Oncology
Aberrant methylation-mediated downregulation of long noncoding RNA LOC100130476 correlates with malignant progression of esophageal squamous cell carcinoma

https://doi.org/10.1016/j.dld.2016.05.010Get rights and content

Abstract

Background

Dysregulated long non-coding RNAs (lncRNAs) are involved in many complicated human diseases including cancer.

Aims

To determine the role and methylation status of a new lncRNA LOC100130476 in the pathogenesis of esophageal squamous cell carcinoma (ESCC).

Methods

One hundred and twenty three ESCC patients with tumor tissues and corresponding adjacent normal tissues were enrolled. The expression level and methylation status of LOC100130476 in esophageal cancer cell lines and primary ESCC samples were respectively detected.

Results

Significant downregulation of LOC100130476 was detected in esophageal cancer cell lines and primary ESCC tumor tissues. Up-regulation of LOC100130476 led to the inhibition of proliferation and invasiveness of the cancer cells. Aberrant hypermethylation of the CpG sites in exon 1 closing to the transcription start site was found to be more tumor-specific and to be more critical for gene silencing. Hypermethylation of these CpG sites was associated with TNM stage and pathological differentiation. ESCC patients in stage III and IV, with low expression or hypermethylation of the CpG sites in exon 1 demonstrated poor patient survival.

Conclusions

LOC100130476 is down-regulated in ESCC at least partly by hypermethylation of CpG sites in exon 1 and its hypermethylation may have prognostic implications for ESCC patients.

Introduction

Esophageal cancer presents symptomatically late in the course of the disease and, despite currently available therapies, carries a poor survival rate [1]. The incidence and mortality of esophageal cancer continue to rise in the United States and United Kingdom during recent years [2]. Adenocarcinoma (AC) and squamous cell carcinoma (SCC) are the two main histological types of esophageal cancer. Esophageal squamous cell carcinoma (ESCC) is the main type of esophageal cancer in China, and has great geographical as well as sociocultural variation in incidence [3]. Some of the high incidence areas of ESCC are in northern China, especially in some counties bordering Hebei, Henan, and Shanxi Provinces [4]. There is a strong tendency toward familial aggregation of ESCC in these high-risk areas; however, a large proportion of the etiology of ESCC in these populations still remains largely unknown for the moment.

As a type of non-coding RNAs (ncRNAs), long non-coding RNAs (lncRNAs) are molecules greater than 200 nt in length, frequently ranging up to 100 kb [5], [6]. Although initially argued to be spurious transcriptional noise, recent evidence suggests that lncRNAs, the proverbial ‘dark matter’ of the genome may play important roles in a wide range of biological processes including stress response, alternative splicing, chromatin remodeling, mRNA decay, cellular development, differentiation, and metabolism [7], [8]. LncRNAs are highly conserved and may function as key regulators in gene regulation via various ways in the cytoplasm and the nucleus. It is not surprising that dysregulated lncRNAs are involved in many complicated human diseases including cancer [9]. LncRNAs have cell-type-specific expression patterns, and increasing evidence has shown altered expression level of lncRNAs in various types of human cancer and dysregulated lncRNAs may function as tumor suppressors or oncogenes, which may be used as potential early tumor diagnostic, metastatic or prognostic markers and molecular-targeted therapy sites in the future [10]. Though only a handful of lncRNAs have been characterized, recent studies have demonstrated that certain lncRNA are specifically associated with ESCC. Plasma levels of POU3F3 were significantly higher in ESCC patients compared with normal controls [11]. Patients with higher depth of invasion, neoplastic grading and TNM usually demonstrated lower lncRNA 91H expression [12]. Upregulation of the lncRNA PlncRNA-1, MALAT1, TUG1 promoted proliferation and migration of ESCC [13], [14], [15]. Furthermore, dysregulated expression of HOTAIR, CCAT2, PCAT-1, UCA1, LOC285194, FOXCUT, and SPRY4-IT1 were associated with prognosis in patients with ESCC [16], [17], [18], [19], [20], [21], [22].

More recently, it has been recognized that the misregulation of lncRNAs is involved in cancer epigenetics [23], [24]. DNA methylation was one of the first epigenetic alterations identified in cancer. Hypermethylation of specific CpG islands can lead to the silencing of tumor suppressor genes involved in key cellular pathways [25]. Promoter methylation may account for the loss of expression of some lncRNAs including H19, MEG3, and SRHC [26], [27], [28]. We and Cao et al. respectively found a new lncRNA, LOC100130476, which demonstrated greatly reduced expression in ESCC by analyzing ESCC lncRNA expression microarrays [29]. LOC100130476 is located on 6q23.3 (GRCh 38/hg38 database, from chr6: 137823670 to 137868233, NCBI: NR_049793.1) (Fig. 1A) and has three CpG islands spanning the regions from +208 to +1539 bp determined by MethPrimer program. We hypothesized that dysregulation of LOC100130476 may be associated with the occurrence and progression of ESCC and aberrant CpG island methylation may be one of the mechanisms to lead to the inactivation of LOC100130476 in ESCC. In the present study, we examined the function and methylation status of LOC100130476 in esophageal cancer cell lines and ESCC tumor tissues, and further elucidate the role of LOC100130476 in the pathogenesis and prognosis of ESCC.

Section snippets

Cell culture and treatment

A total of four human esophageal cancer cell lines TE1, TE13, T.Tn, and Eca109 were examined in this study. Cells were seeded at a low density and incubated for 24 h prior to treatment with DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC). All four esophageal cancer cells (2 × 105/mL) were treated with 5 μmol/L 5-Aza-dC (Sigma, St Louis, MO, USA) for 72 h and medium containing 5-Aza-dC was changed every 24 h. Control cells received no drug treatment.

Patients and specimens

Surgical primary ESCC tissues and

Frequent silencing of LOC100130476 and up-regulation of the gene by 5-Aza-dC treatment in esophageal cancer cell lines

As shown in Fig. 1B and C, the expression of LOC100130476 was remarkably reduced or silenced in four esophageal cancer cell lines. After treatment with DNA methyltransferase inhibitor 5-Aza-dC, the expression level of LOC100130476 was significantly increased in the four esophageal cancer cell lines, indicating the important role of aberrant methylation in the inactivation of LOC100130476 in esophageal cancer cell lines.

Inhibition effect of LOC100130476 on esophageal cancer cell proliferation

CCK-8 assay was used to examine the proliferation of 5-Aza-dC treated or

Discussion

Dysregulation of lncRNAs have been found in a number of cancers, suggesting the potential possibility of lncRNAs as prospective novel therapeutic targets in cancers. Here, we found a new long noncoding RNA LOC100130476 that may play tumor suppressor gene role in ESCC progression. To our best knowledge, the roles and epigenetic inactivation mechanism of LOC100130476 on tumor progression and prognosis have not been investigated and clarified so far. In the present study, we found silenced or

Conflict of interest

None declared.

Funding

This study was supported by grants from the National Natural Science Foundation (No. 81472335), Natural Science Foundation of Hebei Province (No. H2015206196), and Financial Department of Hebei Province [No. (2012) 2056].

References (36)

  • M. Sun et al.

    From discovery to function: the expanding roles of long noncoding RNAs in physiology and disease

    Endocrine Reviews

    (2015)
  • M.T. Qiu et al.

    Long noncoding RNA: an emerging paradigm of cancer research

    Tumour Biology

    (2013)
  • Y.S. Tong et al.

    Identification of the long non-coding RNA POU3F3 in plasma as a novel biomarker for diagnosis of esophageal squamous cell carcinoma

    Molecular Cancer

    (2015)
  • T. Gao et al.

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

    Molecular Carcinogenesis

    (2015)
  • C.M. Wang et al.

    Upregulation of the long non-coding RNA PlncRNA-1 promotes esophageal squamous carcinoma cell proliferation and correlates with advanced clinical stage

    Digestive Diseases and Sciences

    (2014)
  • L. Hu et al.

    Up-regulation of long noncoding RNA MALAT1 contributes to proliferation and metastasis in esophageal squamous cell carcinoma

    Journal of Experimental and Clinical Cancer Research

    (2015)
  • Y. Xu et al.

    Upregulation of the long noncoding RNA TUG1 promotes proliferation and migration of esophageal squamous cell carcinoma

    Tumour Biology

    (2015)
  • F.J. Chen et al.

    Upregulation of the long non-coding RNA HOTAIR promotes esophageal squamous cell carcinoma metastasis and poor prognosis

    Molecular Carcinogenesis

    (2013)
  • Cited by (23)

    • WAKMAR2, a Long Noncoding RNA Downregulated in Human Chronic Wounds, Modulates Keratinocyte Motility and Production of Inflammatory Chemokines

      2019, Journal of Investigative Dermatology
      Citation Excerpt :

      Recently, Tsoi et al. (2015) profiled lncRNA expression in psoriasis skin, revealing LOC100130476 as top downregulated in lesional compared with nonlesional skin of psoriasis patients, which is a chronic inflammatory skin disease sharing some features with wounded skin, for example, epidermis thickening and inflammation (Morhenn et al., 2013; Nickoloff et al., 2006). Also, LOC100130476 was recently reported to be downregulated in esophageal squamous cell carcinoma (Guo et al., 2016a). Among the 53 human tissues characterized in the Genotype-Tissue Expression Project (Lonsdale et al., 2013), the skin has the highest LOC100130476 expression, suggesting its functional role in the skin.

    • LncRNA and mRNA integration network reconstruction reveals novel key regulators in esophageal squamous-cell carcinoma

      2019, Genomics
      Citation Excerpt :

      LOC100130476 is downregulated in ESCC, and its downregulation is attributed to the hypermethylation of CpG sites in exon 1. Its hypermethylation was also associated with malignant progression and poor patient survival in ESCC [83]. In a very recent study, ADAMTS9-AS1 and another lncRNA, AP000696.2, were introduced together as a novel prognostic biomarker for ESCC [84].

    • Advances in esophageal cancer: A new perspective on pathogenesis associated with long non-coding RNAs

      2018, Cancer Letters
      Citation Excerpt :

      LOC100130476 is a lncRNA that contains 3 CpG islands. Guo et al. [32] found that this lncRNA is significantly downregulated in ESCC due to unusual hypermethylation of the CpG sites in exon 1, which is close to the transcription start site of LOC100130476. This finding indicates that LOC100130476 may play a critical role in gene silencing and may lead to an advanced TNM stage and poor differentiation.

    • Mutual interaction of lncRNAs and epigenetics: focusing on cancer

      2023, Egyptian Journal of Medical Human Genetics
    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text