A-to-I RNA editing of the IGFBP7 transcript increases during aging in porcine brain tissues

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Abstract

The IGFBP7 gene encodes insulin-like growth factor protein 7. IGFPB7 is involved in diverse biological functions including cell growth regulation, senescence and apoptosis, and also acts as a tumor suppressor in multiple cancers. The IGFBP7 mRNA is subject to A-to-I RNA editing mediated by adenosine deaminases acting on RNA 1 and 2 (ADAR1 and ADAR2). In the current study we have examined molecular characteristics of the porcine IGFBP7 gene, and determined the mRNA editing in different tissues. The A-to-I RNA editing of human IGFBP7 in positions Arg78 and Lys95 was shown to be conserved in the porcine homologue. In addition, a novel editing site was discovered in position Lys97 in the porcine IGFBP7 transcript. A differential editing was demonstrated at the three positions in the IGFBP7 transcript with very high degrees of editing in frontal cortex, cerebellum and lung. Interestingly, the degree of editing increased during aging in porcine frontal cortex and cerebellum. The IGFBP7 gene was mapped to pig chromosome 8. The porcine IGFBP7 gene was found to be ubiquitously expressed in examined organs and tissues. The methylation status of the IGFBP gene was examined in brain and liver by bisulfate sequencing and a high degree of methylation was found in the two tissues, 52% and 54%, respectively.

Introduction

IGFBP7 belongs to a family of soluble proteins that bind insulin-like growth factors (IGFs) with high affinity. The principal functions of insulin-like growth factors (IGFBPs) are to regulate IGF availability in body fluids and tissues and to modulate IGF binding to its receptors [1]. The IGFBP7 gene encodes a secreted protein of 282 amino acids which binds to IGF-1 and IGF-2 with low affinity. IGFBP7 binds with higher affinity to both insulin and activin [2], [3], [4]. IGFBP7 has diverse functions in various cellular processes such as differentiation, cell growth and survival, senescence and apoptosis [5], [6], [7], [8]. The IGFBP7 gene was originally cloned from mammary epithelial cells and leptomeningeal cells and named mac25 [9], [10]. The human IGFBP7 gene, encoding a 30 kDa polypeptide, is located on chromosome 4 and contains five exons. IGFBP7 mRNA is ubiquitously expressed and also found with low expression in different cancer cells [1]. The down-regulated IGFBP7 expression is observed in many types of malignancies such as colon cancer, prostate cancer and gastric cancer [11], [12], [13], [14] and this has led to a suggested a role of IGFBP7 as a tumor suppressor gene.

A-to-I RNA editing is the post-transcriptional processing of premRNAs and is mediated by adenosine deaminases acting on RNAs (ADARs) that specifically bind to and deaminate their partially double-stranded RNA targets [15], [16]. A-to-I editing is an important mechanism for generating RNA and protein diversity [15]. A-to-I RNA editing can lead to amino acid substitutions, since inosine is interpreted as guanosine by the translational machinery. Human IGFBP7 mRNA is a target of ADARs, and is edited at two positions changing codon 78 from AGG (arginine) to IGG (glycine), and codon 95 from AAG (lysine) to AIG (arginine), also termed R78G and K95R, respectively [16], [17]. The main editing site (K95R) is located in exon 1 within the proposed heparin-binding site of IGFBP7, and is also part of the recognition sequence for proteolytic cleavage. A-to-I editing at the two sites, R78G and K95R, allows for the production of four protein isoforms of IGFBP7 from one allele. Editing of IGFBP7 transcripts has a strong influence on the protein's susceptibility to proteolytic cleavage, and thereby provides a means for modulation of IGFBP7 functionality [18].

In the present study, we report molecular characteristics of the porcine IGFBP7 gene and protein and in particular we describe the IGFBP7 mRNA editing profile in various pig tissues. The degree of IGFBP7 mRNA editing increased during aging in porcine frontal cortex and cerebellum.

Section snippets

Biological materials

Pigs were housed and used in compliance with European Community animal care guidelines. Beforehand, the experimental procedures were submitted to the National Ethical Committee in Denmark. Pig cerebellum, cortex and liver used for RT-PCR cloning of IGFBP7 were obtained from a one year old Danish Landrace pig (id. 147). The following pigs were included in the RNA editing analysis: Danish Landrace pigs (id. 147, 3713 and 9071), three 115 d old embryos, one 7 year old female Danish Landrace, one

Characterization of porcine IGFBP7 cDNA and gene

Using IGFBP7-specific primers and RT-PCR the cDNA representing the entire IGFBP7 open reading frame was amplified, cloned and sequenced. Five porcine IGFBP7 cDNAs were cloned, a full-length completely identical with the published (NM_001163801), and four shorter versions all lacking parts of exon 1, all shown in Fig. S1.

The pig IGFBP7 cDNA was highly homologous (94%) with the human counterpart throughout the coding sequence. The deduced porcine IGFBP7 amino acid sequence contains several

Acknowledgements

The authors wish to thank Bente Flügel and Hanne Jørgensen for excellent technical assistance. We also thank Monica Pettersson from Qiagen for many good advices with pyrosequencing.

References (38)

  • L. Sullivan et al.

    IGFBP7 promoter methylation and gene expression analysis in prostate cancer

    J. Urol.

    (2012)
  • H.Y. Agbemenyah et al.

    Insulin growth factor binding protein 7 is a novel target to treat dementia

    Neurobiol. Dis.

    (2014)
  • M.V. Kato

    A secreted tumor-suppressor, mac25, with activin-binding activity

    Mol. Med.

    (2000)
  • S.M. Firth et al.

    Cellular actions of the insulin-like growth factor binding proteins

    Endocr. Rev.

    (2002)
  • G.E. Walker et al.

    Neuroendocrine-like differentiation of non-small cell lung carcinoma cells: regulation by cAMP and the interaction of mac25/IGFBP-rP1 and 25.1

    Oncogene

    (2006)
  • V. Evdokimova et al.

    IGFBP7 binds to the IGF-1 receptor and blocks its activation by insulin-like growth factors

    Sci. Signal.

    (2012)
  • M. Murphy et al.

    Identification and characterization of genes differentially expressed in meningiomas

    Cell Growth Differ.

    (1993)
  • K. Swisshelm et al.

    Enhanced expression of an insulin growth factor-like binding protein (mac25) in senescent human mammary epithelial cells and induced expression with retinoic acid

    Proc. Natl. Acad. Sci. U. S. A.

    (1995)
  • W.J. Ruan et al.

    IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis with its expression associated with DNA hypomethylation of exon 1

    J. Zhejiang Univ. Sci. B

    (2006)
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