Plasma Exosome Mir-3182 As A Biomarker For Myasthenia Gravis:A Preliminary Study

Background and purpose: Myasthenia gravis(MG)is an acquired autoimmune disease involving cellular immune dependence,antibody-mediated and complement participation,involving transmission dysfunction at the neuromuscular junction,and its pathogenesis has not been fully elucidated.At present,the serological tests of MG are mainly antibody detection,including antiacetylcholine receptor antibodies(anti-ACh R-Ab),anti-skeletal muscle specific receptor tyrosine kinase antibodies(anti-Musk-Ab),anti-striated muscle antibodies(anti-Titin-Ab,anti-Ry R-Ab),anti-low density lipoprotein receptorrelated protein 4 antibody(anti-LRP4-Ab),etc.,but its overall sensitivity is only about 70%,and 5% ～ 10% of the patients with antibodies are all negative.Therefore,looking for new markers is an urgent need for MG clinical diagnosis.mi RNA is a type of non-coding small molecule RNA,which is involved in the post-transcriptional regulation of genes and plays a key role in the development of diseases.Exosomes exist in many biological fluids,including blood(serum,plasma),and are rich in mi RNA,and these mi RNAs have high biological stability and high concentration.Therefore,the main purpose of this study is to find effective plasma exosome mi RNA as a blood marker of MG.Methods: 32 patients with MG who were hospitalized or outpatient in the Department of Neurology,the First Affiliated Hospital of Guangxi Medical University from March 2018 to June 2019 were selected as the MG group,and 32 healthy people who went to the medical center of our hospital for health examination As a control group(Healthy Control,HC).This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Guangxi Medical University,and all patients and controls signed an informed consent.Inclusion criteria for MG patients: a,ages 16-85 years;b,meet the diagnostic criteria of the 2015 version of the Chinese Myasthenia Gravis Diagnosis and Treatment Guidelines.Exclusion criteria: a.Patients with malignant tumors;b.Patients with other autoimmune diseases.First,collect the whole blood sample of the test subject,and collect the plasma after centrifugation.Secondly,use the kit to extract exosomes and exosome mi RNA from plasma samples,identify the plasma-derived exosomes by transmission electron microscopy and flow cytometry,and use high-throughput sequencing to detect exosome mi RNAs in the plasma of MG patients Express and screen out mi RNAs that are significantly differentially expressed.Third,the real-time fluorescence quantitative polymerase chain reaction(q RT-PCR)technique was used to detect the expression level of plasma exosome mi RNA.By making(ROC)curve and calculating the area under the curve(AUC)to evaluate the diagnostic value of mi RNA in MG.Fourth,use bioinformatics methods to analyze the target genes and pathways of the screened mi RNA.Results: First,the plasma exosomes observed by transmission electron microscopy were round or quasi-circular,with the structure of bilayer lipid molecules and the size distribution was uneven,and the diameter ranged from 30 to 100nm.Flow cytometry was used to detect exosome surface specific proteins:CD63 and CD81,with positive rates of 66.9% and 89.1%,respectively,consistent with the characteristics of normal exosomes.Second,high-throughput sequencing results showed that compared with the control group,there were 28 significantly differentially expressed mi RNA in ocular muscle type MG(OMG)patients,and135 significantly differentially expressed mi RNA in systemic MG(GMG)patients,of which mi R-3182 is significantly up-regulated in patients with ocular muscle type and systemic MG.Third,q RT-PCR results showed that compared with the healthy control group,the expression level of plasma exosome mi R-3182 in the MG group was significantly increased,MG: 6.35(4.42,7.22),HC: 2.94(1.80,5.05),P = 0.000,consistent with sequencing results;the expression level of mi R-3182 in the GMG group was significantly higher than that in the OMG group,OMG: 4.83(3.75,6.18),GMG: 6.87(5.46,7.62),nonparametric test P = 0.005,the difference was Statistical significance.ROC curve results show that compared with the healthy control group,the MG group: AUC = 0.837,sensitivity = 71.87%,specificity = 83.33%;for further analysis,the OMG group: AUC = 0.719,sensitivity = 91.67%,specificity = 50%;GMG group: AUC = 0.908,sensitivity= 85%,specificity = 83.33%.Compared with the OMG group,the expression level of mi R-3182 in GMG was AUC = 0.792,sensitivity = 65.00%,and specificity = 91.67%.It shows that mi R-3182 has higher value in the diagnosis of GMG.Fourth,bioinformatics analysis showed that there were 1342 target genes of mir-3182 and 10 key pathways including Th17 cell differentiation and Wnt signaling pathway,among which 11 target genes including m TOR have been verified to be involved in the key pathway of Th17 cell differentiation,suggesting that the target genes of mir-3182 and the bioinformatics pathways involved may be related to the pathogenesis of MG.Conclusion: First,plasma exosome mi R-3182 is of high value in the diagnosis of GMG and may be a serological marker for the diagnosis of GMG.Second,the mechanism of mi R-3182 in the occurrence and development of MG disease needs to be further studied and verified.