biostudies-arrayexpressXia ZhouMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16648Aims This study investigated the impact of hepatocyte exosomes on hepatic stellate cell (HSC) activation and their potential role in liver fibrosis while elucidating the underlying molecular mechanisms. Methods L02 exosomes were extracted, and their influence on LX-2 activation was preliminarily investigated. A mouse liver fibrosis model was established through intraperitoneal injection of 20% carbon tetrachloride (CCl4). Normal and fibrotic hepatocyte exosomes were separately collected to explore their distinct effects on HSC activation. High-throughput sequencing identified differential miRNAs in exosomes from normal and fibrotic hepatocytes. MiR-21-5p, displaying the most substantial expression difference, was selected to assess the correlation between serum exosomal miR-21-5p and liver fibrosis. The target gene of miR-21-5p was validated using a dual luciferase assay. LX-2 cells were transfected with miR-21-5p mimics and inhibitors to clarify the impact and mechanisms of miR-21-5p on HSC activation, proliferation, and collagen synthesis. Results Normal L02 exosomes were internalized by LX-2 cells and inhibited their activation. In comparison to normal hepatocyte exosomes, fibrotic hepatocyte exosomes induced HSC activation. High-throughput sequencing revealed 32 upregulated and 6 downregulated miRNAs in fibrotic hepatocyte exosomes, with the most significant increase observed in miR-21-5p. Serum exosomal miR-21-5p displayed a close association with liver fibrosis. Dual luciferase assays and cell transfection experiments confirmed that miR-21-5p promoted HSC activation, proliferation, and collagen synthesis by targeting Smad7. Conclusion Fibrotic hepatocyte exosomes may trigger HSCs activation through exosomal transfer in liver fibrosis. Exosomal miR-21-5p enhances HSCs activation, proliferation, and collagen synthesis by targeting Smad7, potentially serving as a diagnostic marker and therapeutic target for liver fibrosis.biostudies-arrayexpressNucleic Acid Extraction - ExoRNA samples from both normal and fibrotic hepatocytes, each containing 10 ng, were isolated using the exoEasy Maxi Kit.Library Construction - miRNA libraries were constructed in accordance with the QIAseqTM miRNA Library Kit Handbook.Sequencing - High-throughput small RNA sequencing was performed using the Hiseq 2500 sequencer. Hierarchical cluster analysis was conducted on differential miRNAs present in hepatocyte exosomes using the heatmap function of R software. The target genes of differentially expressed miRNAs were predicted using miRTarBase, TargetScan, and miRDB websites.Sample Collection - Around 100-200ml of supernatant from primary hepatocytes in both normal and liver fibrosis groups were collected, followed by exosome extraction through ultra-high speed centrifugation. The procedure entailed centrifuging hepatocyte supernatants at 2000g for 10 minutes to eliminate dead cells, followed by 10000g centrifugation for 30 minutes to remove cellular debris. The remaining medium underwent ultracentrifugation at 100,000×g for 90 minutes at 4℃. The resultant precipitate was washed with PBS (100,000×g for 90 minutes), and the exosome-containing pellet was suspended in PBS.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - FANse3 ultra-precision sequence alignment algorithm was used. The quantization of gene expression was measured by RPM(RPM=readCount*1,000,000/libsize, libsize: sum of mapped readCount), that is, the number of reads per million from a certain gene. RPM eliminates the influence of sequencing depth on reads count and is the most commonly used method for estimating microRNA expression levels. Due to the high precision of the mapping algorithm, genes with more than 10 reads sequenced were expressed, while genes with less than 10 reads were not.UnknownTranscriptomicsIllumina HiSeq 2500QIAseqTM miRNA Library KitexoEasy Maxi Kit.Beckman ultracentrifugemicroRNA profiling by high throughput sequencingMus musculusXia ZhoufalseHepatocyte exosomal miR-21-5p activates hepatic stellate cells and exacerbates liver fibrosis via targeting Smad7Aims This study investigated the impact of hepatocyte exosomes on hepatic stellate cell (HSC) activation and their potential role in liver fibrosis while elucidating the underlying molecular mechanisms. Methods L02 exosomes were extracted, and their influence on LX-2 activation was preliminarily investigated. A mouse liver fibrosis model was established through intraperitoneal injection of 20% carbon tetrachloride (CCl4). Normal and fibrotic hepatocyte exosomes were separately collected to explore their distinct effects on HSC activation. High-throughput sequencing identified differential miRNAs in exosomes from normal and fibrotic hepatocytes. MiR-21-5p, displaying the most substantial expression difference, was selected to assess the correlation between serum exosomal miR-21-5p and liver fibrosis. The target gene of miR-21-5p was validated using a dual luciferase assay. LX-2 cells were transfected with miR-21-5p mimics and inhibitors to clarify the impact and mechanisms of miR-21-5p on HSC activation, proliferation, and collagen synthesis. Results Normal L02 exosomes were internalized by LX-2 cells and inhibited their activation. In comparison to normal hepatocyte exosomes, fibrotic hepatocyte exosomes induced HSC activation. High-throughput sequencing revealed 32 upregulated and 6 downregulated miRNAs in fibrotic hepatocyte exosomes, with the most significant increase observed in miR-21-5p. Serum exosomal miR-21-5p displayed a close association with liver fibrosis. Dual luciferase assays and cell transfection experiments confirmed that miR-21-5p promoted HSC activation, proliferation, and collagen synthesis by targeting Smad7. Conclusion Fibrotic hepatocyte exosomes may trigger HSCs activation through exosomal transfer in liver fibrosis. Exosomal miR-21-5p enhances HSCs activation, proliferation, and collagen synthesis by targeting Smad7, potentially serving as a diagnostic marker and therapeutic target for liver fibrosis.2025-08-31T00:00:00Z2026-02-13T11:41:19.745Z2026-02-13T11:40:47.82ZE-MTAB-16648ERP189096E-MTAB-4689EFO_0002944EFO_0004170EFO_0002896EFO_0005518EFO_0003816EFO_0004184