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Hepatocyte exosomal miR-21-5p activates hepatic stellate cells and exacerbates liver fibrosis via targeting Smad7

ABSTRACT: Aims 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.

INSTRUMENT(S): Illumina HiSeq 2500, QIAseqTM miRNA Library Kit, exoEasy Maxi Kit., Beckman ultracentrifuge

ORGANISM(S): Mus musculus

SUBMITTER: Xia Zhou

PROVIDER: E-MTAB-16648 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background/Aim: We investigated alterations in the expression of serum exosomal miRNAs with the progression of liver fibrosis and evaluated their clinical applicability as biomarkers. Methods: This study prospectively enrolled 71 patients who underwent liver biopsy at an academic hospital in Korea. Exosomes were extracted from serum samples, followed by next-generation sequencing (NGS) of miRNAs and targeted real-time quantitative polymerase chain reaction. A model was derived to discriminate advanced fibrosis based on miRNA levels and the performance of this model was evaluated. Validation of the effect of miRNA on liver fibrosis in vitro was followed. Methods: This study prospectively enrolled 71 patients who underwent liver biopsy at an academic hospital in Korea. Exosomes were extracted from serum samples, followed by next-generation sequencing (NGS) of miRNAs and targeted real-time quantitative polymerase chain reaction. A model was derived to discriminate advanced fibrosis based on miRNA levels and the performance of this model was evaluated. Validation of the effect of miRNA on liver fibrosis in vitro was followed. Results: NGS data revealed that exosomal miR-660-5p, miR-125a-5p, and miR-122 expression were changed significantly with the progression of liver fibrosis, of which miR-122 exhibited high read counts enough to be used as a biomarker. The level of exosomal miR-122 decreased as the pathologic fibrosis grade progressed and patients with biopsy-proven advanced fibrosis had significantly lower levels of exosomal miR-122 (P < 0.001) than those without advanced fibrosis. Exosomal miR-122 exhibited a fair performance in discriminating advanced fibrosis especially in combination with fibrosis-4 score and transient elastography. In a subgroup of patients with a non-viral etiology of liver disease, the performance of exosomal miR-122 as a biomarker was greatly improved. Inhibition of miR-122 expression increased the proliferation of the human hepatic stellate cell line, LX-2, and upregulated the expression of various fibrosis related proteins. Conclusion: Exosomal miR-122 may serve as a useful non-invasive biomarker for liver fibrosis, especially in patients with non-viral etiologies of chronic liver disease.

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Hepatocyte exosomal miR-21-5p activates hepatic stellate cells and exacerbates liver fibrosis via targeting Smad7

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