The Differentially-expressed LncRNAs And The Bioinformatics Analysis In Myasthenia Gravis

ObjectiveThis study was aimed to explore the difference expression of lncRNAs and mRNA among the myasthenia gravis patients accompany with thymoma or not by lncRNA microarray technology. We will predict the lncRNAs’potential co-expressed target genes, biofuncional clusters and signal transduction pathways by using bioinformatics method. It will utilize the means of cis and trans prediction to analyze the mechanism involved in gene regulation and the networks of TF-lncRNA-target gene. Real-time PCR was used to confirm some of the outstanding differentially expressed lncRNAs. This study will support the foundation theory for the investigation of pathogenesis of myasthenia gravis.Method1. Collection of peripheral blood samples of myasthenia gravis patients and normal controls. Separate the lymphocytes and extract the total RNA.2. Exploration the lncRNA and mRNA expression profile via lncRNA microarray in myasthenia gravis patients and normal controls. Screen the outstanding difference expression of lncRNAs and mRNA（difference ratio>2, p<0.05）.3. Bio-informatics analysis including potential target genes prediction, ENCODE database enriched terms and signal pathways. Construction of networks of TF-lncRNA-target genes. Prediction of differentially expressed lncRNA target genes via the Pearson correlation coefficient. Association analysis for construction of lncRNA-mRNA co-expression network. Analysis of TF-lncRNA-target genes three elementary relationship by Cytoscape.4. Verify the differentially expressed lncRNAs by quantitative real-time PCR:some of the significantly differentially-expressed lncRNAs as detected by microarray were chosen and confirmed by RT-PCR. Comparison the expression status in three groups with each other （myasthenia gravis accompany with thymoma, myasthenia gravis without thymoma and normals）.Results1. Using lncRNA microarray, differently expressed lncRNAs were detected as follows（difference ratio≥2, p≤0.05）:comparison between myasthenia gravis with thymoma and normals, total of1489different lncRNAs were detected, including218up-regulated and1271down-regulated; total of862mRNAs were detected, including249up-regulated and613down-regulated;comparison between myasthenia gravis without thymoma and normals, total of342lncRNAs were detected, including172up-regulated and170down-regulated; total of353mRNAs were detected, including263up-regulated and90down-regulated;comparion between myasthenia gravis with thymoma and without thymoma, total of281lncRNAs were detected, including52up-regulated and229down-regulated. total of204were detected, including59up-regulated and145down-regulated.2. Comparison of myasthenia gravis patients with thymoma and normals: Choose the most significantly differentially expressed up-regulated lncRNA（lncRNA oebiotech₁1933, lncRNA A-24-P927716） and down-regulated lncRNA（lncRNA A-21-P0010030, lncRNA A-21-P0002844） to predict their target genes and bio-function one by one. Confirm the expression by RT-PCR. Perform the gene ontology （GO） analysis and enriched pathway analysis. Found that the cell response to interferon y play the most important role in pathogenesis of MG accompanied by thymoma, followed by positively regulatory cytokine production, regulation of smooth muscle cell proliferation, cytokine receptor biding and so on. Confirm the regulatory mechanism of17lncRNAs on the chromosomes via cis. Meanwhile, construct the two elementary relationship between cytokines（CTCF, TAF1and so on） and diffenentially expressed lncRNAs via trans analysis, build a CTCF-lncRNA oebiotech₂4272-SOST three elementarily mutual regulatory network on the basis of mRNA co-expression. 3. Comparison of myasthenia gravis patients without thymoma and normals:Choose the most significantly differentially expressed up-regulated lncRNA（lncRNA oebiotech₁1933,lncRNA oebiotech₀3926） and down-regulated lncRNA（lncRNA oebiotech₀2627, lncRNA oebiotech22482） to predict their target genes and bio-function one by one. Confirm the expression by RT-PCR. Perform the gene ontology （GO） analysis and enriched pathway analysis. Found that the platelet degramnulation and cell response to interferon y play the most important role in pathogenesis of MG without thymoma, followed by transduction of regulated cytokine, transmission of negatively regulatory ion, inflammatory response, glucocorticoid stimulation, regulation of cytokine production process and so on. Confirm the regulatory mechanism of6lncRNAs on the chromosomes via cis. Meanwhile, construct the two elementary relationship between transcription factors（CTCF, MYC and so on） and diffenentially expressed lncRNAs via trans analysis, build a CTCF-lncRNA A₂1_P0007083-NUS1three elementarily mutual regulatory network on the basis of mRNA co-expression.4. Comparison of myasthenia gravis patients with thymoma and those without thymoma:Choose the most significantly differentially expressed up-regulated lncRNA（lncRNA oebiotech₁3222,lncRNA A-19-P00315959） and down-regulated lncRNA（lncRNA oebiotech₂2652, lncRNA oebiotech₁6223） to predict their target genes and bio-function one by one. Confirm the expression by RT-PCR. Perform the gene ontology （GO） analysis and enriched pathway analysis. Found that the chermokine receptor binding and the interaction between cytokine and its receptor play the most important role in the pathogenesis, followed by migration of positively regulatory leukocyte, cytokine transduction, ion transport and so on. Confirm the regulatory mechanism of6lncRNAs on the chromosomes via cis. Meanwhile, construct a CTCF-lncRNA oebiotech₂2642-SMCR7L three elementarily mutual regulatory network on the basis of mRNA co-expression.Conclusion 1. Explore the differentially-expressed lncRNAs and mRNAs via lncRNA microarray in myasthenia gravis patients with thymoma, MG patients without thymoma and normal controls.2. Confirm the regulatory mechanism of three groups of lncRNAs on the chromosomes via cis and TF analysis.3. Construct three elementarily mutual regulatory networks such as lncRNA CTCF-oebiotech22642-SMCR7L on the basis of trans analysis.