The role of MicroRNAs in hepatic fibrosis progression and therapeutic implications

Introduction

Hepatic fibrosis represents a wound-healing response to chronic liver injury characterized by excessive accumulation of extracellular matrix components, particularly collagens and fibronectin [2] . Progressive fibrosis leads to architectural distortion of the liver parenchyma, ultimately resulting in cirrhosis with its associated complications including portal hypertension, hepatocellular carcinoma, and liver failure [3] . The global burden of chronic liver disease continues to rise, driven by increasing prevalence of viral hepatitis, nonalcoholic fatty liver disease, and alcohol-related liver injury. Understanding the molecular mechanisms governing fibrogenesis has become essential for developing effective therapeutic interventions. Hepatic stellate cells (HSCs) serve as the primary effector cells in liver fibrosis, undergoing activation and transdifferentiation into myofibroblast-like cells upon liver injury [4] . Activated HSCs produce excessive extracellular matrix proteins and secrete proinflammatory cytokines that perpetuate the fibrotic response. The activation process involves complex signaling networks, with transforming growth factor-beta (TGF-β) recognized as the master regulator of fibrogenesis [5] . However, therapeutic targeting of TGF-β has proven challenging due to its pleiotropic functions in maintaining tissue homeostasis and immune regulation. MicroRNAs are small non-coding RNA molecules of approximately 22 nucleotides that regulate gene expression through post-transcriptional mechanisms [6] . By binding to complementary sequences in target messenger RNAs, miRNAs can induce transcript degradation or translational repression. A single miRNA may regulate hundreds of target genes, while individual genes may be regulated by multiple miRNAs, creating complex regulatory networks [7] . The discovery of miRNA dysregulation in various pathological conditions has generated considerable interest in their potential as both biomarkers and therapeutic targets. Research conducted over the past decade has revealed that specific miRNAs play essential roles in hepatic fibrosis development and progression [8] . Pro-fibrotic miRNAs such as miR-21, miR-34a, and miR-221 promote HSC activation and extracellular matrix production, while anti-fibrotic miRNAs including miR-29 and miR-200 family members suppress fibrogenic pathways [9] . The balance between these opposing regulatory forces appears to determine the trajectory of fibrosis progression or resolution. Understanding these miRNA-mediated regulatory mechanisms offers opportunities for therapeutic intervention that could complement or replace current treatment approaches. Despite substantial progress in identifying fibrosisassociated miRNAs, comprehensive profiling across the full spectrum of fibrosis stages in human liver tissue remains limited [10] . Most previous investigations have focused on animal models or cell culture systems, which may not fully recapitulate human disease pathophysiology. Additionally, the therapeutic potential of miRNA-based interventions requires validation in clinically relevant experimental systems. This research aimed to characterize miRNA expression patterns across human liver fibrosis stages, identify key regulatory networks, and evaluate the therapeutic potential of targeting specific miRNAs in hepatic stellate cell models [11] . The findings contribute to understanding fibrogenesis mechanisms and support development of miRNA-based diagnostic and therapeutic strategies for chronic liver disease.

Material and Methods

Material

This research enrolled 186 patients with chronic liver disease attending the Hepatology Division at Hospital das Clínicas, University of São Paulo, between January 2022 and August 2024. Patients were categorized according to METAVIR fibrosis staging: F0 (n=32), F1 (n=38), F2 (n=41), F3 (n=39), and F4 (n=36). Inclusion criteria comprised adults aged 18-70 years with documented chronic liver disease requiring diagnostic liver biopsy for clinical indications. Exclusion criteria included hepatocellular carcinoma, prior liver transplantation, concurrent malignancy, or immunosuppressive therapy within six months. Liver biopsy specimens were obtained using 16-gauge Tru-Cut needles under ultrasound guidance by experienced hepatologists. Each specimen was divided into portions for histopathological assessment and molecular analysis. RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, USA) and miRNeasy Mini Kit (Qiagen, Hilden, Germany) following manufacturer protocols. Human hepatic stellate cell line LX-2 was obtained from American Type Culture Collection and maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum under standard culture conditions.

Methods

MicroRNA profiling employed Illumina small RNA sequencing on NovaSeq 6000 platform with minimum read depth of 10 million reads per sample. Raw sequencing data underwent quality filtering using Trimmomatic and alignment to human genome reference (GRCh38) using Bowtie. Differential expression analysis utilized DESeq2 with significance threshold of adjusted p-value < 0.05 and fold change > 2. Validation of selected miRNAs employed TaqMan Advanced miRNA Assays (Applied Biosystems) with U6 snRNA as endogenous control. Target prediction and pathway analysis integrated results from TargetScan, miRDB, and DIANA-microT databases, requiring consensus prediction from at least two algorithms. Gene ontology and KEGG pathway enrichment analyses were performed using DAVID Bioinformatics Resources and Enrichr platforms. Network visualization employed Cytoscape software with ClueGO plugin for functional annotation clustering. Functional validation experiments utilized LX-2 cells transfected with miRNA mimics or inhibitors (Dharmacon, Lafayette, USA) using Lipofectamine RNAiMAX (Thermo Fisher). Cells were treated with TGF-β1 (10 ng/mL) for 48 hours to induce fibrogenic activation. Gene expression was assessed by quantitative RT-PCR using SYBR Green chemistry with GAPDH normalization. Protein expression was evaluated by Western blotting with antibodies against collagen type I, α-smooth muscle actin, and TIMP-1 (Abcam, Cambridge, UK). Statistical analyses employed GraphPad Prism 9.0 with significance threshold p<0.05.

Ethical Considerations

This research received approval from the Ethics Committee of Hospital das Clínicas, University of São Paulo (Protocol CAAE 45892318.7.0000.0068, approved March 2022). All participants provided written informed consent following detailed explanation of research procedures and objectives. Patient confidentiality was maintained through deidentification of samples and assignment of unique coded identifiers. Data storage followed institutional security protocols with restricted access limited to authorized research personnel. The research was conducted in accordance with the Declaration of Helsinki principles for medical research involving human subjects. The research cohort demonstrated progressive age increase across fibrosis stages, with mean age ranging from 42.3 years in F0 to 56.1 years in F4 (Table 1). Hepatitis C virus infection and non-alcoholic fatty liver disease represented the predominant etiologies across all groups. Serum alanine aminotransferase levels showed positive correlation with fibrosis stage, reflecting ongoing hepatocellular injury. Sequencing analysis identified 47 miRNAs with significant differential expression across fibrosis stages. Functional classification revealed that pro-fibrotic miRNAs comprised 34.7% of identified species, while anti-fibrotic miRNAs represented 28.3% (Figure 1). The remaining miRNAs exhibited dual functions depending on cellular context or regulatory relationships with multiple target pathways. Expression profiling revealed distinctive patterns correlating with fibrosis severity (Figure 2, Table 2). Pro-fibrotic miRNAs including miR-21, miR-34a, miR-155, and miR-221 demonstrated progressive upregulation from F0 to F4 stages, with F4/F0 ratios ranging from 7.1 to 9.2. Strong positive correlations were observed between these miRNAs and METAVIR fibrosis scores (r = 0.82-0.91). Anti-fibrotic miRNAs showed inverse patterns, with miR-29a and miR-122 declining to approximately 20% of normal levels in cirrhotic samples. Conversely, inhibition of miR-21 using antisense oligonucleotides reduced activation markers by 47.3%, supporting bidirectional therapeutic approaches targeting pro-fibrotic and anti-fibrotic miRNAs.

Results

Discussion

This investigation provides comprehensive characterization of miRNA expression dynamics across the full spectrum of human hepatic fibrosis and demonstrates therapeutic potential of miRNA modulation in hepatic stellate cells. The findings establish strong correlations between specific miRNA species and fibrosis severity, supporting their utility as diagnostic biomarkers and therapeutic targets [12][13][14] .

The progressive upregulation of miR-21, miR-34a, miR-155, and miR-221 with advancing fibrosis aligns with their established roles in promoting TGF-β signaling and hepatic stellate cell activation. MiR-21 directly targets SMAD7, a negative regulator of TGF-β signaling, thereby amplifying pro-fibrotic cascades [15] . The consistent downregulation of miR-29 family members confirms their protective function in maintaining extracellular matrix homeostasis through direct targeting of collagen transcripts. Restoration of miR-29 expression through mimic delivery effectively suppressed fibrogenic markers, supporting therapeutic development strategies. The inverse correlation between miR-122 expression and fibrosis stage reflects progressive loss of differentiated hepatocyte populations as parenchyma is replaced by fibrotic tissue [16] . This liver-specific miRNA serves dual functions as both a biomarker of hepatocellular damage and a potential therapeutic agent for maintaining hepatocyte phenotype. The identification of dual-function miRNAs highlights the complexity of regulatory networks and suggests that therapeutic targeting must consider context-dependent effects. Pathway analysis revealed convergence of multiple miRNA species on TGF-β/SMAD signaling and extracellular matrix regulation pathways. This network architecture suggests that combination approaches targeting multiple miRNAs may achieve superior therapeutic efficacy compared to singleagent interventions [17] . The synergistic interactions between pro-fibrotic and anti-fibrotic miRNAs create opportunities for rational combination strategies that simultaneously suppress fibrogenic pathways while promoting resolution mechanisms.

Clinical Implications

The strong correlations observed between miRNA expression and histological fibrosis staging suggest potential for developing non-invasive diagnostic approaches based on circulating miRNA profiles. Several identified miRNAs, particularly miR-122 and miR-21, are detectable in peripheral blood and may serve as liquid biopsy markers for fibrosis assessment [18] .

Acknowledgement

Acknowledgements Funding SourcesThis research was supported by the São Paulo Research Foundation (FAPESP) and the National Council for Scientific and Technological Development (CNPq), Brazil.

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