Elsevier

Clinica Chimica Acta

Volume 431, 20 April 2014, Pages 255-259
Clinica Chimica Acta

Investigation of circulating lncRNAs in B-cell neoplasms

https://doi.org/10.1016/j.cca.2014.02.010Get rights and content

Highlights

  • Expression of the lncRNA molecules is tissue- and disease-specific.

  • This is the first study analyzing plasma lncRNA levels in B-cell malignancies.

  • TUG1 expression is high and HOTAIR levels are low in patients with multiple myeloma.

  • Lower GAS5 and lincRNA-p21 levels are observed in CLL.

Abstract

Long non-coding RNAs (lncRNA) which are longer than 200 base pairs in length, play an important role in cellular machinery. Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are neoplasms of B-cells. In our study we aimed to investigate circulating lncRNA levels of CLL and MM patients. For this purpose we selected 5 candidate lncRNAs (TUG1, LincRNA-p21, MALAT1, HOTAIR, and GAS5) where the first two are regulated by p53. Analyses were performed by real-time PCR using cDNA synthesized from plasma RNAs. In both disease groups differential levels of plasma lncRNAs were observed. LincRNA-p21 was the only molecule displaying significant changes in the CLL group while all remaining lncRNAs showed significant differences in the MM group. In the MM group only TUG1 showed higher levels than the healthy volunteers. In conclusion, the expression levels of the candidate lncRNA molecules display a general trend for tissue- and disease-specific expression which can provide important potential biomarkers specific to the particular disease type. However, further studies are necessary to elucidate their involvement in disease development and progression.

Introduction

CLL being the disease of the elderly, is the most frequently observed leukemia in the western world, with a heterogeneous clinical outcome due to the genetic alterations in the leukemic cells. Leukemic cells display a wide diversity in their morphology, immunophenotype and genetic material. The chromosomal abnormalities most frequently observed in CLL are deletion in chromosome 13q, trisomy 12, and deletions in 11q, 17p, and 6q (4). In addition to these, expression levels of the zeta-chain associated (TCR) protein kinase 70 kDa (ZAP-70) or CD38 (adverse prognostic marker) and the presence of somatic IGHV mutations (patients carrying mutations respond better to the therapy) are widely investigated and are promising prognostic markers of the disease [1].

Multiple myeloma (MM) is another example of B-cell malignancies and is characterized by uncontrolled proliferation of plasma cell clones in the bone marrow. The disease may involve diffuse marrow infiltration, focal bone lesions and soft tissue (extramedullary) disease. MM is incurable and the main biomarker used for its diagnosis is monoclonal gammopathy in serum or urine samples of the patients. Staging of the disease is performed according to the International Staging System [2].

Gene expression data obtained from high throughput DNA microarrays have provided valuable information on the genes aberrantly expressed in MM. Furthermore, gene expression profiling and gene copy number alterations have shown a promising prognostic role that needs to be validated in larger studies. Besides, interactions between myeloma cells and bone marrow cells or extracellular matrix proteins that are mediated through cell surface receptors influence tumor growth, survival, migration, and drug resistance [2].

Protein-coding genes constitute only about 2% of the human genome. However, up to 70% of the human genome is transcribed into RNA [3]. Therefore, the human genome contains much more non-coding information than coding, in the form of a wide variety of non-coding RNA transcripts. In addition to classical “housekeeping” RNAs (e.g., ribosomal RNAs, transfer RNAs, and others) and the more recently discovered and well-defined microRNAs (miRNAs), the genome is also packed with long non-coding RNAs (lncRNAs). It has been shown that the mammalian genome encodes more than a thousand functional lncRNAs that have been mostly conserved across the mammals [3], [4]. In a recent study 5446 lncRNA genes have been identified in the human genome and, combined with the previously published data this adds up to 6736 lncRNAs [5].

Due to their various structural and biochemical characteristics the lncRNAs are implicated in diverse biological functions, including nuclear architecture, regulation of gene expression, immune surveillance, or pluripotency of the embryonic stem cells. Recent studies provide evidence that lncRNAs are also involved in cancer development and maintenance of tumorigenesis [6], [7]. However, the mechanism by which these lncRNAs function, and their regulation and role in cancer is unknown at present.

Contrary to the conventional belief that RNA is labile in nature, cell-free RNA is present and relatively stable in circulation [8]. It has been suggested that RNA in plasma or serum is associated with particulate matter such as apoptotic bodies or lipid vesicles and thus protected from degradation by nucleases [9], [10]. Although a variety of RNA molecules including messenger RNA, and miRNAs have been investigated in the circulation so far, no data is available on the circulating lncRNAs and their relevance in disease.

The subject of the present study is cell-free lncRNAs in blood circulation and their significance in B-cell neoplasias. Since apoptosis and/or necrosis is a frequent event common to malignant tumors we hypothesized that lncRNA molecules are shed into circulation via vesicles and exosomes. To select the most appropriate candidate lncRNA molecules in B-cell neoplasia we analyzed the information available in the databases on the expression levels of the various lncRNA molecules in different cell and tissue types. As a result, five lncRNA molecules were selected according to their different functions in the tumor suppressor (LincRNA-p21 and GAS5), oncogenic (MALAT1 and TUG1) and epigenetic (HOTAIR) processes. HOTAIR is among the best-studied non-coding RNAs, and acts either as a cis- or trans-acting epigenetic regulator of chromatin [4]. It has recently been shown to promote invasiveness and metastasis in cancer [11], [12] by reprogramming the chromatin state [13], [11], [14] and making it an important target for diagnosis and therapy. The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is broadly expressed in human tissues [15] and known to be dysregulated in many human cancers. Expression of MALAT1 correlates with tumor development, and progression or survival in lung, liver and breast cancer [16]. The taurine upregulated gene 1 (TUG1) has recently been shown to be transcriptionally regulated by p53 in response to DNA damage [13].

LincRNA-p21 and GAS5 exhibit tumor suppressor activities [17], [18]. LincRNA-p21 has been named after its proximity to the p21 gene and acts as a transcriptional repressor. It forms a complex with the hnRNP-K ribonucleoprotein complex, and is involved in suppression of the cell cycle regulatory genes upon stimulation by the tumor suppressor protein p53 [17]. Expression of LincRNA-p21 is altered in certain conditions [19]. GAS5 (Growth Arrest Specific 5) has been first discovered in growth-arrested mouse NIH3T3 fibroblasts due to a significant increase in its expression level [18]. In humans it is transcribed from a small open reading frame on chromosome 1q25.1 which does not code for a functional protein [20], [21]. Interaction of GAS5 with the DNA binding domain of glucocorticoid receptors leads to the suppression of several antiapoptotic genes, rendering the cells sensitive to apoptosis [22]. Expression of GAS5 induces apoptosis in prostate and breast cancer cell lines. Lower GAS5 expression has been reported in breast tumors [23]. GAS5 is also a significant determinant in the mTOR pathway [24].

In this study, we investigated the circulating levels of the lncRNA molecules in plasma of the patients with chronic myeloid leukemia (CLL) and multiple myeloma (MM) and compared the levels with healthy subjects. This is the first report investigating the circulating long non-coding RNA levels in plasma of patients with chronic lymphocytic leukemia (CLL) and multiple myeloma (MM).

Section snippets

Patients and healthy controls

68 patients with CLL, 62 patients with MM and 40 healthy individuals were enrolled in the study. The study was approved by the local ethics committee and written informed consent was obtained from the participants. Characteristics of the patients are given in Table 1. Venous blood was drawn from patients and total RNA from 200 μl plasma was extracted using the TriPure Isolation Reagent (Roche, Germany).

RNA extraction from plasma was performed according to the protocol recommended by the

Results

Plasma lncRNA levels were analyzed in 68 patients with CLL, 62 patients with MM and 36 healthy controls. We observed a significant difference in the expression levels of TUG1, MALAT1, HOTAIR and GAS5 lncRNAs between the patients with MM and the healthy controls. Interestingly, only LincRNA-p21 was differentially expressed in the CLL group (Fig. 1, Table 3a, Table 3b). Cell-free lncRNA expression levels in the patients were lower than the healthy controls except for TUG1 expression which was

Discussion

lncRNAs are implicated in numerous biological functions such as cell cycle control, apoptosis, imprinting and epigenetic regulation. They act in the modulation of transcription and play regulatory roles analogous to the proteins in controlling the localization and activity of proteins. LncRNAs function as scaffolds, microRNA sponges, competitors or activators for transcription, and take part in paraspeckles, splicing and endo si-RNA formation [26].

In this study we investigated expression levels

Conclusion

In conclusion, the expression levels of the candidate lncRNA molecules display a general trend for tissue- and disease specific expression which can provide important potential biomarkers specific to the particular disease type. However, further studies are necessary to elucidate their involvement in disease development and progression.

Acknowledgement

This study was supported by the Scientific Research Projects Coordination Unit of Istanbul University, Project Number: 22012.

References (49)

  • J.L. Rinn et al.

    Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs

    Cell

    (2007)
  • A. Palumbo et al.

    Multiple myeloma

    N Engl J Med

    (2011)
  • Y. Sanchez et al.

    Long non-coding RNAs: challenges for diagnosis and therapies

    Nucleic Acid Ther

    (2013)
  • J.S. Mattick

    The genetic signatures of noncoding RNAs

    PLoS Genet

    (2009)
  • H. Jia et al.

    Genome-wide computational identification and manual annotation of human long noncoding RNA genes

    RNA

    (2010)
  • E.A. Gibb et al.

    Human cancer long non-coding RNA transcriptomes

    PLoS One

    (2011)
  • M. Huarte et al.

    Large non-coding RNAs: missing links in cancer?

    Hum Mol Genet

    (2010)
  • B.C. Wong et al.

    Plasma RNA integrity analysis: methodology and validation

    Ann N Y Acad Sci

    (2006)
  • E.K. Ng et al.

    Presence of filterable and nonfilterable mRNA in the plasma of cancer patients and healthy individuals

    Clin Chem

    (2002)
  • R.A. Gupta et al.

    Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis

    Nature

    (2010)
  • Y. Wan et al.

    HOTAIR: flight of noncoding RNAs in cancer metastasis

    Cell Cycle

    (2010)
  • A.M. Khalil et al.

    Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression

    Proc Natl Acad Sci U S A

    (2009)
  • M.C. Tsai et al.

    Long noncoding RNA as modular scaffold of histone modification complexes

    Science

    (2010)
  • R. Lin et al.

    A large noncoding RNA is a marker for murine hepatocellular carcinomas and a spectrum of human carcinomas

    Oncogene

    (2007)
  • Cited by (156)

    • Applications of noncoding ribonucleic acids in multiple myeloma patients

      2022, Clinical Applications of Noncoding RNAs in Cancer
    View all citing articles on Scopus
    View full text