Fig 1.
The effects of E. multilocularis infection on hepatic fibrosis in mouse.
(A) Representative pictures of Hematoxylin and Eosin (H&E), Masson’s Trichrome, and Picrosirius Red staining for liver fibrogenesis in control mice, high-fat diet induced mice, and E. multilocularis-infected mice. Arrows indicate cysts. (B) The birefringent collagen content was quantified in Masson’s Trichrome- or Picrosirius Red-stained sections by color thresholding-based segmentation using ImageJ.
Fig 2.
The activation of HSCs induced by E. multilocularis infection.
(A) Representative pictures of immunofluorescence staining for α-SMA in the livers of E. multilocularis-infected and uninfected mice. Arrows indicate cysts. (B) Quantification of α-SMA-positive cells in the livers of E. multilocularis-infected and uninfected mice. (C) Vitamin A lipid droplet autofluorescence in isolated primary HSCs was detected at a wavelength of 328 nm using a fluorescence microscope. (D) Flow cytometry analysis of isolated mouse HSCs using Gfap. (E) The expression of α-sma, col1α1, and vimentin in the HSCs from E. multilocularis-infected mice 30-, 60-, and 90-day post-infection (dpi) by qRT-PCR. (F) The expression of α-SMA in the HSCs from E. multilocularis-infected mice 30-, 60-, and 90-day post-infection (dpi) by Western blotting. Data for final statistical analysis were taken from 3 independent experiments. *p <0.05, **p < 0.01, ***p < 0.001.
Fig 3.
Dysregulation of mmu-miR-342-3p in liver HSCs during E. multilocularis infection.
(A) A volcano plot of the differentially expressed miRNAs between two liver HSCs from E. multilocularis-infected mice at 60 and 90-day post infection (dpi), where the red and green, indicated significantly upregulated miRNAs (Up) and downregulated miRNAs (Down), respectively. (B) KEGG pathway enrichment analysis of target genes. (C) The expression of mmu-miR-342-3p in hepatocyte cells (HCs), Kupffer cells (KCs), and hepatic stellate cells (HSCs). (D) The expression of mmu-miR-342-3p in liver HSCs from E. multilocularis-infected mice at 30-, 60-, and 90-day post-infection (dpi). The uninfected mice at each sampling time point were used as controls. Data for final statistical analysis were taken from 3 independent experiments. ***p < 0.001.
Fig 4.
Upregulation of mmu-miR-342-3p in the culture-activated HSCs.
(A) The qRT-PCR analysis of α-Sma, Col1α1, Gfap in in vitro culture-activated HSCs. (B) Immunofluorescence staining of α-SMA in in vitro culture-activated HSCs. Scale bar = 50 μm (C) The expression of mmu-miR-342-3p in in vitro culture-activated HSCs. Data for final statistical analysis were taken from 3 independent experiments. **p < 0.01, ***p < 0.001.
Fig 5.
Inhibition Zbtb7a by mmu-miR-342-3p via directly binding to its 3′UTR.
(A) Schematic representation of a putative binding site of mmu-miR-342-3p in the 3’UTR of Zbtb7a (up) and the dual-Glo Luciferase assay for the interaction between mmu-miR-342-3p-Mimics and Zbtb7a (down). The miRNA ‘seed’ sequences are indicated in bold in black. WT: WT- Zbtb7a or Mut- Zbtb7a construct was co-transfected into 293T cells with either mimic negative control (mimic NC) or mmu-miR-342-3p mimic (miR-342-3p mimic) and luciferase activities were measured after 24 h transfection. (B) The expression of mmu-miR-342-3p in the HSCs from E. multilocularis-infected mice transfected with inhibitor negative control (NC) or mmu-miR-342-3p inhibitor (Inhibitor) by RT-qPCR. (C) The expression of Zbtb7a at mRNA and protein levels in the HSCs from E. multilocularis-infected mice transfected with mmu-miR-342-3p inhibitor (Inhibitor). (D) The expression of zbtb7a in primary HSCs from E. multilocularis-infected mice at 30-, 60-, and 90-day post-infection (dpi). (E) Correlation between the expression of Zbtb7a and mmu-miR-342-3p. (n = 9, Spearman’s correlation analysis; r, correlation coefficient) Data for final statistical analysis were taken from 3 independent experiments. **p < 0.01; ***p < 0.001.
Fig 6.
The effect of down-regulated mmu-miR-342-3p on HSC activation during E. multilocularis infection.
(A) The expression of mmu-miR-342-3p in the HSCs from E. multilocularis-infected mice after transfecting with inhibitor negative control (NC) or mmu-miR-342-3p inhibitor (inhibitor). (B) Effects of downregulated mmu-miR-342-3p on the expression of HSC activation marker (α-Sma and Vimentin) and quiescent marker Gfap in the HSCs from E. multilocularis-infected mice. (C) The mmu-miR-342-3p expression was significantly downregulated in the mouse livers by CS-mmu-miR-342-3p inhibitor nanoparticles (CSNP-Inhibitor) compared with that of CS-inhibitor negative control nanoparticles (CSNP-NC). (D) The Zbtb7a expression both at mRNA and protein levels were significant upregulated in the livers of E. multilocularis-infected mice by injecting with CS-mmu-miR-342-3p inhibitor nanoparticles. (F) Effect of mmu-miR-342-3p on the expression of α-Sma, Col1α1, Vimentin, and TGF-β in the in the livers of E. multilocularis-infected mice by injecting with CS-mmu-miR-342-3p inhibitor nanoparticles. Data for final statistical analysis were taken from 3 independent experiments. ***p < 0.001.