Differential Expression And Clinical Value Of MiR-410-3p In Serum From Primary Biliary Cholangitis Patients

Aims:Differentially expressed micro RNAs(miRNAs)in serum from primary biliary cholangitis(PBC)patients with anti-gp210 antibodies positive were screened.This study investigated the clinical value of differentially expressed miRNAs in PBC and predicted their downstream target genes and function and biological processes of target genes.Methods:Comprehensive deep miRNA sequencing technique was used to evaluate the expression profiles of serum miRNAs from 3 patients with anti-gp210 antibodies positive PBC and 3 patients with anti-gp210 antibodies negative PBC and 3 normal controls which were randomly selected from the study subjects.We first selected significantly differentially expressed candidate miRNAs.Then,quantitative real-time PCR(qRT-PCR)was further used for experimental verification in serum from PBC group(36 patients with anti-gp210 antibodies positive PBC and 46 patients with anti-gp210 antibodies negative PBC)and control group(20 healthy subjects and 20 patients with chronic hepatitis B).Receiver operating characteristic(ROC)analysis was used to investigate the clinical diagnostic value of differentially expressed miRNAs in serum from patients with anti-gp210 antibodies positive PBC.This study compared their serum levels difference between cirrhotic PBC patients and non-cirrhotic PBC patients.The relationship between the change of their serum expression levels and the natural course,cholangitis activity score was evaluated.This study compared their serum levels difference between PBC patients responding to UDCA treatment and PBC patients showing non-response to UDCA treatment.Finally,the target genes of differentially expressed miRNAs were predicted using TargetScan and miRDB databases.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were conducted to explore the biological function of target genes.Results:1.By deep sequencing,we found 192 miRNAs were differentially expressed in PBC patients compared to the normal controls in serum.The expression levels of 60 miRNAs were found to differ significantly between anti-gp210 antibodies positive PBC patients and anti-gp210 antibodies negative PBC patients.2.It was verified by qRT-PCR experiment that the expression of serum miR-410-3p was significantly up-regulated in PBC patients compared with the normal controls(P<0.0001)and the CHB patients(P<0.0001).Expression of serum miR-410-3p in patients with anti-gp210 antibodies positive PBC has shown a 6.9-fold increase compared to normal controls(P<0.0001)and 8.2-fold increase compared to patients with anti-gp210 antibodies negative PBC(P<0.0001).3.Receiver operating characteristic(ROC)analysis revealed that serum miR-410-3p yielded area under the curve(AUC)values of 0.775(0.688～0.862),with a corresponding sensitivity of 64.6%and a specificity of 95.0%in discriminating PBC from normal controls when the optimal critical value was 1.44.Serum miR-410-3p yielded AUC values of 0.791(0.695～0.887),and when the cut-off value was 1.31,a sensitivity was 80.5%and a specificity was 70.0%in distinguishing PBC from CHB patients.Serum miR-410-3p yielded AUC values of 0.969(0.926～1.000),and when the cut-off value was 1.45,a sensitivity was 92.4%and a specificity was 90.0%in distinguishing anti-gp210 antibodies positive PBC from normal controls.Serum miR-410-3p yielded AUC values of 0.810(0.710～0.911),and when the cut-off value was 3.56,a sensitivity was 94.4%and a specificity was 69.6%in distinguishing anti-gp210 antibodies positive PBC from anti-gp210 antibodies negative PBC.4.The serum level of miR-410-3p was higher in cirrhotic patients in comparison to non-cirrhotic PBC patients(P<0.01).In patients with anti-gp210 antibodies positive PBC,these patients with nature course phase Ⅲ-Ⅳ had a higher level of miR-410-3p compared to patients with nature course phase Ⅰ-Ⅱ(P<0.001).When miR-410-3p expression was compared in relation to CA,the miR-410-3p expression level was substantially enhanced in PBC patients with a CA of 2-3 than in those with a CA of 0-1(P<0.05).5.In 15 patients with PBC showing non-response to UDCA treatment,the expression of miR-410-3p was significantly increased compared to that in normal controls(P<0.0001)and PBC patients responding to UDCA treatment(P<0.05).6.GO analysis showed that underlying target genes of miR-410-3p were mainly involved in cell communication,cell metabolism,signal transduction,cell apoptosis,cell differentiation,cell proliferation and so on.The potential target genes in relation to differentiation,activation,proliferation,and homeostasis of T cell or B cell included PPP3CB,ETSI,TNFSF11,TNFSF9,FOXF1 and RUNXIT1,CBFB,SP3.KEGG analysis suggested that some key target genes of miR-410-3p were involved in many important signaling pathways,mainly including TGF-p signaling pathway,MAPK signaling pathway and Wnt signaling pathway,PI3K-Akt signaling pathway and NF-kB signaling pathway.Conclusions:1.The serum miRNAs expression profiles of patients with anti-gp210 antibodies positive PBC are obviously different from those of patients with anti-gp210 antibodies negative PBC.2.Serum miR-410-3p may be a potential diagnostic biomarker of anti-gp210 antibodies positive PBC;the over-expression of serum miR-410-3p may reflect the occurrence of cirrhosis,cholangitis activity,non-response to UDCA treatment and natural course phaseⅢ-Ⅳ in PBC patients.3.Bioinformatics analysis shows that the target genes of miR-410-3p may be involved in the occurrence and development of PBC.