Analysis Of Differential Expression Of Non-coding RNA In Peripheral Serum Secretion Of Patients With Primary Biliary Cholangitis

Background Primary biliary cholangitis(PBC)is a kind of autoimmune liver disease with complex pathogenesis.At present,there are few studies on the non-coding RNA(nc RNA)of exocrine in PBC.ObjectiveIn this paper,we will explore the possible role of nc RNA in the disease by looking for the nc RNA with differential expression in PBC and deeply understanding the disease from the molecular mechanism level.MethodsPeripheral blood serum samples from 6 healthy patients and 6 PBC patients were collected,total RNA in exosome was extracted and exosomal RNA library was established,then analyzed and searched for exosomal nc RNA with differential expression by RNA-sequencing(RNA-Seq).Quantitative real-time polymerase chain reaction(q RT-PCR)was used in 30 patients with PBC,rheumatoid arthritis(RA)and normal control(NC)group were verified.On this basis,correlation analysis of clinical data,prediction of target genes and related pathways,ROC curve analysis were performed for nc RNAs whose verification results were in line with expectations.Results1.exosomal LINC00472 and exosomal NEAT1 are highly expressed long non-coding RNA(lnc RNA)in PBC group,and the difference of exosomal LINC00472 in PBC group and NC group was statistically significant(P<0.05).The difference between PBC group and RA group was also statistically significant(P<0.05).There was no significant difference in exosomal NEAT1 between PBC group and NC group(P=0.246).2.In the PBC group,The relative expression level of exosomal LINC00472 was positively correlated with alkaline phosphatase(ALP)and gamma-glutamyl transpeptidase(γ-GT)(P=0.022,P=0.044).According to the anti-mitochondrial antibody(AMA),anti-SP100 antibody and anti-GP210 antibody are divided into positive and negative groups,respectively.There was no significant difference in the relative expression level of exosome LINC00472 among all groups(P=0.859,P=0.711,P=0.267).3.Exosomal mi R-431-5p,mi R-214-3p and mi R-149-5p were micro RNAs(mi RNAs)with low expression in PBC group,and the expression levels in PBC group were statistically significant compared with NC group(P< 0.05).However,in the comparison between the PBC group and the RA group,only the expression level of mi R-214-3p showed statistically significant difference between the two groups(P=0.003).In addition,PBC group was divided into antibody positive group and antibody negative group according to the positive of AMA,anti-SP100 antibody and gp210 antibody.There were no statistical differences in the relative expression levels of exosomal mi R-431-5p,mi R-214-3p and mi R-149-5p among all groups(P > 0.05).4.According to the RNA library and ce RNA analysis constructed in this study,there was a negative correlation between the expression of exosomal mi R-214-3p(r=-0.404,P=0.027)and exosomal LINC00472.The expression of exosomal mi R-149-5p(r=-0.451,P=0.012)was also negatively correlated with that of LINC00472.5.Through ROC curve analysis,the AUC of the exosomal LINC00472 for diagnosis of PBC is 0.798(95% CI: 0.681～0.916,sensitivity 0.767,specificity 0.8,P<0.05),and the AUC of the combined diagnosis with AMA is 0.930(95% CI: 0.8520 to 1.000,sensitivity 0.933,specificity 0.867,P<0.05).The AUC of secretions mi R-431-5p in diagnosis of PBC=0.826(95% CI: 0.714～0.937,sensitivity 0.931,specificity 0.724,P<0.05),and the combined diagnosis with AMA is 0.958(95% CI: 0.852～1.000,sensitivity 0.793,specificity 1.0,P<0.05).The AUC of secretions mi R-214-3p in diagnosis of PBC is 0.758(95% CI: 0.623,～0.893,sensitivity 0.815,specificity 0.667,P<0.05),and the combined diagnosis with AMA is 0.927(95% CI: 0.863～0.992,sensitivity 0.778,specificity 0.926,P<0.05).6.According to KEEG pathway analysis,the target genes related to exosomal LINC00472 and mi R-214-3p were enriched in 26 pathways,including MAPK pathway,Wnt pathway,T cell-related pathway,EB virus infection-related pathway,etc.Conclusions1.In PBC,the expression of exosomal LINC00472 was up-regulated and the expression of exosomal mi R-431-5p was down-regulated.The different expression between groups suggested that they might be involved in the pathogenesis of the disease,and both had the ability to diagnose PBC.2.Compared with exosomal mi R-431-5p,the differential expression of exosomal LINC00472 is more specific,which may affect the conduction of inflammatory pathways(MAPK pathway,Wnt pathway,T cell-related pathway,etc.)and participate in the occurrence of cholestasis,providing new possibilities for future diagnosis and targeted therapy.