*3.3. miRNA Expression Analysis in the CCl4-Mouse Model*

Next, we analyzed the expression of miRNA-451a, miRNA-142-5p, Let-7f-5p, and miRNA-378a-3p in a well-studied mouse model of liver fibrosis, with repeated injections of carbon tetrachloride (CCl4) [20]. When mice are exposed to the CCl4-toxin two times a week, for four weeks, significant hepatocyte-damage and HSC-activation can be seen (Figure 3A,B). An analysis of total liver tissue from sick mice, compared to healthy controls, revealed significant changing levels for miRNA-451a, miRNA-142-5p, Let-7f-5p, and miRNA-378a-3p (Figure 3C), with overlapping expression patterns, as found in activating HSCs. The expression of two miRNAs extensively characterized in chronic liver diseases, miRNA-122-5p [21] and miRNA-29a-3p [22], was used as positive controls. Plasma obtained from the CCl4-mouse model identified significant changing expression levels of all analyzed miRNAs (Figure 3D). Interestingly, all miRNAs had a plasma expression pattern opposite to what was found in total liver tissue or activating HSCs. Altogether, these results suggest the potential use of these circulating miRNAs, without the need to distinguish between miRNAs packaged into extracellular vesicles or bound to (lipo-)proteins, as markers for HSC activation and fibrosis progression.

**Figure 1.** miRNA expressioninmouse in vitro activated hepatic stellate cells (HSCs). (**A**) Immunofluorescence staining of quiescent (24 h of culture) and activated (10 days of culture) primary mouse HSCs for activation markers Desmin, αSMA, and Vimentin. 4 ,6-Diamidino-2-phenylindole (DAPI) was used as nuclear staining. Representative images are shown. (**B**) mRNA expression levels determined by quantitative polymerase chain reaction (qPCR) of HSC-activation markers *Acta2*, *Col1a1*, and *Lox* in freshly isolated HSCs (0 h), as compared to HSCs activated by 10 days of culture (D10). (**C**) Heatmap of relative expression levels, as determined by qPCR, for selected candidate miRNAs in activated HSCs (D10), as compared to freshly isolated HSCs (0 h). (**D**) miRNA-451a, miRNA-142-5p, Let-7f-5p, and miRNA-378a-3p were found to be significantly dysregulated upon HSC activation. One-tailed unpaired t-test analysis was used to determine statistical significance. Results are shown as mean ± standard error of the mean (SEM); *n* = 5.
