School of Medicine and Health Sciences Poster Presentations
Target validation of differentially expressed miRNA in ectopic germinal centers of myasthenia gravis thymus
Document Type
Poster
Status
Postdoc
Abstract Category
Immunology/Infectious Diseases
Keywords
myasthenia gravis, germinal center, thymus, miRNA, reciprocal pairing
Publication Date
Spring 2018
Abstract
Myasthenia gravis (MG) is an autoimmune neuromuscular disorder resulting in weakness of voluntary muscles. The autoantibodies are directed against proteins present at the post-synaptic surface of neuromuscular junction (NMJ). A characteristic pathology of patients with early onset MG (EOMG) is thymic hyperplasia with ectopic germinal centers (GC). However, mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. Previous study assessed the central mechanisms involved in the formation of GCs in the thymus through analysis of the microRNA (miRNA) and mRNA expression. Briefly, thymus samples from EOMG patients were grouped based on appearance of GC. MiRNA and mRNA were evaluated using GeneChip® miRNA 4.0 Array and GeneChip® Human Transcriptome Array 2.0, respectively. Partek Genomic Suite 6.6 and Transcript Analysis Console 2.0 programs were used for further analysis. Thirty-four mature miRNAs and forty-six annotated mRNA transcripts were differentially expressed between the two groups (>1.5 fold change, ANOVA p<0.05). Reciprocal pairing analysis of miRNA and mRNA identified 11 pairs of miRNA and target mRNA. Target Scan identified Regulator of G protein Signalling 13 (RGS13), known to be involved in GC regulation, as potential target for miR139-5p and miR 452-5p. Interferon Regulatory Factor 8 (IRF8) expressed in GC positive samples was identified as another target for miR-452-5p. Both RGS13 and IRF8 are upregulated in GC positive samples.
In this study, we validated the reciprocal pairing of miRNA (miR139-5p and miR452-5p) and the target mRNAs (RGS13 and IRF8). Dual luciferase assay was used to validate target binding. Putative miRNA binding region from 3’ untranslated region (UTR) of these genes were cloned into pmirGlo vector downstream of firefly luciferase gene. The constructs were co-transfected with respective miRNA mimics and negative controls in 293T cell lines. Luciferase assay was performed 48 hours post transfection. Firefly luciferase activity was normalized to the Renilla luciferase activity as internal control. There was significant decrease in firefly luciferase activity on transfection of miRNA mimic as compared to negative control. Our results shown that target genes RGS13 and IRF8 are regulated by the miRNA.
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Open Access
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Target validation of differentially expressed miRNA in ectopic germinal centers of myasthenia gravis thymus
Myasthenia gravis (MG) is an autoimmune neuromuscular disorder resulting in weakness of voluntary muscles. The autoantibodies are directed against proteins present at the post-synaptic surface of neuromuscular junction (NMJ). A characteristic pathology of patients with early onset MG (EOMG) is thymic hyperplasia with ectopic germinal centers (GC). However, mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. Previous study assessed the central mechanisms involved in the formation of GCs in the thymus through analysis of the microRNA (miRNA) and mRNA expression. Briefly, thymus samples from EOMG patients were grouped based on appearance of GC. MiRNA and mRNA were evaluated using GeneChip® miRNA 4.0 Array and GeneChip® Human Transcriptome Array 2.0, respectively. Partek Genomic Suite 6.6 and Transcript Analysis Console 2.0 programs were used for further analysis. Thirty-four mature miRNAs and forty-six annotated mRNA transcripts were differentially expressed between the two groups (>1.5 fold change, ANOVA p<0.05). Reciprocal pairing analysis of miRNA and mRNA identified 11 pairs of miRNA and target mRNA. Target Scan identified Regulator of G protein Signalling 13 (RGS13), known to be involved in GC regulation, as potential target for miR139-5p and miR 452-5p. Interferon Regulatory Factor 8 (IRF8) expressed in GC positive samples was identified as another target for miR-452-5p. Both RGS13 and IRF8 are upregulated in GC positive samples.
In this study, we validated the reciprocal pairing of miRNA (miR139-5p and miR452-5p) and the target mRNAs (RGS13 and IRF8). Dual luciferase assay was used to validate target binding. Putative miRNA binding region from 3’ untranslated region (UTR) of these genes were cloned into pmirGlo vector downstream of firefly luciferase gene. The constructs were co-transfected with respective miRNA mimics and negative controls in 293T cell lines. Luciferase assay was performed 48 hours post transfection. Firefly luciferase activity was normalized to the Renilla luciferase activity as internal control. There was significant decrease in firefly luciferase activity on transfection of miRNA mimic as compared to negative control. Our results shown that target genes RGS13 and IRF8 are regulated by the miRNA.