Epigenetic regulation of gga-microRNA-126 during a virus-induced lymphoma in chicken Integrated veterinary unit research (IVRU) I. Gennart; D. Coupeau; L. Parissi; S. Pejakovic; B. Muylkens Introduction Gga-miR-126 was undetectable in the infected cell lines while in the nine organs it was expressed. Gga-miR-126 expression was the most abundant in the lung, the heart, the liver and the kidney. It can be concluded that gga-miR-126 is not detectable in tumor-induced latently infected cells. Then, to characterize this low expression level, DNA methylation pattern was assessed in MSB-1 cell line and in the nine organs through Bisulfite Genomic Sequencing Assay (BGSA). Gga-miR-126 is located in the chromosome 17 into the 6th intron of the cellular gene EGFL-7 (Epidermal Growth Factor Like-7). By an in silico analysis, two CpG islands were found in this gene. One CpG island (CpG1) is located in the first intron and the other one (CpG2) is located around the precursor of gga-miR-126 (gga-pre-miR-126). The DNA methylation pattern was assessed in these two CpG islands. In MSB-1 cell line, the level of DNA methylation was very high at the two CpG islands (CpG1, 85 % and CpG2, 95 %). In the nine organs, DNA methylation was also observed in CpG1 and CpG2 with a different percentage. Indeed, a lower level of methylation was observed in CpG1 (from 7 % to 31 %) while CpG2 presented a high level of methylation (from 53 % to 85 %). The repression of gga-miR-126 in latently infected cells (MSB-1) might be explained by the high percentage of methylation at the two CpG islands. In human, these two CpG islands are also present in EGLF-7 and DNA methylation was assessed at CpG1 site in normal and tumoral cells (Watanabe et al., 2012). In tumoral cells CpG1 site presented a high percentage of methylation while in normal cells a low level of DNA methylation was observed. Watanabe et al., quantified then hsa-miR-126 in tumoral and normal cells and observed that a low and a high expression were observed, respectively. These results showed that there is a relation between CpG1 site and the regulation of hsa-miR-126 in cancerous and normal cells. For now in the chicken, it was observed a low level of gga-miR-126 in tumoral cells (MSB-1). This repression of gga-miR-126 could be explained by the high level of DNA methylation in the two CpG islands (CpG1 and 2) present into EGFL-7. It would be interesting to find transcriptional start site(s) to assess if it has an independent or dependent expression from EGFL-7 and to assess the exact impact of DNA methylation on gga-miR-126 expression. Both in human and animal, some of the herpesvirus latent infections progress to cancer. Gallid herpesvirus-2 (GaHV-2), an oncogenic α-herpesvirus, modulates viral and cellular gene expression and triggers transformation of T CD4+ latently infected cells. This avian pathogen, responsible for the Marek’s disease (MD), naturally infects chickens and provides a unique model for studying virus induced lymphoma development. This study focus on a microRNA (gga-miR-126) downregulated during the viral life cycle. Originally described as a miRNA mediating proper angiogenesis and vascular integrity, gga-miR-126 has been reported to impair cancer progression through signaling pathways that control tumor cell proliferation, migration and survival. Results Gga-miR-126 is not expressed in transformed T cells latently infected with GaHV-2 The two CpG islands of gga-miR-126 locus are highly methylated in virus transformed T cells N=3 Organs Brain Thymus gland Testis Liver MSB-1 Cerebellum Kidney Heart Spleen Lung Gga-miR-126 is undetectable in tumor-induced latently infected cells (MSB-1 cells) Gga-miR-126 expression is the most abundant in the lung and the heart Methylation siganture is different for MSB-1 cells compare to the 9 organs Figure 1: Graphic representing gga-miR-126 expression level in tumoral infected cell line (MSB-1) and nine organs of three uninfected chicken. In the X axis, relative quantification of gga-miR-126. In the Y axis, cell line and organs used for the quantification. Q-RT-PCR (quantitative-reverse transcription-polymerase chain reaction) was performed for gga-miR-126 quantification. Results were standardized with 3 housekeeping genes (18s ribosomal RNA, β-actine, succinate dehydrogenase subunit A) selected with the GENORM software. N represents the number of independent experimentation performed. The error barrs are the standard deviation calculated from relative quantification of gga-miR-126 from three independent well. Figure 2: Graphic representing gga-miR-126 global DNA methylation level in tumoral infected cell line (MSB-1) and 9 organs of three healthy chicken at two CpG islands (see detailed locations in fig. 3). In the X axis, DNA methylation level at the loci CpG 1 (percentage). In the Y axis, DNA methylation level at the loci CpG2 (percentage). DNA methylation pattern of two CpG islands in gga-EGFL-7/miR-126 gene A B C RE In MSB-1 cell line, the level of DNA methylation is very high at the two CpG islands (CpG1, 86 % and CpG2, 95 %) In the nine organs, a lower level of methylation is observed in CpG1 (from 7 % to 29 %) while CPG 2 presented a high level of methylation (from 53 % to 86 %) Figure 3: (A) Structure of gga-EGFL-7 (Endothelial Growth Factor Like-7)/miR-126 gene. Gga-EGFL-7 is composed of 9 exons (grey boxes) and 8 introns (black barrs). Gga-miR-126 (yellow box) is located in the 6th intron of the cellular gene. Two CpG islands are present in the first (CpG1) and the 6th intron (CpG2). (B) (C) Precise location of methylated cytosine (rectangle) at cytosine/guanine (CG) dinucleotides in CpG island 1 (CpG1, B) and CpG island 2 (CpG2, C) . DNA methylation pattern was assessed in MSB-1 cells and in the 9 organs of three healthy chicken through Bisulfite Genomic Sequencing Assay (BGSA). Inside each rectangle there is black rectangle representing the average level of DNA methylation. RE indicate the location of response element for major transcription factors found by bioinformatic analysis (genomatix software). Percentage at the right of figures B and C are the global DNA methylation pattern for CpG1 and CpG2 in the different condition. Conclusion Altogether these results show a low expression of miR-126 in tumoral cells (MSB-1) (Fig.1). This repression of gga-miR-126 could be explained by the high level of DNA methylation in the two CpG islands (CpG1 and CpG2) present into gga-EGFL-7/miR-126 gene (Fig.3B and 3C). It would be interesting to find transcriptional start site(s) to assess if gga-miR-126 has an independent or dependent expression from gga-EGFL-7 and to identify a promoter region to determine which transcription factors are implicated in gga-miR-126 expression.