MiRNA-29b-3p was highly expressed in peripheral blood exosomes of children with BA. Exosomes overexpressing miRNA-29b-3p hypomethylated the DNA promoter region of Fibronectin and Snail in hepatocytes, which contributed to the high expression of Fibronectin and Snail and the occurrence of EMT, which further led to an increase of α-SMA and accelerated the fibrosis of BA. The progress of BA fibrosis was accelerated by the increase of α-SMA. This study, by exploring the pathogenesis of BA hepatic fibrosis, thus suggests new ideas for the diagnosis and treatment of BA liver fibrosis, and provides a basis for further understanding of BA hepatic fibrosis from the perspective of exosomes, epigenetics and EMT.
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Figure 1 Validation of miR-29b-3p overexpression and detection of the methylation level in patients with BA. a. peripheral blood exosomes were collected from children with BA and CC, and the exosomes were observed under transmission electron microscope, and disc-shaped exosome morphology was seen; b. the expression levels of miR-29b-3p in peripheral blood exosomes of 30 children with BA and 30 children with CC were verified by qPCR; c. the expression levels of miR-29b-3p in liver tissues of 30 children with BA and 30 children with CC were verified by qPCR; d. e. DNMT3a and DNMT3b expression in liver tissues of BA and CC groups were detected by qPCR and Western-blot; f-h. qPCR detected methylation levels in liver tissues of BA and CC groups(*P < 0.05, **P < 0.01.)
Figure 2 Construction of stable transcripts and validation of exosomes. a. Left, Jurkat cells were infected with lentivirus to obtain a negative null control; right, Jurkat cells overexpressing miRNA-29b-3p stable transcripts. b. Three sets of exosomes were observed by transmission electron microscopy, and all of them were disc-shaped in morphology. c. Nanoparticle tracking technology was used to analyze the particle size of the collected exosomes. d. Expression of miRNA-29b-3p in exosomes obtained from Jurkat cells transfected with miRNA-29b-3p mimic. e. Western blotting to detect exosomal surface proteins (CD9, CD81). Control: blank control of the untreated Jurkat cell group; nc: negative control empty lentivirus-infected Jurkat cell group; miRNA-29b-3p mimic: overexpressing miRNA-29b-3p lentivirus-infected Jurkat cell group. (*P < 0.05).
Figure 3 Effect of miRNA-29b-3p-overexpressing exosomes on L02 cells after addition of DNA methyltransferase inhibitor 5-AzaC. a. qPCR detection of miRNA-29b-3p, DNMT3a and DNMT3b expression in L02 cells after co-culture with different groups of exosomes. b. Western blot detection of DNMT3a, DNMT3b and cell cycle-related indices in L02 cells after co-culture with exosomes. c. Dual-luciferase assay to demonstrate that DNMT3a and DNMT3b are target genes of miR-29b-3p. d, e. Western blot and qPCR detection of EMT-related indicators in L02 cells after co-culture with exosomes. Control: blank control group; miR-29b-3p exosome: overexpression of miRNA-29b-3p lentiviral co-culture cell group; Exosome: normal exosome co-culture cell group; NC-Exosome: null control lentiviral co-culture cell group; 5-AzaC: 5-AzaC inhibitor group. (*P < 0.05).
Figure 4 Regulation of methylation of the DNA promoter regions of Snail, Fibronectin and α-SMA in L02 cells by exosomes + 5-AzaC. a. Methylation of CpG sites in the DNA promoter region of Snail, Fibronectin and α-SMA genes in each group. b. Quantification of methylation level. Control: blank control group; miR-29b-3p exosome: miRNA-29b-3p-overexpressing lentiviral co-culture cell group; Exosome: normal exosome co-culture cell group; NC-Exosome: null control lentiviral co-culture cell group; 5-AzaC: 5-AzaC inhibitor group. (*P < 0.05).
Figure 5 Construction of the BA animal model and detecting the expression of relevant indices after adding miRNA-29b-3p inhibitor. a. Western blot detection of the expression of relevant proteins 7 days and 14 days after injection of RRV. b. HE staining results. c. Immunohistochemical detection of the expression of DDR2. d. Histogram of DDR2 expression at 14 days. e. Immunohistochemical detection of FSPI expression. f. Histogram of FSP1 expression at 14 days. (*P < 0.05).
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