Differential Expression And Correlation Analysis Of MicroRNA In Bile Duct Occlusive Liver Fibrosis

Objective To study the differential expression of micro RNA in liver tissues of bile duct obstructive liver fibrosis and the GO and KEGG analysis,providing new directions and targets for the later study of bile duct obstructive liver fibrosis.Method1.Research subjects: Strictly following the enrollment criteria,patients with normal liver tissue and patients with bile duct obstructive diseases who were hospitalized in Qingdao Municipal Hospital affiliated to Qingdao University from January 2018 to June 2020 were selected for inclusion in the study.2.Specimen processing: Divide 3 normal liver tissues and 3 bile duct obstructive liver fibrosis tissue samples into two,one is stained by HE and Masson to confirm the presence or absence of liver fibrosis,and the other is cryopreserved for gene sequencing.3.Gene sequencing and differential micro RNA screening: After quality testing,library construction,library quality testing,computer sequencing,and micro RNA detection,known and unknown micro RNAs are obtained,and edge R software is further used to screen differential micro RNAs.4.Target gene prediction and correlation analysis: use miRanda and RNAhybrid software to predict target genes,and then use top GO software and KOBAS analysis to complete target gene GO enrichment analysis and KEGG signal pathway analysis.5.Establishment of animal model: select male Wistar rats,ligate their common bile ducts for2 weeks for modeling,obtain liver fibrous tissue and perform Masson staining for verification.6.Real-time fluorescent quantitative PCR: to verify the expression of miR-483-3p in Wistar rats with obstructive liver fibrosis of the bile ducts,for preliminary screening in later experiments.Result1.HE and Masson staining: the selected liver tissue after pathological verification meets the criteria of normal liver tissue and bile duct obstructive liver fibrosis,and statistical analysis of the area of collagen fibers in the two groups of Masson staining has significant differences(P< 0.05).2.Gene sequencing and differential micro RNA screening: A total of 1575 cases of micro RNA were screened in the experiment,of which a total of 1550 cases of known micro RNAs,a total of 25 cases of newly discovered micro RNAs,and a total of 44 cases of differential micro RNAs were screened from known micro RNAs.There were 18 cases of up-regulated micro RNAs and 26 cases of down-regulated micro RNAs.Among them,5 cases of significantly differentially expressed micro RNAs were down-regulated in fibrotic tissues,namely miR-483-3p,miR-1271-5p,miR-5589-3p,miR-1295b-3p,miR-7977.3.Target gene prediction and correlation analysis: predict the potential target genes corresponding to the differentially expressed micro RNAs,and perform GO analysis to show the molecular functions of the relevant genes,the cell location,the biological processes involved,and the KEGG signal pathway analysis of target genes mainly acts on the Metabolic pathway.4.Establishment of animal model: After 2 weeks of bile duct ligation in male Wistar rats,the normal structure of liver lobules was found to be disordered,collagen fibers increased,and liver fibrosis was obvious after Masson staining verification.5.Low expression of miR-483-3p: The down-regulation of miR-483-3p in Wistar rat bile duct obstructive liver fibrosis was evaluated in this study,which can be used in later experimental studies.Conclusion1.After comparing the two groups of liver tissues,a total of 5 micro RNAs with significant differential expression were screened: miR-483-3p,miR-5589-3p,miR-1271-5p,miR-1295b-3p,miR-7977,It suggests that these genes may be involved in the process of liver fibrosis.2.The research has clarified the molecular functions,cellular components and biological processes involved in the target gene.The pathways involved mainly include metabolic pathways,tumor pathways,PI3K-Akt signaling pathways,MAPK signaling pathways,Ras signaling pathways,and Rap1 signaling.Pathways and other signal transduction pathways.Micro RNA may regulate target genes through the above pathways and affect bile duct obstructive liver fibrosis.3.MiR-483-3p was further verified to be down-regulated in bile duct obstructive liver fibrosis by PCR,and the specific mechanism needs to be further verified by later experiments.