Genome-Wide Identification Of Regulating Network And Functional Mechanism Research Of MiRNAs In Rheumatoid Arthritis

Rheumatoid Arthritis(RA)is an immune-mediated chronic inflammatory disease characterized by persistent multiarticular synovitis,cartilage destruction,and loss of joint function.The disease causes a huge pain on patients and the high recurrence and incurability severely reduces the life quality and increases the social burden of patients.Due to the combined effects of multiple factors,the pathogenesis of RA is still not clear.To identify the potential biomarkers for RA and explore the functional mechanisms is extremely important,which will improve the clinical diagnosis in RA and provide basic data for developing the methods and technologies of RA early diagnosis and intervention.Previous studies showed microRNAs,acted as the important post-transcriptional regulator,are involved in regulation of the immune system during the development of RA.The present study aimed to identify the significant differential expressed miRNAs and set up association network of miRNA and RA as well as regulating network between miRNAs and their target mRNAs.The molecular and cellular experiments will also be performed to detect the functional mechanism underlying the association between biomarkers and RA.In part I,through using high-throughput technology the study will identify significant differential expressed miRNAs and mRNAs in peripheral blood mononuclear cells(PBMCs)from RA patients and health controls.The regulating network between miRNAs and their target mRNAs will be established and relevant bioinformatics analysis will be performed.The detected miRNAs will be replicated the associations and evaluated the diagnostic value in another independent population.In part II,the cell lines of endogenous high expression of miR-99b,which get the support from part I that may be potential biomarkers for RA,will be established within Jurkat T cells.The affections of miR-99b on biological characteristics of Jurkat cells will be evaluated and the expressions oftarget genes and cytokines related to immune response will be evaluated as well.PartIObjectiveTo systematically identify potential miRNA biomarkers in peripheral blood mononuclear cells(PBMCs)for RA based on the high-throughput chip sequencing technology.MethodsThe study will sequence miRNA and mRNA from PBMCs for each subjects using high-throughput technology in RA patients and controls.Significantly differential expressed miRNAs will be detected from the corresponding expression profiles and the regulating network between miRNAs and the target mRNAs will be established.The bioinformatics analysis of targets will be performed including functional annotation clustering analysis and protein-protein interaction analysis.By performing the causal inference literature,the study screened and selected the potential targets and signal pathway with statistical significancefor detected miRNAs.The detected miRNA biomarkers will be verified in another independent population and the diagnostic value of the biomarkers will be estimated by receiver operating characteristic curve analysis.ResultsThe study identified a total of 18 RA associated differential expressed miRNAs,17 of them were the novel discovered miRNAs for RA.By the performance of target prediction and evaluation of pearson correlation coefficient between miRNAs and their targets,the regulating network between both were set up.The results of RT-PCR conducted in another independent population showed that hsa-miR-1184 and hsa-miR-99b-5p were replicated the associations with RA.Causal inference literature analysis detected hsa-miR-99b-5p regulated the interleukin 6 signal transducer(IL6ST)through targeting mTOR,which may involved in the development of RA.The bioinformatics analysis also verified the hypothesis that mTOR showed to be RA susceptibility genes and affected the pathogenesis of RA.The results of ROC analysis showed that the specificityand sensitivityof hsa-miR-1184 and hsa-miR-99b-5p were relatively high and the combination of both miRNAs could increase the diagnostic value for RA.ConclusionsThis study screened and identified 18 novel and replicable potential miRNA biomarkers for RA.The integration of the evidences from RT-PCR experiment,causal inference literature and ROC analysis highlighted the significance of hsa-miR-99b-5p in the pathogenesis of RA and suggested hsa-miR-99b-5p could be acted as a potential epigenetic factor for RA.Part IIObjectiveTo explore the biological characteristics of miR-99b-5p on Jurkat T cells and detect the functional mechanisms between miR-99b-5p and RA.MethodsLentiviral vector was constructed with miR-99b eukaryotic expression vector using pCDH.Using quantitative PCR method the expression of miR-99b targets and cytokines related to immune system were estimated in Jurkat cells after infected by recombinant lentivirus infection.The expression of miR-99b associated targets and cytokines in PBMCs were also detected in the populations with RA or not.CCK-8 for cell proliferation detection,Annexin V/PI double staining for cell apoptosis detection and cell cycle detection were performed to study the affection of miR-99b on biological characteristics of Jurkat T cells.ResultsFrom the results of RT-PCR between RA patients and controls,the target mTOR and interleukin 6 signal transducer(IL6ST)of miR-99b deregulated in PBMCs.The expression of IL-2,IL-6,IL6ST and IFN-? showed significantly lower in Jurkat after infection by miR-99b lentivirus than the negative controls,while TNF-a showed the increased changes in the expression.The expression of mTOR showed no obvious decreased trend but the expression of AKT1 decreased obviously between infection and no infection group.The overexpression of miR-99b could inhibit cell apoptosis and promote cell proliferation obviously.ConclusionsThe overexpression of miR-99b decreased the expression of cytokines that related to immune system,including IL6 and IL6ST,and promoted the secretion of TNF-a through targeting the AKT/mTOR signal pathway,which could promote Jurkat cell proliferation and inhibit cell apoptosis,suggesting the promotion on the development of RA.