Study On Expression Of Non-coding RNAs In Peripheral Blood Exosomes And Molecular Mechanism Of MiR-122-5p In Primary Biliary Cholangitis

Primary biliary cholangitis(PBC)is an autoimmune-mediated chronic cholestatic liver disease.Weakness and pruritus are the most common clinical symptoms.The main pathological features of PBC are chronic progressive damage,destruction,loss,and fibrosis of bile duct epithelial cells(small bile ducts),eventually leading to cirrhosis.The exact etiology and pathogenesis of PBC are unknown,but ample evidence suggests that a highly complex interaction between genetic and environmental factors may lead to the development and progression of PBC through immune disorders and inflammatory responses.Exosomes are small vesicles formed from endosomal membrane and released outside cells by fusion with plasma membrane.They can contain a variety of bioactive nucleic acids,such as micro RNA/mi RNA,long non-coding RNAs(lnc RNAs),lipids and proteins play a role in information exchange between cells.Different tissues and cells,or the same tissues and cells in different states,various components of exosomes can be significantly changed,and is closely related to the occurrence and development of a variety of human diseases.Exosomes interfere with the production and maintenance of a variety of autoantibodies and are associated with the development and progression of autoimmune responses.Non-coding RNAs(nc RNAs)are functional RNA molecules that cannot be translated into proteins and perform their biological functions at the RNA level.With the development of sequencing technology and bioinformatics,many studies have shown that nc RNAs play an important role in regulating autoimmune and inflammation,including participating in the occurrence and development of PBC and other autoimmune diseases.However,there are few studies on exosomal nc RNAs and PBC so far.This study aims to explore the possible role of nc RNAs in peripheral blood exosomes of PBC patients in the pathogenesis of PBC.The study consists of two parts.The first part analyzes differential expression of nc RNAs(including mi RNAs and lnc RNAs)in peripheral blood exosomes of PBC patients by transcriptome sequencing technology(RNA-seq).The signaling pathways involved in these differentially expressed Genomes were analyzed by bioinformatics,and based on the results of exosomal RNA-seq and Kyoto Encyclopedia of Genes and Genomes(KEGG),their genomes were analyzed jointly.Differential expression of exosomal nc RNAs(especially mi RNAs)was screened and verified in expanded samples.According to the verification results and previous literature,exosomal mi R-122-5p was finally selected as the research object,and the correlation between exosome mi R-122-5p and clinical indicators was further analyzed,as well as the potential application value of exosome mi R-122-5p in the clinical diagnosis of PBC.The second part mainly explores the effects of mi R-122-5p on the phenotypes,epithelial mesenchymal transformation and fibrosis indexes of primary human intrahepatic biliary epithelial cells(HIBECs)by in vitro experiments.To explore the molecular mechanism of mi R-122-5p in the occurrence and development of PBC.Part I: Differential expression of nc RNAs in peripheral blood exosomes of PBC and its clinical significanceObjective To explore the differential expression of nc RNAs in peripheral blood exosomes of PBC patients,and to analyze the major signaling pathways involved in differential expression of nc RNAs through bioinformatics.The differential expression of exosomal mi RNAs was screened and the sample size was expanded for verification,so as to further analyze the relationship between them and the clinical indicators of PBC and explore their potential clinical application value.Methods1.Six patients diagnosed with PBC between November 2020 and January 2023 were randomly selected,and six healthy controls with the same age and gender were randomly selected in the physical examination center during the same period.Peripheral blood of the subjects was collected,and serum exosomes were extracted by overspeed centrifugal method.The expression profiles of RNAs in serum exosomes of PBC and normal population were analyzed by RNA-seq.Six differential expression mi RNAs(DE mi RNAs)and 4 DE lnc RNAs were screened according to RNA-seq results.2.A total of 21 patients with PBC and 22 normal controls matched in age and sex were randomly selected.General data(age,sex,medication history,family history),biochemical and immunological indexes of all subjects were recorded,and peripheral blood serum exosomes were extracted.3.Mi R-122-5p was selected according to the verification results and previous literature,and the correlation between exosome mi R-122-5p and clinical indicators was analyzed.According to receiver operating characteristic(ROC)curve,the potential application value of exosomal mi R-122-5p in the clinical diagnosis of PBC was explored.Results1.General Information of enrolled subjects: In the detection of PBC patients by RNA-seq,the positive rate of anti-mitochondrial antibody M2(AMA-M2)and the levels of total bilirubin(TBIL),direct bilirubin(DBIL),alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),γ-glutamyl transferase(γ-GT),and total bile acid(TBA)were all higher than in the normal control population.The difference was statistically significant(p<0.05).In PBC patients with expanded sample validation,positive rates of AMA-M2,anti-gp210 antibody,antinuclear antibody(ANA)and the levels of peripheral blood albumin(ALB),TBIL,indirect bilirubin,DBIL,ALT,AST,ALP,γ-GT and TBA were higher than those of normal control group,and the difference was statistically significant(p<0.05).2.Results of RNA-seq and verification of primary screening genes: In peripheral blood exosomes of PBC patients,8531 DE lnc RNAs(829 up-regulated and 7702down-regulated)and 263 DE mi RNAs(96 up-regulated and 167 down-regulated)were preliminatively identified.KEGG analysis showed that the concentration of DE lnc RNAs and DE mi RNAs in serum exosomes was associated with Erb B,bacterial invasion of epithelial cells,fatty acid degradation,toll and imd,mitogen activated protein kinase(MAPK)and other signaling pathways.Based on RNA-seq and previous literature,six exosomal DE mi RNAs(mi R-122-5p,mi R-29a-3p,mi R-744-5p,mi R-34a-5p,mi R-149-5p,mi R-339-5p)and four DE lnc RNAs(NEAT1,LINC004472,PRKCQ-AS1,PCBP1-AS1)were screened and conducted expanded sample size verification,and the results showed that exosomal mi R-29a-3p and mi R-122-5p and LINC004472 were consistent with the results of previous full transcriptional sequencing.Combined with the results of KEGG analysis,mi R-122-5p was finally selected as the target gene for further study.3.Potential clinical value of mi R-122-5p: Exosomal mi R-122-5p was positively correlated with liver function injury and cholecystasis indexes(ALT,AST,DBIL,gamma-GT,ALP,TBA).Exosomal mi R-122-5p combined with gp210 and sp100 could improve the sensitivity of PBC diagnosis,but had no significant effect on the specificity(sensitivity and specificity were 71.43% and 90.91%,respectively).Conclusions1.The cotarget genes of DE lnc RNAs and DE mi RNAs in PBC exosomes are mainly concentrated in the signaling pathways related to ErbB,bacterial invasion of epithelial cells,fatty acid degradation,toll and imd,and MAPK.2.Exosomal mi R-122-5p was positively correlated with liver injury and cholestasis.3.Exosomal mi R-122-5p combined with anti-gp210 antibody and anti-sp100 antibody can significantly improve the diagnostic sensitivity of PBC,and can be used as a potential biomarker for clinical diagnosis of PBC.Part II: Molecular mechanism of mi R-122-5p in the pathogenesis of PBCObjectiveBased on the results of the part I,the molecular mechanism of mi R-122-5p in the occurrence and development of PBC was preliminarily explored through the prediction and verification of mi R-122-5p target genes and the study of p38 MAPK signaling pathway.Methods1.Primary human intrahepatic biliary epithelial cells(HIBECs)were cultured in vitro.HIBECs was transfected with the mimics,inhibitor and negative sequence controls(NC)of mi R-122-5p,tumor necrosis factor receptor superfamily member 19(TNFRSF19),apoptotic signal-regulated kinase 1(ASK1)by liposome 2000(lip2000).2.Enzyme linked immunosorbent assay(ELISA)detected the inflammatory cytokine interleukin(IL)-1β,IL-4,IL-6,IL-8,IL-10,IL-12,IL-17A/IL-17,tumor necrosis factor-α,transforming growth factor(TNF-α),transforming growth factor(TGF-β1)and interferon-γ(IFN-γ)in peripheral blood of PBC and HIBECs supernatant3.HIBECs were treated with different concentrations and time gradients of lipopolysaccharide(LPS)and estradiol(E2),respectively.The best inducer was selected according to the level of inflammatory factors in the cell supernatant to induce the inflammatory injury model of HIBECs.4.CCK8 kit was used to detect the cell viability of HIBECs.The cell cycle and apoptosis kit were used to detect cell cycle and apoptosis by flow cytometry,and the proliferation ability of HIBECs was observed by combining cell viability to the ratio of cell cycle(G2+S).5.Real-time quantitative fluorescent polymerase chain reaction(RT-qPCR)and western blotting(WB)were used to detect the m RNA and protein levels of epithelialmesenchymal transition(EMT)markers vimentin,e-cadherin,n-cadherin,and fibrosis markers α-smooth muscle actin(α-SMA),type I collagen,TNFRSF19,ASK1,and p38.6.KEGG enrichment analysis of target genes predicted by mi R-122-5p was performed using Dr.Tom(biosys.bgi.com/),an online biological information platform provided by BGI,and related signaling pathways were screened out in pre-experiments.7.The signaling pathway was blocked with the signaling pathway blocker,and the influence on HIBECs was observed.8.Target Scan,mi RDB,mi RTarbase and Tarbase software were used to predict the target genes of mi R-122-5p,and the intersection with the differentially expressed m RNAs in exosomal m RNAs sequencing results was used for Venn diagram.The target genes of mi R-122-5p were determined by mitogen-activated protein kinase(MAPK)signaling pathway map(https://www.kegg.jp/pathway/map04010 + K04441).9.Dual fluorescein reporter assays were used to verify the binding of mi R-122-5p to target genes.10.Small interfering RNA(si RNA)were designed with three different target genes,the si RNA with the highest interference efficiency was selected to transfect HIBECs,and the interaction between target genes and p38 MAPK signaling pathway was observed.Results1.ELISA results showed that the levels of pro-inflammatory cytokines IL-1β,IL-6,IL-8,IL-12,IL-17 A,TNF-α,IFN-γ and pro-fibrosis factor TGF-β1 in peripheral blood of PBC patients were higher than those of normal controls,and the levels of antiinflammatory factors IL-4 and IL-10 were lower than those of normal controls.2.After lip2000 transfection with mi R-122-5p mimics and inhibitor,the levels of mi R-122-5p in HIBECs were increased and decreased,respectively.The high expression of mi R-122-5p promoted the proliferation of HIBECs and inhibited the apoptosis,EMT and fibrosis of HIBECs.The low expression of mi R-122-5p inhibited the proliferation of HIBECs and promoted apoptosis,EMT and fibrosis of HIBECs(p<0.05).3.When HIBECs was treated with LPS of 0.1μg/ml for 24 h,the levels of inflammatory cytokines IL-1β,IL-6 and IL-8 in HIBECs supernatant increased,so LPS was selected as the inducer of HIBECs inflammatory injury model.4.LPS inhibited proliferation of HIBECs and promoted apoptosis,EMT and fibrosis of HIBECs,which could be largely reversed by mi R-122-5.LPS induced HIBECs to produce inflammatory damage while inhibited the expression of mi R-122-5p,which further inhibited the proliferation of HIBECs,promoted the apoptosis of HIBECs,and aggravated the EMT and fibrosis of HIBECs.5.KEGG enrichment analysis showed that,the target genes predicted by mi R-122-5p are mainly enriched in adrenergic signaling in cardiomyocytes,endocytosis,Hippo,and tumor pathways in cardiomyocytes cancer,muctin type O-glycan biosynthesis,mucsaminoglycan biosynthesis,MAPK,glycosaminoglycan biosynthesis,other type of glycan biosynthesis,Wnt and other signaling pathways.The p38 MAPK signaling pathway was selected based on the preliminary experimental results.6.Blocking the p38 MAPK signaling pathway increased the proliferation,decreased apoptosis,decreased EMT and decreased fibrosis of HIBECs,and decreased the levels of IL-1β,IL-6,IL-8,IL-12,IL-17 A,TNF-α,IFN-γ and TGF-β1.Elevated levels of the IL-4 and IL-10.Moreover,blocking p38 MAPK pathway can partially reverse the effects of LPS on inhibiting proliferation,promoting apoptosis,proinflammation,promoting EMT and fibrosis of HIBECs,which has a synergistic effect with mi R-122-5p.In addition,LPS can also up-regulate the expression of p38 and p-p38 proteins.7.Venn diagram showed a total of 1,488 overlapping genes,from which 891 downregulated genes were screened out(mi R-122-5p was up-regulated in the results of RNA-seq).RT-q PCR and WB confirmed that TNFRSF19 was the target gene of mi R-122-5p.8.Results of dual fluorescein reporter assays showed that mi R-122-5p had binding sites with TNFRSF19.9.Si-1(referred to as si-TNFRSF19)has the highest interference efficiency.SiTNFRSF19 decreased the expression of p38 and p-p38 proteins in HIBECs,and could partially counteract the effect of increased p38 and p-p38 proteins caused by the low expression of mi R-122-5p.In addition,TNFRSF19 can also regulate the level of ASK1 m RNA and protein,and then regulate the downstream p38 MAPK signaling pathway.Conclusions1.Mi R-122-5p promoted the proliferation of HIBECs,and inhibited the expression of apoptosis,EMT,fibrosis and some inflammatory cytokines of HIBECs.2.Mi R-122-5p can inhibit p38 MAPK signaling pathway and has synergistic effect with p38 MAPK blockers.3.Mi R-122-5p targeting TNFRSF19 regulates ASK1 to affect HIBECs proliferation,apoptosis,EMT,fibrosis and inflammatory cytokine levels through p38 MAPK signaling pathway.4.Mi R-122-5p is expected to be a potential target for treatment of PBC.