Clinical Significance Of Differentially Expressed Plasma Exosomal Long Non-coding RNA （LncRNAs） In Patients With MPO-AAV

ObjectiveTo investigate the role of differentially expressed plasma exosomal（long non-coding RNA）lnc RNAs in patients with MPO-AAV,and to analyze their clinical significance.MethodsPatients with active MPO-AAV who were diagnosed in the Department of Rheumatology and Immunology of hospital from November 2019 to November 2021were selected as the case group,and clinical documents and Birmingham Vasculitis Activity Score（BVAS）of each patient were recorded.And healthy donors with matching age and sex were randomly selected from the hospital medical examination center as the healthy control group.Exosomes were isolated from plasma in MPO-AAV patients and healthy donors using ultracentrifugation.Transmission electron microscopy（TEM）and nanoparticle tracking analysis（NTA）were used to identify the morphology and size distribution of exosomes.Total RNA from plasma exosomes was extracted by RNeasy Mini Kit.In the first phase,high-throughput sequencing technology was used to detect the expression levels of plasma exosomal lnc RNAs in active MPO-AAV patients（6 cases）and healthy controls（6 cases）to construct lnc RNAs expression profiles,then differentially expressed lnc RNAs profiles were obtained.In the second phase,five selected differentially expressed plasma exosomal lnc RNAs in MPO-AAV were verified by real-time quantitative PCR（RT-q PCR）（20 active MPO-AAV and 20healthy controls）.Prediction and functional analysis of target genes of differentially expressed plasma exosomal lnc RNAs of patients with MPO-AAV were performed.Correlation analysis were used to explore the correlation between five selected lnc RNAs and clinical indexes,and the diagnostic values of these lnc RNAs in MPO-AAV were analyzed by ROC curves.Results⒈In the 20 selected patients with MPO-AAV,18 patients were diagnosed as MPA and 2 as GPA.They were composed of 7（35%）males and 13（65%）females,their ages ranged from 44 to 88（63±11）years old.Kidney and lung were the most common in multiple damaged organs,with 18 cases（90%）of the kidney and 17 cases（85%）of the lung,respectively.The BVAS scores of patients ranged from 12.00 to 26.00（17.85±4.03）.The healthy control group were consisted of 11 males（55%）and 9 females（45%）,their ages ranged from 52 to 85（68±8）years old.There was no significant difference between MPO-AAV group and healthy control group in terms of sex（χ~2=1.616,P=0.206）and age（t=-1.857,P=0.071）.⒉The vesicular structure with irregular spherical shape of the isolated plasma exosomes were observed by TEM.The diameter of plasma exosomes detected by NTA were ranged from 50 to 120nm,and were mainly in 80nm.⒊The results of high-throughput sequencing showed that there were2711differentially expressed lnc RNAs in the MPO-AAV compared with the healthy control group,of which 862 were up-regulated and 1849 were down-regulated.⒋The KEGG pathway enrichment analysis results showed that the target genes for differentially expressed exosomal lnc RNAs in MPO-AAV were mainly related to TNF,adipocytokines and human virus infection-related signaling pathways.The GO enrichment analysis showed that the target genes for differentially expressed exosomal lnc RNAs in MPO-AAV were mainly related to components of cell and organelles,binding of proteins and nucleic acids and neutrophil degranulation,transport of proteins.⒌The verification results of five randomly selected lnc RNAs by q RT-PCR were consistent with sequencing results.⒍The relative expression level of plasma exosomal LINC00705 in MPO-AAV was significantly higher than that in the healthy control group[7.82（2.26,30.59）vs 0.79（0.31,4.81）,P=0.001],and the AUC of LINC00705 was 0.790（95%CI:0.650～0.930,P=0.002）.⒎The relative expression levels of plasma exosomal RPS14P3 in MPO-AAV was significantly higher than that in the healthy control group[7.20（1.45,17.35）vs 1.33（0.37,2.75）,P=0.001)],and the AUC of RPS14P3was 0.788（95%CI:0.649～0.926,P=0.002）.⒏The relative expression levels of plasma exosomal SUZ12P1 in MPO-AAV was significantly higher than that in the healthy control group[8.29（3.07,13.17）vs 1.07（0.13,4.78）,P=0.001],and the AUC of SUZ12P1was 0.780（95%CI:0.629～0.931,P=0.002）.⒐The relative expression levels of plasma exosomal DANCR in MPO-AAV was significantly higher than that in the healthy control group[4.52（1.48,15.89）vs 0.80（0.38,2.89）,P=0.001],and the AUC of DANCR was 0.795（95%CI:0.657～0.933,P=0.001）.⒑The relative expression levels of plasma exosomal SNHG1 in MPO-AAV was significantly higher than that in the healthy control group[4.98（1.92,38.16）vs 1.93（0.14,6.37）,P=0.007],and the AUC of SNHG1was 0.745（95%CI:0.593～0.897,P=0.008）.⒒The AUC of plasma exosomal LINC00705,RPS14P3,SUZ12P1,DANCR and SNHG1 multi-indicator combination was 0.925（95%CI:0.833～0.999,P=0.000）.⒓In MPO-AAV,the relative expression level of plasma exosomal LINC00705,RPS14P3,SUZ12P1,DANCR and SNHG1 were positively correlated with BVAS（r_s=0.707,P=0.000;r_s=0.590,P=0.006;r_s=0.469,P=0.037;r_s=0.511,P=0.021;r_s=0.476,P=0.034,respectively）,had no significantly correlation with age,disease course,ESR,CRP and Cr（P>0.05）.Whereas multiple regression analysis suggested that BVAs was only positively correlated with the relative expression level of RPS14P3（β=0.478,P=0.049）.Conclusion1.The differentially expressed plasma exosomal lnc RNAs may be involved in the vascular endothelial injury,inflammatory and immune response of MPO-AAV.2.The higher expression ofplasma exosomal lnc RNAs LINC00705,RPS14P3,SUZ12P1,DANCR in patients with MPO-AAV suggests that these lnc RNAs might be involved in the pathogenesis of MPO-AAV,and their correlation with disease activity suggests that they may be potential predictive biological markers for assessing disease activity.It also provides new ideas for the treatment of MPO-AAV.3.The combination of plasma exosomal LINC00705,RPS14P3,SUZ12P1,DANCR and SNHG1 can improve the diagnostic value of MPO-AAV compared with single lnc RNAs,and these lnc RNAs may be potential biomarkers for diagnosing MPO-AAV.