Abstract
Marek's disease is an oncogenic and lymphoproliferative disease of chickens caused by Marek's disease virus. Hypermethylation or hypomethylation of CpG islands in gene promoter region are involved in the initiation and progression of carcinogenesis. In this study, we analyzed differential methylation levels of upstream region of gga-miR-130b-3p gene between Marek's disease virus-infected tumorous and non-infected spleens. Around the upstream 1 kb of gga-miR-130b-3p gene, two amplicons were designed that covered 616 bp. There were forty-eight CpG sites in this region. CpG sites in this region presented higher methylation level in tumorous spleens compared with that in non-infected ones. There were eight CpG sites significantly hypermethylated in tumorous spleens. The expression level of three DNA methyltransferases including DNMT1, DNMT3a and DNMT3b increased and the expression level of Tet ten-eleven translocation protein 2 remarkably decreased in tumorous spleens. Hypermethylation in the upstream region of gga-miR-130b-3p gene might be a direct reason for its downregulation in MD tumorous tissues. Moreover, cell proliferation of Marek's disease lymphoblastoid cell line MDCC-MSB1 was remarkably inhibited at 24, 36, 48, 60 and 72 h post-gga-miR-130b-3p-agomir transfection. The transwell migration assay indicated cell number of migration was significantly lower in miRNA agomir transfection group. Matrix metalloproteinases MMP2 and MMP9 are involved in tumor invasion, and their protein levels were significantly downregulated at 72 h post-miRNA-agomir transfection. Collectively, these results indicated that hypermethylation in upstream region of gga-miR-130b-3p gene contributed to its downregulation in tumorous tissues. Gga-miR-130b-3p plays an inhibitory role in lymphomatous cell transformation.
Highlights
Marek’s disease (MD) in chickens is a critical disease that threatens the development of poultry, which is caused by oncogenic herpes virus Marek’s disease virus (MDV)
The upstream region of gga-miR-130b-3p gene was hypermethylated in MDV-infected tumorous spleens
The stimulating protein 1 (SP1) binding site was located at the CpG site 24, 25, 26, 27 in the upstream region of gga-miR-130b-3p gene and their methylation rate was 0.575, 0.900, 0.800, 0.675, respectively in tumorous spleens
Summary
Marek’s disease (MD) in chickens is a critical disease that threatens the development of poultry, which is caused by oncogenic herpes virus Marek’s disease virus (MDV) This disease results in various clinical syndromes and is mainly characterized by immunosuppression, neuritis, and rapid-onset neoplastic T-cell lymphomas formation in multiple visceral organs [1,2,3,4,5]. DNA methylation is a type of epigenetic modification that entails the covalent addition of a methyl group to the cytosine residue within the CpG context through the catalysis of DNA methyltransferases, which affects gene expression without genetic alterations [11]. Both miRNA and DNA methylation are crucial regulators of gene expression. MiRNAs can target DNMT mRNA and inhibit their expression, which results in variations on genome-wide methylation patterns [11, 15, 16]
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