Preliminary Analysis And Functional Study Of Differential Expression Profiles Of MicroRNAs Associated With Nonalcoholic Fatty Liver Disease

Background Nonalcoholic fatty liver disease(NAFLD)has become the most common chronic liver disease in the world.However,the exact pathogenesis remains unclear,non-invasive specific biomarkers have not been found,and specific drugs have not yet been developed.Studies have shown that microRNAs,which are important parts of epigenetics,have significance in the pathogenesis,diagnosis and treatment of NAFLD.Exosomal microRNAs are more stable than free microRNAs under the protection of phospholipid bilayer structure.However,little research has been done on the differential expression profiles and functional characteristics of human blood exosomal microRNAs in NAFLD.Objective To screen the expression profiles of serum exosome microRNAs in patients with NAFLD,to study the similarities and differences of microRNAs expression between serum exosomes of patients with NAFLD and liver tissues of animal models,and to and perform target gene prediction and bioinformatics analysis to preliminarily explore the significance of microRNAs in the occurrence,development,diagnosis and treatment of NAFLD.Methods(1)After extracting and identifying exosomes from 4 cases of moderate to severe NAFLD and 4 healthy men's serum with similar demographic characteristics,Illumina high-throughput sequencing was used to screen differential expression profiles of microRNAs.The 4 most differentially expressed microRNAs were verified by qRT-PCR in 20 cases of mild NAFLD,moderate to severe NAFLD and Control in each group.(2)The animal model of NAFLD was established by feeding C57 BL/6 mice with high fat diet(HFD).Blood and liver samples of HFD and CTL mice were collected at 4,8 and 12 weeks respectively.Blood biochemistry,liver pathology and the relative expression of the above 4 microRNAs were detected.(3)Target gene prediction was performed on the above 4 microRNAs.Target Scan,miRanda,and Mireap were used to predict the intersection of the target gene results,and the results obtained from the miRTarBase database were combined as the final prediction results.Results(1)A total of 19 serum exosomal microRNAs with significant differences(P<0.05)and differential expression of more than 2 times were screened out,of which 8 were up-regulated and 11 were down-regulated.(2)The relative expression of hsa-miR-122-5p,hsa-miR-146b-5p and hsa-miR-197-3p:moderate to severe NAFLD>mild NAFLD>Control.The relative expression of hsa-miR-483-3p:NAFLD>Control.There was no significant difference between mild and moderate to severe of NAFLD.(3)Fat droplets appeared in some hepatocytes of mice fed with high-fat diet for 8 weeks.After 12 weeks of feeding,diffuse steatosis appeared,showing a mixed steatosis dominated by vesicles.(4)After 12 weeks of feeding,the relative expression of microRNAs:mmu-miR-122-5p(HFD<CTL),mmu-miR-146b-5p(HFD>CTL),and the relative expression of mmu-miR-483-3p showed no significant difference.(5)A total of 2623 target genes were predicted for the 4 microRNAs.GO functional analysis showed that the target genes were extensively annotated in molecular function,cell composition,and biological processes.There were 84 pathways with significant enrichment of target genes,and 20 of them most relevant to NAFLD were selected.5 target genes related to at least 10 pathways were screened,namely PIK3R2,AKT2.AKT3,MAPK1 and NFKB1.Conclusion(1)Serum exosomal hsa-miR-122-5p,hsa-miR-146b-5p,hsa-miR-197-3p and hsa-miR-483-3p have different significance in identifying NAFLD and healthy people.They are involved in fat metabolism and the occurrence and development of various liver diseases.(2)The mature NAFLD model could be established by feeding C57 BL/6 mice with high-fat diet for 12 weeks.(3)Mmu-miR-122-5p and mmu-miR-146b-5p may play an important role in the process of liver steatosis in mice.(4)Those 4 microRNAs are hoped to be used as biomarkers for non-invasive diagnosis of NAFLD and new targets for specific therapy.