Effect and mechanism of Csi-miR-125a induced liver fibrosis in the exosomes of Clonorchis sinensis

doi:10.12140/j.issn.1000-7423.2025.02.008

Abstract

Abstract:

Objective To examine the role of hub miRNAs from Clonorchis sinensis exosomes in hepatic fibrosis, so as to provide new insights into deciphering the mechanisms underlying hepatic fibrosis induced by C. sinensis. Methods Adult worms of C. sinensis was collected from liver specimens of a domestic cat infected with C. sinensis, cultured in vitro, and excretory/secretory products were collected, centrifuged and filtered. Exosomes were extracted and observed under a transmission electron microscope, and their particle size distribution was analyzed. Total RNA was extracted from exosomes, and sequenced, and miRNA was predicted using the miRDeep2 software. The miRNA sequences were aligned with those in the miRBase database, and the expression of each miRNA was quantified and screened. The target genes of selected miRNAs were predicted using the miRanda, miRDB, and PITA software, and the Gene Ontology (GO) and signaling pathways of target genes were subjected to enrichment analyses using the topGO and clusterProfiler software. Hepatic stellate cells (HSCs) were incubated with exosomes at concentrations of 5, 10, and 20 μg/ml for 24 and 48 h, and a blank control group was assigned. HSCs were transfected with a selected miRNA (Csi-miR-125a) at a final concentration of 50 nmol/L for 48 h, and a negative control group was assigned. The proliferation of HSCs was measured using the cell counting kit-8 (CCK-8) assay, and the expression of HSC activation-related protein α-smooth muscle actin (α-SMA) and type Ⅲ collagen, autophagy-related proteins microtubule associated protein 1A/1B light chain 3 (LC3) and p62, and the phosphorylation of the signaling pathway-related extracellular signal regulated kinase (ERK) protein was determined using Western blotting with mouse anti-human β-actin, rabbit anti-human α-SMA, rabbit anti-human type Ⅲ collagen, rabbit an-human LC3, rabbit anti-human p62, rabbit anti-human ERK, and rabbit anti-human p-ERK primary antibodies (1∶1 000 dilution), and the secondary horseradish peroxidase (HRP) conjugated goat anti-mouse or anti-rabbit IgG antibody (1∶1 000 dilution). Results The C. sinensis exosomes appeared a small vesicular shape with a phospholipid bilayer structure, and the main peak particle size was 72 nm (range, 30 to 150 nm). The C. sinensis exosomes contained multiple miRNAs, and Csi-miR-125a, which had the second highest expression and a high similarity to known miRNA sequences, was selected for subsequent experiment. Csi-miR-125a had overlapping of 301 predicted sites of target genes, and these target genes were significantly enriched in cellular components such as cytoplasm, involved in molecular functions of protein binding and ion binding, and biological processes of biological regulation, systematic development, and nervous system development, and significantly enriched in Hedgehog signaling pathways, extracellular matrix- receptor interaction related pathways, and NOD like receptor signaling pathways. The CCK-8 assay measured the highest A₄₅₀ value (1.707) at an exosome concentration of 10 μg/ml for 24 h incubation, and the A₄₅₀ value was higher in the Csi-miR-125a co-culture group (1.122) than in the negative control group (0.492). Western blotting determined that the grayscale values of the α-SMA bands were 0.588 and 0.904 in the negative control group and the Csi-miR-125a group (t = 10.560, P < 0.01), and the grayscale values of type Ⅲ collagen bands were 0.783 and 0.997 in the negative control group and the Csi-miR-125a group (t = 6.483, P < 0.05).The grayscale ratios of LC3 Ⅱ/LC3 Ⅰ bands were 1.093 and 2.215 in the negative control group and the Csi-miR-125a group (t = 5.206, P < 0.05), and the grayscale values of p62 bands were 0.537 and 1.369 in the negative control group and the Csi-miR-125a group (t = 13.100, P < 0.01), while the grayscale ratios of pERK/ERK bands were 0.791 and 1.037 in the negative control group and the Csi-miR-125a group, respectively (t = 5.067, P < 0.05). Conclusion There are diverse miRNAs in C. sinensis exosomes, which are involved in multiple cellular functions and signaling pathways. Csi-miR-125a may contribute to hepatic fibrosis through blocking the autophagic flux of HSCs and activating the ERK signaling pathway to promote HSCs proliferation and activation.

Key words: Clonorchis sinensis, Hepatic fibrosis, Exosomes, Csi-miR-125a, ERK signaling pathway

CLC Number:

R383.22

WU Yinjuan, YI Xueqing, LI Meiyu, XU Anyuan, WU Aoxun, ZHONG Zifeng, LI Xuerong. Effect and mechanism of Csi-miR-125a induced liver fibrosis in the exosomes of Clonorchis sinensis[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2025, 43(2): 198-204.

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