Verification,Molecular Regulation And Target Gene Prediction Of MiR-125b-5p In Patients With Alveolar Echinococcosis

Objectives:1)To detect the expression of hsa-miR-125b-5p in the serum and the tissues of the marginal zone of liver lesions of patients with alveolar echinococcosis;2)Huh7 cells were transfected with lentivirus in vitro to over-express hsa-miR-125b-5p and observe changes in cell viability;3)Preliminary prediction and analysis of target genes of hsa-miR-125b-5p,providing a certain theoretical basis for further searching for molecular markers for early diagnosis of alveolar echinococcosis.Methods:1)The relative expression levels of hsa-miR-125b-5p in the serum of 22 patients with alveolar echinococcosis and 22 healthy controls were detected by realtime quantitative PCR;2)Detection of the relative expression of hsa-miR-125b-5p in the marginal bands of liver lesions and liver tissues far away from the lesions by fluorescence quantitative PCR;3)The LV-hsa-miR-125b-5p lentiviral vector was constructed to transfect Huh7 cells,and the transfected Huh7 cells were treated with cck-8 kit.Then the OD value at the wavelength of 450 nm was detected with an enzyme marker machine,and the proliferation activity was determined by statistical analysis;4)Four database resources of miRDB,star Base,miRwalk and miRTarbase were used to predict the possible target genes of hsa-miR-125b-5p.Then use Enrichr database,KEGG database and STRING database for GO analysis,pathway analysis and PPI network(protein-protein interaction)of target proteins.Database analysis was used to verify the association between hsa-miR-125b-5p and alveolar echinococcosis.Results:1)The expression of hsa-miR-125b-5p was significantly up-regulated in the serum of patients with alveolar echinococcosis(P<0.0001);2)The expression of hsamiR-125b-5p in the marginal tissues of patients with alveolar echinococcosis was significantly up-regulated(P<0.0001);3)After over-expression of hsa-miR-125b-5p,the liver cell proliferation activity was reduced(P< 0.05);4)GO analysis showed that the target genes of hsa-miR-125b-5p were significantly enriched in the regulation of apoptotic processes and the regulation of cell proliferation in biological processes(P<0.05),and were significantly in DNA binding and MAP kinase activity in molecular functions(P<0.05),and were significantly enriched in chromatin RNA,ribosome,and mitochondria in cell components(P<0.05).KEGG pathway analysis predicts signal pathways closely related to liver fibrosis: hepatocellular carcinoma pathway,MAPK pathway;The protein interaction network analysis identified eight target genes that are important in protein interactions: TP53,STAT3,AKT1,JUN,JAK2,MAPK14,SMAD4,EGFR.Conclusions:1)Compared with the control group,the expression of hsa-miR-125b-5p in the serum of patients with alveolar echinococcosis and in the tissues of the liver lesion marginal zone is different,and all of them are up-regulated;2)Preliminary verification at the cell level that hsa-miR-125b-5p overexpression can negatively affect the proliferation and viability of Huh7 cells;3)The preliminary prediction and analysis of the target gene function of hsa-miR-125b-5p provides a certain basis for further searching for molecular markers for early diagnosis of alveolar echinococcosis.