The Functional Mechanism Investigation Of Gga-miR-103-3p, Gga-miR-130a, And Linc-GALMD3 In Chicken Marek’s Disease Lymphoma Tumorigenesis

Marek’s disease (MD) is a highly contagious T-cell lymphoid neoplasia induced by Marek’s disease virus (MDV), which causes huge economic losses to the poultry industry. In this study, we used the MD model to analyze the mechanism of miRNAs and lincRNAs in MDV tumorigenesis.In a previous study, we performed Solexa deep sequencing and discovered gga-miR-103-3p and gga-miR-130a were both remarkably downregulated in MDV-infected tissues. Currently, we further verified the expression of gga-miR-103-3p and gga-miR-130a among MDV-infected spleen, MD lymphoma from liver, non-infected spleen, and non-infected liver by qPCR. The target genes of gga-miR-103-3p and gga-miR-130a were predicted using TargetScan and miRDB, and clustered through Gene Ontology analysis. Six candidate target genes of gga-miR-103-3p and nine candidate target genes of gga-miR-130a were validated by dual luciferase reporter assay. Results showed that gga-miR-103-3p could combine the 3’UTR of CCNE1 and TFDP2 mRNA, and gga-miR-130a could bind to the 3’UTR of INSIG1, ACVR1, HOXA3, and MDFIC mRNA. Furthermore, results of the qPCR and Western Blot assays demonstrated that gga-miR-103-3p represses the protein expression of CCNE1 and TFDP2, inhibits the mRNA expression of CCNE1, but can not modulate the expression of TFDP2 mRNA; and gga-miR-130a regulates HOXA3 and MDFIC at the protein level but not at the mRNA level. In a word, CCNE1 and TFDP2 were the target genes of gga-miR-103-3p, and the target genes of gga-miR-130a were HOXA3 and MDFIC. Subsequent cell proliferation and migration assays suggested that gga-miR-103-3p suppresses MDCC-MSB1 migration, but can not obviously modulate the cell proliferation; and gga-miR-130a shows an inhibitory effect on MDCC-MSB1 cell proliferation and migration.Linc-GALMD3 was first identified and validated highly expressed in MDV-infected CD4+T cells in both two reciprocal cross lines, which were line 63 (MD resistant) × line 72 (MD susceptible), and line 72× line 63. The sequence of linc-GALMD3 in CD4+T and MDCC-MSB1 cells were totally the same, which were confirmed by Touchdown-PCR and Sanger Sequencing assays. Therefore, MDCC-MSB1 was used to explore the biological significance of linc-GALMD3 in MD tumor transformation. The shRNA was used to knockdown the expression of linc-GALMD3 in MDCC-MSB1 cells, and the highest interference efficiency was 53% (shRNA3-1657). The RNA-Seq was performed before and after linc-GALMD3 knockdown by shRNA3-1657, and 748 differentially expressed genes (DEGs) were found (FDR<0.01). We used the GO and KEGG pathway analysis to explore the biological function of the 748 DEGs. These genes were significantly clustered into 12 biological processes,19 cellular components, and 2 molecular functions, including cell cycle, mitochondrion, and nucleotide binding. Most of the DEGs were clustered into Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease, which are all neurodegenerative disease. Eight common genes shared in these three pathways were all participated in oxidative phosphorylation associated with Mitochondrial dysfunction that related to various cancers.In summary, this study reveals that gga-miR-103-3p and gga-miR-130a inhibits lympfoma cell proliferation and migration by targeting their corresponding target genes. Linc-GALMD3 was first identified in MD, and participates in MDV tumorigenesis with its 748 related genes. Our findings provide data resources and theoretical basis for functional mechanism investigation of non-coding RNAs in MD tumor transformation, and lay a foundation for resistance inheritance research.