNA methylation available by free download from http://dna.leeds.ac.uk/cpgviewer/ download.php. Statistical Analysis Differences in 91H expression, IGF2 expression and the methylation status of H19 ICR were compared with a chi-square test, Student’s t-test or ANOVA test. All analyses were performed using SPSS 18.0 for Windows and P-values of 0.05). The association with lncRNA 91H Then ESCC cell lines TE-1 and Eca-109 were treated with 5-aza-CdR which were both determined hypermethylated by methylation test. After 48 h, the methylation status of theses two cell lines were tested to confirm demethylation in both of them. Then the 91H expression of cells treated with different density were detected. The results of two cell lines all showed that 91H expression was associated with the density of demethylation agent (Figure 1). The lncRNA levels of 91H not only inTE-1 but also in Eca-109 at 10 mM was lower than at neither 5 mM nor 2.5 mM as the results shown in Table 4 (P < 0.05). Then the 91H expression of patients with different H19 ICR methylation status were also compared which demonstrated that the 91H expression in patients whose methylation status >0.5 (1.57  0.52) were also lower than patients whose methylation status 0.5 (2.00  1.47, P ¼ 0.014). IGF2 Expression IGF2 expression in patients The IGF2 expression in tumor tissues of all the 232 ESCC patients were also examined to assess the relationship between 91H and IGF2. The results verified that IGF2 expression was associated with patients which declared that patients with higher lymph nodes involvement, depth of invasion, neoplastic grading and TNM usually demonstrated higher IGF2 expression (Table 3, P < 0.05).

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Table 1. The Association Between 91H Expression and ESCC Patients' Clinical Features

Sex Male Female Age group 45 46–64 65 Cigarettea Smoking Non-smoking Family history of ESCC With history Without- history Depth of invasion T1 T2 T3 T4 Lymph nodes involvement N0 N1 N2 Neoplastic grade G1 G2–G3 TNM I–II III–IV Metastasis With metastasis Without metastasis a

N

91H

152 80

1.67  0.62 1.72  1.20

16 112 104

1.95  0.25 1.76  0.92 1.56  0.83

95 137

1.62  0.55 1.73  1.02

46 186

1.72  1.18 1.68  0.76

56 67 106 3

1.99  0.52 1.82  1.3 1.45  0.54 1.36  0.15

135 84 13

1.57  0.71 1.84  1.00 1.88  1.01

46 186

2.05  1.51 1.60  0.57

123 109

1.88  1.02 1.47  0.57

160 72

1.63  0.74 1.81  1.08

x2/t-value

P

0.47

0.641

2.30

0.101

0.93

0.356

0.28

0.778

6.07

0.001

3.02

0.051

3.32

0.001

3.74

0.001

1.47

0.144

Definitions of smoker: more than 100 cigarettes in entire lifetime.

The correlation to lncRNA 91H ESCC cell lines TE-1 and Eca-109 were also used in exploring the relationship between IGF2 and H19. After RNA interference, the expression of 91H in all

cell lines were confirmed reduced than negative control (P < 0.05, Table 4). Then the expression of IGF2 in cell lines were examined by real-time PCR and IF. The results of real -time PCR showed that the mRNA levels of IGF2 in two cell lines were

Table 2. The Association Between H19 ICR Methylation and ESCC Patients' Pathological Features

Depth of invasion T1 T2 T3 T4 Lymph nodes involvement N0 N1 N2 Neoplastic grade G1 G2–G3 TNM I–II III–IV

Molecular Carcinogenesis

Number

91H

56 67 106 3

56.41  16.71 58.3  9.39 57.48  7.71 59.01  5.19

135 84 13

56.27  12.15 58.18  9.49 57.77  6.27

46 186

58.02  10.03 56.80  11.24

123 109

56.93  13.19 57.17  7.88

x2/t-value

P

1.299

0.276

0.811

0.446

0.673

0.502

0.171

0.865

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lncRNA 91H CONTRIBUTES TO THE OCCURRENCE AND PROGRESSION OF ESCC

Figure 1. The expression of 91H in cell lines treated with 5-aza-CdR by 48 h. (a) The lncRNA levels of 91H in TE-1 at 10 mM (0.44  0.08) was lower than the lncRNA levels of 91H at 5 mM (0.53  0.15) and 2.5 mM (0.82  0.07, P ¼ 0.001). (b) The lncRNA levels of 91H in Eca-109 at 10 mM (0.10  0.05) was lower than the lncRNA levels of 91H at 5 mM (0.22  0.06) and 2.5 mM (0.42  0.14, P ¼ 0.001).

significantly higher than those treated with negative control (P < 0.05, Figure 2, Table 5). Furthermore, Significant differences were also found by IF that the IOD of IGF2 in both cells lines treated with siRNA were higher than negative controls (P < 0.05, Figure 2, Table 5). Moreover, the expression of IGF2 in cell lines treated with 5-aza-CdR were also detected. The mRNA levels of IGF2 in TE-1 and Eca-109 at 10 mM were higher than the mRNA levels of IGF2 at 5 mM and 2.5 mM (P < 0.05, Figure 3, Table 5). Then IF was also applied to the comparison between mRNA levels of IGF2 in cell lines treated with different density of 5aza-CdR and negative control. The IOD of IGF2 in both cell lines treated with 10 mM 5-aza-CdR were higher than the others (P < 0.05, Figure 3, Table 5). DISCUSSION Recently, the presence of extracellular or cell-free RNA in circulation has become a promising diagnostic

and prognostic tool for human cancers. Although much success has been achieved using circulating mRNAs as potential biomarkers for cancer diagnosis and prognosis [20–23], lncRNAs have not yet been studied in this context. This is the first systematic study of the characteristics of lncRNA 91H in ESCC and its association with tumor progression. In the present study, we demonstrate that 91H expression is significantly associated with ESCC patients’ tumor progression which is regulated by H19 ICR and correlated to the IGF2 overexpression in vivo and in vitro. Earlier studies had suggested that the IGF axis comprised two growth factors (IGF1 and IGF2), several high affinity binding proteins (IGFBPs1-6), IGFBP-related proteins, and two principal cell membrane receptors (IGF1R and IGF2R) homologous to the insulin receptor (IR) [24,25]. In many tumor systems, IGFs had been found to stimulate the cell proliferation, protect cells from apoptosis, and to be

Table 3. The Association Between IGF2 mRNA Expression and ESCC Patients' Pathological Features N Total Depth of invasion T1 T2 T3 T4 Lymph nodes involvement N0 N1 N2 Neoplastic grade G1 G2-G3 TNM I–II III–IV

Molecular Carcinogenesis

IGF2 mRNA

x2/t-value

P

22.757

0.001

14.024

0.001

3.56

0.001

8.358

0.001

92 56 67 106 3

2.65  1.43 3.05  1.09 6.74  4.95 5.15  1.98

135 84 13

3.67  2.68 6.36  5.26 5.04  2.27

46 186

2.28  1.56 5.12  4.28

36 56

2.85  1.52 6.72  4.06

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Table 4. 91H Expression in TE-1 and Eca-109 After Being Treated With siRNA and Demethylation Agent TE-1

siRNA With siRNAs Negative control 5-aza-CdR (mM) 2.5 5 10

Eca-109

Expression

x /t-value

P

Expression

x2/t-value

P

0.33  0.10 0.77  0.26

3.532

0.008

0.33  0.26 0.98  0.29

3.753

0.006

0.82  0.07 0.53  0.15 0.44  0.08

17.950

0.001

0.42  0.14 0.22  0.06 0.10  0.05

14.998

0.001

2

associated to tumor metastasis mediated via paracrine or autocrine mechanisms [26]. Increased IGF2 expression resulting from lost of imprint (LOI) of IGF2 had been reported in murine models [27], and in patients with Wilms’ tumor [28] and colorectal cancer [29], likely a consequence of abnormal activation of the normally silenced maternal allele. However, the human transcriptome has been found to be more complex than a collection of proteincoding genes and their splice variants [30,31]. Recent evidence suggests that the transcriptional noise of the genome may play a major biological role in cellular development and human diseases [32]. The lncRNA 91H, a new antisense transcript produced from the maternal allele, has been verified to be correlated with IGF2 expression in breast cancer [18] which then proposed a model to explain the 91H trans effect on

IGF2 expression in which cooperation between two sets of enhancer sequences would be required for full expression of the gene. In addition, both parental alleles would be competing for a common limiting pool of regulatory factors. In this model, 91H would be essentially involved in modifying the accessibility of upstream conserved regions to regulating proteins. Indeed, maternal expression of 91H would preferentially direct regulatory elements on the paternal allele within the HUC region. This region would cooperate with ENH sequences. On the maternal allele, 91H would block the locus and prevent the HUC sequences from interaction with any regulatory factors resulting in only paternal IGF2 enhanced expression. In this study, the expression of IGF2 and 91H were both examined in ESCC patients. The results declared that the expression of 91H and IGF2 were both

Figure 2. The expression of 91H and IGF2 in cell lines treated with siRNA. (a) The expression of 91H and IGF2 in TE-1 detected by real-time PCR. The 91H expression in TE-1 treated with siRNA was lower than negative control and IGF2 expression in TE-1 treated with siRNA was higher than those treated with negative control (P < 0.05). (b) The expression of 91H and IGF2 in Eca-109 detected by real-time PCR. The 91H expression in TE-1 treated with siRNA was lower than negative

control and IGF2 expression in Eca-109 treated with siRNA were higher than those treated with negative control (P < 0.05). (c) The expression of IGF2 in TE-1 examined by IF (400). (1): The expression of IGF2 in TE1 treated with siRNA. (2): The expression of IGF2 in TE-1 treated with negative control. (d) The expression of IGF2 in Eca-109 examined by IF (400). (1): The expression of IGF2 in TE-1 treated with siRNA. (2): The expression of IGF2 in TE-1 treated with negative control.

Molecular Carcinogenesis

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lncRNA 91H CONTRIBUTES TO THE OCCURRENCE AND PROGRESSION OF ESCC

Table 5. IGF2 Expression in TE-1 and Eca-109 After Being Treated With siRNA and Demethylation Agent TE-1

Real-time siRNA With siRNAs Negative control 5-aza-CdR (mM) 2.5 5 10 IF (104) siRNA With siRNAs Negative control 5-aza-CdR (mM) 0 2.5 5 10

x /t-value

P

Expression

x2/t-value

P

4.91  1.68 1.38  0.61

4.393

0.002

3.62  1.44 1.75  1.00

2.389

0.044

1.46  0.38 2.25  0.80 2.98  0.76

6.331

0.013

1.66  0.24 1.99  0.41 3.41  0.77

15.832

0.001

7.60  0.66 2.01  0.43

12.242

0.001

8.64  1.74 3.71  0.68

4.567

0.01

1.70  0.11 2.25  0.40 3.13  0.37 3.71  0.94

8.135

0.008

1.71  0.55 5.16  0.32 6.73  1.04 11.25  2.61

22.749

0.001

Figure 3. The expression of IGF2 after treating with 5-aza-CdR by 48 h. (a) IGF2 expression in TE-1 examined by real-time PCR. The mRNA levels of IGF2 at 10 mM was higher than the mRNA levels of IGF2 at 5 mM and 2.5 mM (P < 0.05). (b) IGF2 expression in Eca-109 examined by real-time PCR. The mRNA levels of IGF2 at 10 mM was also higher than the mRNA levels of IGF2 at 5 mM and 2.5 mM (P < 0.05). (c) IGF2 expression in TE-1 treated with different density

Molecular Carcinogenesis

Eca-109

Expression

2

of agent detected by IF. (1) 10 mM, (2) 5.0 mM, (3) 2.5 mM, (4) negative control. The expression of IGF2 in TE-1 treated at 10 mM was higher than the others (P < 0.05). (d) IGF2 expression in Eca109 treated with different density of agent detected by IF. (1) 10 mM, (2) 5.0 mM, (3) 2.5 mM, (4) negative control. The expression of IGF2 in Eca-109 treated at 10 mM was higher than the others too (P < 0.05).

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associated with tumor progression. However, patients with higher depth of invasion, neoplastic grading and TNM usually showed lower 91H but higher IGF2 expression which were consisted with the model of 91H trans effect on IGF2 expression. Then the results of vitro research on ESCC cell lines also confirmed that by siRNA treatment and 91H knockdown, the competition would be lost and, as a consequence, both IGF2 alleles would be now able to interact with the regulatory factors with similar efficiencies which lead to the overexpression of IGF2 in TE-1 and Eca-109 cell lines. In the early study of Berteaux, the results showed that the 91H RNA favours IGF2 expression which was contrary to our work [18]. It may due to the different methylation status of H19 ICR which would change the structure of maternal allele in IGF2 and H19. Indeed, 91H RNA corresponds to a short-lived and 120-kb-long transcript, overlapping the H19 imprinted gene and intergenic regions including the ICR [33]. Elsewhere, it has been demonstrated that lncRNAs are able to guide the epigenetic regulation of genes [34,35]. So the relationship of H19 ICR and 91H expression was also explored in ESCC patients and cell lines. Patients with different depth of invasion, neoplastic grading and TNM have not demonstrated any differences in H19 ICR methylation status which are not consisted with 91H expression. But in vitro study, we discovered that 91H expression significantly reduced when the cell was treated with demethylation agent which declared that the expression of 91H may be regulated by H19 methylation and H19 methylation may be an early event in tumor development. This is the first report investigating the expression of the 91H lncRNA extensively in ESCC patients. But there still existed some limitations in this study. First of all, the results in this study demonstrated that the lncRNA 91H depressed the IGF2 expression which was different with early study of Berteaux which might due to the different methylation status of H19 ICR in different tumors. Much more studies should be involved in this area. Then, metastasis formation was not found to be associated with 91H expression in this study but the p value of the comparison in lymph nodes involvement, a pathological factor which was usually correlated to tumor metastasis was 0.051 which suggested the probably association between tumor and 91H expression. But in this study, the results were not significant for the small number of patients which required confirmation through adequately designed prospective studies. In summary, our results suggested that lncRNA 91H was associated with H19 ICR methylation and inhibited IGF2 expression of ESCC patients which may optimize the mechanism of IGF2 regulation in tumor development. Patients with higher depth of invasion, neoplastic grading and TNM usually demonstrated lower 91H expression potentially represents Molecular Carcinogenesis

a novel clinically relevant event to identify individuals at increased risk for the occurrence, progression and prognosis of ESCC. ACKNOWLEDGEMENTS This study was supported by grants from the National Nature Science Foundation of China (no. 81172141), the Nanjing Science and Technology Committee project (no. 201108025), the Nanjing Medical Technology Development Project (no. ZKX11025) to Shukui Wang. REFERENCES 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69–90. 2. Parkin DM, Bray FI, Devesa SS. Cancer burden in the year 2000. The global picture. Eur J Cancer 2011;37:S4–S66. 3. Talukdar FR, Ghosh SK, Laskar RS, Mondal R. Epigenetic, genetic and environmental interactions in esophageal squamous cell carcinoma from northeast India. PLoS ONE 2013;8: e60996. 4. Reik W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 2007;447:425–432. 5. Peters J, Beechey C. Identification and characterisation of imprinted genes in the mouse. Brief Funct Genomic Proteomic 2004;2:320–333. 6. Leighton PA, Saam JR, Ingram RS, Stewart CL, Tilghman SM. An enhancer deletion affects both H19 and Igf2 expression. Genes Dev 1995;9:2079–2089. 7. Leighton PA, Ingram RS, Eggenschwiler J, Efstratiadis A, Tilghman SM. Disruption of imprinting caused by deletion of the H19 gene region in mice. Nature 1995;375:34–39. 8. Ripoche MA, Kress C, Poirier F, Dandolo L. Deletion of the H19 transcription unit reveals the existence of a putative imprinting control element. Genes Dev 1997;11:1596– 1604. 9. Kaaks R, Stattin P, Villar S, et al. Insulin-like growth factor-II methylation status in lymphocyte DNA and colon cancer risk in the Northern Sweden Health and Disease cohort. Cancer Res 2009;69:5400–5405. 10. Liou JM, Wu MS, Lin JT. Loss of imprinting of insulin-like growth factor II is associated with increased risk of proximal colon cancer. Eur J Cancer 2007;43:1276–1282. 11. Lai MC, Yang Z, Zhou L, et al. Long non-coding RNA MALAT-1 overexpression predicts tumor recurrence of hepatocellular carcinoma after liver transplantation. Med Oncol 2012;29: 1810–1816. 12. Matouk IJ, Mezan S, Mizrahi A, et al. The oncofetal H19 RNA connection: hypoxia, p53 and cancer. Biochim Biophys 2010;1803:443–451. 13. Benetatos L, Vartholomatos G, Hatzimichael E. MEG3 imprinted gene contribution in tumorigenesis. Int. J. Cancer 2011;129:773–779. 14. Rougeulle C, Heard E. Antisense RNA in imprinting: Spreading silence through Air. Trends Genet 2002;18:434– 437. 15. Rougeulle C, Cardoso C, Font es M, Colleaux L, Lalande M. An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript. Nat Genet 1998;19:15–16. 16. Sleutels F, Zwart R, Barlow DP. The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature 2002;415:810–813. 17. Wutz A, Smrzka OW, Schweifer N, Schellander K, Wagner EF, Barlow DP. Imprinted expression of the Igf2r gene depends on an intronic CpG island. Nature 1997;389:745–749. 18. Berteaux N, Aptel N, Cathala G, et al. A novel H19 antisense RNA overexpressed in breast cancer contributes to paternal IGF2 expression. Mol Cell Biol 2008;28:6731–6745.

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Long non-coding RNA 91H contributes to the occurrence and progression of esophageal squamous cell carcinoma by inhibiting IGF2 expression.

Long non-coding RNAs (lncRNAs) have been recently recognized as a major class of regulators in mammalian systems. 91H, a novel long noncoding antisens...
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