*3.4. Patient Characteristics and Plasma miRNA Alterations During Liver Fibrosis Progression*

Next, we investigated whether the plasma levels of these six miRNAs can be correlated to liver fibrosis severity in patients suffering from different chronic liver diseases. Patient characteristics are summarized in Table 1. A total of 208 patients were included, of which 92 patients were diagnosed with no or minimal fibrosis (F0–1) and 116 patients with significant fibrosis (F ≥ 2), as staged by elastography. Patients with various aetiologies of liver disease were recruited—chronic alcohol abuse (*n* = 33), chronic HBV/HCV infection (*n* = 74), and NAFLD (*n* = 101). Patients with chronic alcohol abuse or viral infection underwent transient elastography (FibroScan®) to distinguish significant liver fibrosis (F <sup>≥</sup> 2) from no or minimal fibrosis (F0–1); (median (25th; 75th percentile)) 12 (9.1; 33.6) kPa versus 5.2 (4.0;

6.1) kPa, respectively. Patients who presented with NAFLD all suffered from Diabetes Mellitus type 2 and underwent ARFI to distinguish significant liver fibrosis (F ≥ 2) from no or minimal fibrosis (F0–1); (median (25th; 75th percentile)) 1.525 (1.30; 1.68) m/s versus 1.15 (1.12; 1.20) m/s, respectively. Various clinical scoring algorithms, such as the AST/ALT ratio, Fib-4 score, APRI, and the recently developed PRTA-score [17], were calculated. All liver-related laboratory parameters, except for ALT and Creatinine values, and all fibrosis scoring tools are significantly different between the F0–1 and F ≥ 2 patient cohorts, validating the early- or late disease character of the included patients (Table 1).

**Figure 2.** miRNA target prediction. Bioinformatics-based target prediction was carried out, using four different predictive algorithms: TargetScan, miRDB, starBase, and miRTarBase. (**A**) Venn diagram showing putative target genes for the differentially expressed miRNAs in culture activated primary mouse HSCs. A list of target genes mutual among all four miRNAs is shown. mRNA expression analysis of the identified mutual target genes in activated (D10) versus quiescent (0 h) HSCs identified (**B**) three genes with no significant difference, (**C**) three genes to be significantly up-regulated, and (**D**) seven genes to be significantly down-regulated. One-tailed unpaired t-test analysis was used to determine statistical significance. Results are shown as mean ± SEM; *n* = 5.

**Figure 3.** miRNA expression analysis in a CCl4-induced mouse model of liver fibrosis. (**A**) Total liver tissue of mice that received CCl4-injections two times a week, for a period of four weeks, and healthy controls, was used to visualize hepatocyte damage and inflammation (H&E), and cross-linked collagen deposition (Sirius Red). Arrows indicate infiltrated inflammatory cells. Representative images are shown. (**B**) The area of Sirius Red positive staining was calculated using Orbit analysis software and is plotted as percentage of the total area (*n* = 7 mice per group). (**C**) miRNA expression analysis of total liver tissue extracted from CCl4-injected mice, as compared to healthy controls (*n* = 7 mice per group). Results are shown as mean ± SEM. (**D**) Tukey boxplots represent miRNA expression values in plasma obtained from CCl4-injected mice, as compared to healthy controls (*n* = 7 mice per group). Obtained Ct levels were normalized by use of spiked-in Cel-miRNA-39. One-tailed unpaired t-test analysis was used to determine statistical significance.


**Table 1.** Baseline characteristics of the patient cohort.

n: number; NAFLD: non-alcoholic fatty liver disease; IQR: interquartile range; BMI: body mass index; AST: aspartate aminotransferase; ALT: alanine aminotransferase; Alk Phos: alkaline phosphatase; GGT: gamma-glutamyl transferase; AST/ALT ratio: aspartate aminotransferase/alanine aminotransferase ratio; APRI: AST to platelet ratio index; Fib-4: Fibrosis-4; PRTA-score: PDGFRβ-thrombocytes-albumin score; ns: not significant.

Analysis of the plasma of these patients showed that miRNA-451a and miRNA-142-5p were significantly up-regulated, while Let-7f-5p was significantly down-regulated, in patients with significant liver fibrosis (F ≥ 2). miRNA-378a-3p remained stable during fibrosis progression (Figure 4A). Area under receiver operating characteristic (AUROC) analysis identified comparable diagnostic utility among miRNA-451a (AUC = 0.6065), miRNA-142-5p (AUC = 0.6220), and Let-7f-5p (AUC = 0.6485) (Figure 4A and Supplementary Table S3). Late-stage fibrosis markers miRNA-122-5p (AUC = 0.5969) and miRNA-29a-3p (AUC = 0.5922) (Figure 4B,C and Supplementary Table S3) were found to have lower diagnostic utility. While APRI (AUC = 0.6481) and AST/ALT (AUC = 0.5956) were comparable to or were outperformed by the analyzed miRNAs, FIB-4 (AUC = 0.6879) and the PRTA-score (AUC = 0.7732) remained superior for the diagnosis of significant liver fibrosis (Figure 4D and Supplementary Table S3).
