Abstract Background Liver fibrosis is a progressive pathological process primarily driven by the transdifferentiation of hepatic stellate cells (HSCs) into myofibroblast-like cells which secrete excessive extracellular matrix (ECM). Although microRNAs (miRNAs) have emerged as key regulators of fibrogenesis, the therapeutic potential and mechanistic specificity of miR-214-3p (miR-214) in liver fibrosis remain insufficiently defined. Methods An adeno-associated virus (AAV)-based system was used to achieve either whole-liver or HSC-specific overexpression of miR-214 (via GFAP promoter) in a mouse model of alcohol-associated liver fibrosis induced by Lieber-DeCarli ethanol diet combined with low-dose CCl₄ injection. Liver fibrosis, steatosis, and inflammation were evaluated by biochemical assays, histology, immunostaining, and gene expression analyses. In vitro, stable miR-214 overexpression and knockdown in LX-2 cells were performed to assess effects on HSC proliferation, transdifferentiation, and ECM gene expression. MECP2 was identified as a direct functional target of miR-214 by bioinformatics and luciferase reporter assays. Results miR-214 expression was significantly downregulated during HSC activation in vitro and in fibrotic livers. Whole-liver overexpression of miR-214 alleviated liver fibrosis but caused undesirable steatosis and inflammation. Notably, HSC-specific miR-214 overexpression ameliorated liver fibrosis without inducing these adverse effects. Functionally, miR-214 inhibited HSC proliferation and ECM gene expression, while its inhibition promoted this process. Mechanistically, miR-214 exerts its anti-fibrosis function at least in part by directing targeting MECP2, a critical regulator for HSC activation. Conclusions These findings not only identify miR-214 as a promising antifibrotic agent, but also highlight the translational advantage of cell-specific miRNA delivery. HSC-targeted miR-214 gene therapy may offer a promising and safer approach for treating liver fibrosis. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-025-06880-x. Keywords: AAV, MiR-214, Hepatic stellate cell, Liver fibrosis, Gene therapy Introduction Liver fibrosis is characterized by excess deposition of extracellular matrix (ECM) in the liver, resulting from chronic liver injury caused by any aetiology like virus infections, alcohol abuse, and non-alcoholic steatohepatitis, and other etiologies [[40]1]. The severity of liver fibrosis is highly associated with the increased mortality in patients with chronic liver disease, including non-alcoholic and alcohol-related liver disease [[41]2]. As hepatic fibrosis disease has become a global health problem, it is essential to decode the underlying pathogenic mechanisms and identify effective therapeutic interventions. However, current effective antifibrotic therapies are still limited [[42]3]. Hepatic stellate cells (HSCs) distribute in the subendothelial space of Disse and account for ~ 10% of liver resident cell [[43]4]. Excessive activation of HSCs is considered as a central driver of liver fibrogenesis [[44]5–[45]7]. Chronic liver injury promotes HSCs transdifferentiation from a quiescent state into a proliferative myofibroblast-like phenotype, leading to the accumulation of extracellular matrix [[46]8]. Therefore, understanding the molecular mechanisms regulating HSC activation is crucial for the development of targeted antifibrotic strategies [[47]9]. For instance, nuclear receptors like PPARγ negatively regulate HSC activation and fibrosis progression. Agonists against these nuclear receptors are under clinical trials for liver fibrosis treatment [[48]10–[49]12]. These findings support the concept that HSC-directed therapies can serve as promising approaches for liver fibrosis treatment. Recent advances have highlighted the critical roles of epigenetic mechanisms in the initiation and progression of liver fibrosis [[50]13–[51]15], such as histone modification [[52]16, [53]17], DNA methylation [[54]18, [55]19], and noncoding RNAs. Among these, miRNA is a class of single-strand small noncoding RNA with ~ 22 nucleotides in length, which generally inhibits gene expression at post-transcriptional level. Dysregulated expression of certain miRNA had been linked to various human diseases, including fibrosis. Specially, some miRNAs have been implicated in the pathogenesis and progression of liver fibrosis. For example, downregulation of miR-145a-5p promoted steatosis-to-fibrosis progression by targeting Nr4a2 [[56]20]. miR-378 was reported to limit HSC activation and liver fibrosis by suppressing Gli3 expression [[57]21]. And miR-190b-5p and miR-296-3p were recently identified as novel therapeutic attenuators of liver fibrosis [[58]22]. These studies underscore the therapeutic potential of targeting miRNAs in liver fibrosis. miR-214 has been implicated in various pathophysiology processes [[59]23–[60]28], yet its precise role in liver fibrosis remains unclear. A previous study reported that miR-214 induced apoptosis in rat fibroblastic cells. Some reports showed that miR-214 downregulation promoted the expression of connective tissue growth factor (CTGF) [[61]29, [62]30], suggesting a possible antifibrotic role of miR-214. However, another study described that synthetic miR-214 inhibitors alleviated ECM deposition in a hepatocellular carcinoma mouse model [[63]31]. These discrepancies may arise from differences in mouse models, delivery methods, or lack of cell-type specificity in miRNA modulation. Therefore, a more refined, cell-targeted approach is needed to dissect the function of miR-214 in liver fibrosis. AAV-based gene therapy represents a promising clinical strategy for many human diseases, which offers durable gene expression with high tissue specificity and minimal immunogenicity [[64]32]. In this study, we employed an AAV-based delivery system to restore miR-214 expression either in the whole liver or specifically in HSCs via the GFAP promoter in a mouse model of liver fibrosis. Furthermore, in vitro studies were also conducted to investigate the functional and mechanistic effects of miR-214 on HSC activation. Our findings reveal the importance of cell-type-specific gene delivery in maximizing therapeutic efficacy while minimizing off-target effects, thereby offering a viable precision medicine strategy for liver fibrosis. Methods Chemicals and reagents The following chemicals and reagents are from commercial sources: DMEM, Opti-MEM, and Trypsin, BasalMedia (Shanghai, China); FBS and the collagenase type IV, Gibco (Grand Island, NY, USA); Protease, DNase I, NaH[2]PO[4], HEPES, NaHCO[3], EGTA, d-(+)-Glucose, CaCl[2], MgCl[2], MgSO[4], KH[2]PO[4], NaCl, KCl, and Nycodenz, Sigma-Aldrich (St. Louis, MO, USA); CCl[4], Sinopharm (Shanghai, China); Cell Counting Kit-8, MCE (New Jersey, USA); Hydroxyproline (HYP) Colorimetric Assay Kit, Elabscience (Wuhan, China); Total cholesterol assay kit, Triglyceride assay kit, Alanine aminotransferase Assay Kit, and Aspartate aminotransferase Assay Kit, Nanjing Jiancheng Bioengineering Institute (Nanjing, China); Adeno-associated virus, GeneChem (Shanghai, China); Primers, Tsingke (Beijing, China); Lieber-DeCarli diet, Readydietech (Shenzhen, China). Alcohol-related liver fibrosis (ALF) model 7–8-week-old male C57BL/6J mice were acclimated to a Lieber-DeCarli liquid diet containing 4% (w/w) ethanol (28% ethanol-derived calories) [[65]33] over a period of 1 week, and then maintained on 4% (w/w) ethanol diet for another 6 weeks, accompanied by i.p. injections of 0.6 ml/kg b.w. CCl[4] twice a week. The control mice were pair-fed with control Lieber-DeCarli diet and i.p. injections with corn oil. All animal experiments were approved by the Institutional Animal Care and Use Committee of Qingdao University (No. 20230901C577220240121151). AAV8-based miR-214 delivery Adeno-associated virus serotype 8 (AAV8) exhibits strong hepatotropism which can efficiently facilitate liver-specific expression of exogenous genes. For overexpression of miR-214 in the whole liver, AAV8-miR-214 driven by CMV promoter was constructed by inserting a short hairpin sequence containing mature miR-214 sequence. AAV8-miR-214 driven by GFAP promoter was constructed to achieve HSC-specific overexpression of miR-214. After 2-weeks of 4% ethanol diet feeding and CCl[4] treatment, a dose of 1.5 × 10^11 v.g. purified AAV were given into each mouse by tail vein injection and CCl[4] injections were held for 1 week. Mice were sacrificed after following 5 weeks feeding. Serum and liver biochemical examination Mouse serum samples were used to determine the levels of total cholesterol (TC) and triglyceride (TG), as well as the activity of alanine aminotransferase and aspartate aminotransferase. In addition, the supernatant which extracted from homogenized mouse liver tissue using anhydrous ethanol was used to assess the liver TC and TG levels, and the content of hydroxyproline. Immunohistochemical analysis (IHC) F4/80 staining were performed to specifically label liver macrophages. Briefly, fresh liver tissues were fixed with 4% PFA, and then embedded in paraffin and cut into 5 μm slices. Then these slices were deparaffinized, rehydrated and removed antigen. After blocking, liver slices were incubated with the F4/80 antibody followed by incubation with HRP-conjugated secondary antibodies. Finally, the slices were developed using a DAB chromogenic Kit. Primary HSCs isolation Normal 12-week male BALB/c mice were used for primary HSCs isolation. After in vivo enzyme digestion, primary HSCs were isolated based on retrograde pronase-collagenase perfusion of the liver and subsequent density-gradient centrifugation as previously described [[66]34]. Isolated primary HSCs were cultured in high glucose DMEM supplemented 10% FBS and incubated at 37 °C with 5% CO[2]. LX2 cell transfection MECP2 siRNAs were purchased from Tsingke (Beijing, China). Lipofectamine 2000 reagent was used for LX2 cell transfection. Cells were incubated for another 48 h after MECP2 siRNA (50 nM) transfection. Plasmid construction for miR-214 overexpression and inhibition To obtain a miR-214 expressing plasmid (miR-214 OE), a hairpin structure containing miR-214 mature sequence was cloned into EcoR I and Age I sites of the lentiviral pLKO.1-puro vector. In addition, to inhibit endogenous miR-214 function, a Tough Decoy structure for miR-214 (miR-214 TuD) was designed by cloned a long hairpin structure containing two miR-214 reverse complementary sequence into the pLKO.1-puro vector. The sequences of oligos used in plasmid construction were listed in Table S1. All these two recombinant plasmids were sequenced by sanger sequencing. Lentivirus packaging and LX2 cell infection Lentivirus for miR-214 overexpression or inhibition was packaged by transfected HEK 293FT cells with psPAX2, pMD2G, and miR-214 OE/TuD plasmids using Lipofectamine 2000 reagent. 48 h later, cell culture medium was collected and added into LX2 cells supplemented with polybrene. Then positive infected cells were selected by puromycin treatment. RNA isolation and RT-qPCR Total RNA of cells and liver tissues was extracted using a RNAiso Plus reagent. Then mRNA and miRNA reverse transcription were performed using HiScript III RT SuperMix for qPCR (+gDNA wiper) and miRNA 1st Strand cDNA Synthesis Kit (by stem-loop), respectively. Finally, QuantiNova™ SYBR® Green PCR kit was used for following quantitative PCRs as described in previous study [[67]35]. Western blotting Immunoblotting was conducted as described in our previous study [[68]36]. Total proteins were extracted from cells and tissues using RIPA lysis buffer. Protein concentrations were determined by using a BCA kit (Thermo Scientific). 10 μg total proteins were subjected to SDS-PAGE electrophoresis, and transferred to a PVDF membrane. After blocking in 5% skim milk, membranes were incubated with following primary antibodies: αSMA (HUABIO, cat#ET1607-53, 1:5000), p-Smad2 (Abways, cat#CY5857, 1:1000), Smad2 (Abways, cat#CY5090, 1:1000), p-Smad3 (Abways, cat#CY5140, 1:1000), Smad3 (Abways, cat#CY5013, 1:5000). Then HRP-conjugated secondary antibodies were uses and the targeted bands were detected by using an enhanced chemiluminescence substrate. Cell proliferation assay Equal amount of miR-214 overexpressing LX2 cells and scramble control cells were seeded in a 96-well plate. The cell viability on the 1-to-6 day was measured using a CCK8 reagent. Absorbance at 450 nm was measured by using a microplate reader. Colony formation assay LX-2 cells with stable miR-214 overexpression or knockdown were used to perform the colony formation assay. 1000 cells per well were plated in a 6-well plate, and incubated for 14 days at 37 °C cell incubator. Culture medium was replaced every 3 days. After fix by methanol, cell colonies were stained by 0.5% crystal violet solution at room temperature for 1 h. Images were acquired and the number and the area of colonies were then quantified by ImageJ. Cell cycle detection The cell cycle and apoptosis detection kit from Beyotime (Beijing, China) was used. Single cell suspensions of LX-2 cells with stable miR-214 overexpression or knockdown were prepared and fixed by pre-cooled 90% ethanol overnight at 4 °C. After PBS wash, cells were resuspended in the PI staining solution and incubated at 37 °C for 30 min. Then flow cytometry was performed and data was analyzed by using the ModFit LT 5.0 software. Cell fractionation assay NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific) was used to separate the nuclear and cytoplasmic fractions of LX-2 cells. To detect the distribution of miR-214 in LX-2 cells, RNA from the nuclear and cytoplasmic fractions was extracted, and reverse transcribed to cDNA products, followed by RT-qPCR detection of the expression levels of miR-214, U6 (nuclear marker), and GAPDH (cytoplasmic marker) in the nucleus and cytoplasm [[69]35]. The fold enrichment was calculated by the 2^−dCt method. Statistical analysis The results were expressed as mean ± standard deviation. Student’s t-test was used to analyze the difference between two groups. Ordinary one-way ANOVA analysis with Dunnett’s multiple comparisons test was used to analyze comparisons between multiple groups. Pearson correlation analysis was used to analyze the correlation between miR-214 and Acta2 or Fn1 mRNA expression levels. GraphPad Prism8.0 software was used for statistical analysis and plotting. P values less than 0.05 was considered statistically significant. Statistical significance was showed as *p < 0.05 and **p < 0.01. Results miR-214 is downregulated in activated primary HSCs and LX2 cells To determine the expression changes of miR-214 upon HSCs activation, we first isolated and purified mouse primary HSCs via in situ liver perfusion. And during in vitro culture, autoactivation of primary HSCs was observed which was accompanied with obvious morphological changes from round weakly adherent quiescent cells into a highly adherent myoblast-like cells (Fig. [70]1A). And the activation of HSC was further confirmed by the increased expression of Fn1, Col1a1 and Acta2 mRNAs (Fig. S1A). Notably, the expression of miR-214 decreased steadily upon primary HSC autoactivation (Fig. [71]1B). LX2 is an immortalized human hepatic stellate cell line and can be activated by TGFβ treatment (Fig. S1B-D). Our data showed that miR-214 expression was significantly downregulated by TGFβ in both a time- and dose-dependent manner (Fig. [72]1C, D). Taken together, our data indicated that the expression of miR-214 was downregulated during HSC activation. Fig. 1. [73]Fig. 1 [74]Open in a new tab miR-214 is downregulated in activated HSCs and fibrotic liver. A Morphological images of primary HSCs during spontaneous activation from day 0 to day 2. B RT-qPCR analysis of miR-214 expression in primary HSCs during day 0 to day 2. C RT-qPCR analysis of miR-214 expression in LX2 cells treated with 3 ng/ml TGFβ for 6 h and 12 h. D RT-qPCR analysis of miR-214 expression in LX2 cells treated with 0.3 and 3 ng/ml TGFβ for 12 h. E Experimental design scheme of the alcohol-related fibrosis (ALF) model, which was established by combining Lieber-DeCarli liquid ethanol diet and i.p. injection of low dose of CCl[4] (0.6 ml/kg·b.w.). F RT-qPCR analysis of miR-214 expression in the liver of Ctrl and ALF group. G, H Correlation analysis between miR-214 expression and the expression of Acta2 (G) and Fn1 (H). Ctrl, pair-treated group; ALF, alcohol-related fibrosis. **, p < 0.01 Downregulation of miR-214 in the liver of alcohol-associated fibrosis mice To further assess the expression level of miR-214 in the early-stage fibrotic liver, a chemical-induced fibrosis model was established by combining Lieber-DeCarli liquid ethanol diet and i.p. injection of low dose CCl[4] (Fig. [75]1E). Serum ALT and AST levels and liver TG levels were increased in the alcohol-related fibrosis (ALF) model (Fig. S2A, B). Positive Sirus Red staining was observed (Fig. S2C) and αSMA protein was accumulated in the liver of the model mice (Fig. S[76]2D), indicating the successful establishment of the liver fibrosis model. Importantly, the expression of miR-214 was significantly decreased in this fibrosis model (Fig. [77]1F) and showed strong negative- correlations with the HSC activation markers Acta2 and Fn1 (Fig. [78]1G, H), suggesting its potential regulatory role in liver fibrosis. Fig. 2. [79]Fig. 2 [80]Open in a new tab Whole-liver miR-214 overexpression ameliorates liver fibrosis. A Schemic illustration of the experiment design. The fibrosis model was established by combining Lieber-DeCarli liquid ethanol diet and i.p. injection of CCl[4]. After 2 weeks, mice were administered with AAV8-miR-214 and AAV8-Ctrl via tail vain injection, and CCl[4] i.p. was hold for 1 week. B RT-qPCR analysis of the expression of miR-214 in the liver. C Serum ALT and AST levels. D Liver hydroxyproline levels. E Sirius Red staining and αSMA immunohistochemical staining of the liver tissues. F RT-qPCR analysis of the expression of Acta2 and Fn1 genes. n.s., non-significant; *, p < 0.05; **, p < 0.01 Whole-liver miR-214 overexpression ameliorates liver fibrosis but promotes liver steatosis and inflammation To evaluate the therapeutic potential of miR-214 in vivo, AAV8-mediated delivery of miR-214 driven by the CMV promoter was used to achieve whole-liver miR-214 overexpression in fibrotic mice (Fig. [81]2A, B). Although serum ALT and ALT levels were not changed (Fig. [82]2C), the liver hydroxyproline levels were significantly lowered by miR-214 overexpression (Fig. [83]2D). Besides, the liver fibrosis as shown by αSMA-positive cells and Sirius Red staining was decreased in mice overexpressing miR-214 (Fig. [84]2E). And the key signaling pathway, Smad pathway, in HSC activation was inhibited by miR-214 overexpression (Fig. S3). The mRNA expression levels of ECM gene Acta2 and Fn1 were also significantly suppressed (Fig. [85]2F). However, increased liver weight and hepatic TG levels were observed in the miR-214 overexpression mice (Fig. [86]3A, B). And severe macrosteatosis and lipid accumulation were observed in the liver of miR-214 overexpression mice (Fig. [87]3C). Moreover, the expression levels of mRNA involved in lipogenesis, such as Fasn and Acc1, were markedly upregulated by miR-214 (Fig. [88]3D), which may responsible for the occurrence of liver steatosis. Besides, H&E staining also showed that obvious inflammatory infiltration was promoted by miR-214 (Fig. [89]3C) and more F4/80 positive inflammatory cells appeared (Fig. [90]3E). Consistently, the hepatic expression of inflammatory genes, such as Tnfa, Adgre1, Ccl2, and Cxcl5, were increased in mice with miR-214 overexpression (Fig. [91]3F). Taken together, whole-liver overexpression of miR-214 could attenuate liver fibrosis but aggravate hepatic steatosis and inflammation. Fig. 3. [92]Fig. 3 [93]Open in a new tab Liver-wide miR-214 overexpression boosts liver steatosis and inflammation. A Liver weight. B Liver TG and TC levels. C H&E and Oil-Red O staining of the liver tissues. D RT-qPCR analysis of the gene expression related to fatty acid uptake and lipid metabolism. E F4/80 staining showing liver macrophage infiltration in the liver. F RT-qPCR analysis of the expression of inflammation-related genes. n.s., non-significant; *, p < 0.05; **, p < 0.01 HSC-specific miR-214 overexpression alleviates liver fibrosis without boosting steatosis and inflammation To avoid the drawbacks of non-specific overexpression, HSC-specific overexpression of miR-214 was performed by i.v. injection AAV driven by GFAP promoter (Fig. [94]4A). Increased miR-214 was observed in the livers of mice administrated with AAV8-GFAP-miR-214 (Fig. [95]4B). Serum ALT and AST levels were not affected (Fig. [96]4C). But the expressions of fibrosis marker genes were significantly decreased by HSC-specific expression of miR-214 (Fig. [97]4D). Accordingly, positive area of Sirius Red staining was also reduced (Fig. [98]4E). Immunoblotting results showed that αSMA expressions and Smad signaling were inhibited upon miR-214 overexpression (Fig. [99]4F, G). Fig. 4. [100]Fig. 4 [101]Open in a new tab HSC-specific miR-214 overexpression alleviates liver fibrosis. A Experimental design scheme. The fibrosis model was established by combining Lieber-DeCarli liquid ethanol diet and i.p. injection of CCl[4]. After 2 weeks, mice were administered with AAV8-GFAP-miR-214 and AAV8-GFAP-Ctrl via tail vain injection. B The expression level of miR-214 in the liver. C Serum ALT and AST levels. D The expression level of the fibrosis marker genes. E Sirius Red staining of liver tissues. F, G Western blotting showing the expression of proteins in the Smad signal pathway. n.s., non-significant; *, p < 0.05; **, p < 0.01 Interestingly, HSC-specific overexpression of miR-214 significantly decreased the liver TC and TG levels (Fig. [102]5A), and hepatic lipid accumulation was also ameliorated (Fig. [103]5B). And the adverse upregulation of lipogenesis genes by whole-liver miR-214 expression was not detected in the liver of HSC-specific miR-214 expression (Fig. [104]5C). Additionally, HSC-specific expression of miR-214 did not affect the infiltration of F4/80-positive cells (Fig. [105]5D). And the expression of Ifng and Cxcl1 was decreased while the expression of Ccl2 and Adgre1 was not changed (Fig. [106]5E). Overall, our findings suggested that HSC-restricted overexpression of miR-214 could alleviate liver fibrosis without boosting steatosis and inflammation. Fig. 5. [107]Fig. 5 [108]Open in a new tab HSC-specific miR-214 overexpression does not promote steatosis and inflammation. A Liver TC and TG levels. B Oil-Red O staining of liver tissues. C RT-qPCR analysis of the expression of genes related to lipid metabolism. D F4/80 staining showing liver macrophages. E RT-qPCR analysis of the expression of inflammation-related genes. n.s., non-significant; *, p < 0.05; **, p < 0.01 miR-214 inhibits HSC proliferation and ECM gene expression To explore the functional role of miR-214 in vitro, we constructed relevant plasmids (Fig. [109]6A) and established LX2 cell lines with stable miR-214 overexpression or knockdown (Fig. [110]6B). Overexpression of miR-214 significantly inhibited LX2 proliferation (Fig. [111]6C) and suppressed colony formation (Fig. [112]6E, F, Fig. S4A). Additionally, inhibition of endogenous miR-214 promoted LX2 cell proliferation (Fig. [113]6D) and obviously increased the colony numbers and areas (Fig. [114]6E, F). Cell cycle analysis data showed that miR-214 inhibition increased the percentage of cells in S and G2/M phase (Fig. [115]6G), while miR-214 overexpression reduced these cell cycle distributions (Fig. [116]6H). Fig. 6. [117]Fig. 6 [118]Open in a new tab miR-214 inhibits HSC proliferation. A Schematic illustrating the construction of miR-214 overexpression and inhibition plasmid. B RT-qPCR analysis of the expression of miR-214 in LX2 cells stably expressing (miR-214 OE) or inhibiting miR-214 (miR-214 TuD). miR-214 OE and miR-214 TuD lentivirus were packaged and LX2 cells were infected. Positive cells were selected by puromycin screening. C, D Cell proliferation ability of LX2 cells with miR-214 overexpression (C) or inhibition (D). E, F Colony images (E) and number/area analysis (F) of LX2 cells with miR-214-OE and miR-214 TuD. G, H Cell cycle analysis showing the cell cycle distribution of LX2 cells with miR-214 inhibition (G) or overexpression (H) group. **, p < 0.01 Furthermore, using the mirPath v.3 tool, we identified that ECM-receptor pathway was the most significantly enriched (Fig. [119]7A), indicating potential roles of miR-214 in ECM deposition. Further RT-qPCR and immunoblotting data showed that miR-214 overexpression inhibiting the expression of ECM-related genes in LX2 cells (Fig. [120]7B, C). And inhibition of endogenous miR-214 upregulated relevant gene expressions (Fig. S4B). Taken together, our finding indicated that miR-214 could inhibit HSC proliferation and ECM gene expression. Fig. 7. [121]Fig. 7 [122]Open in a new tab miR-214 inhibits ECM gene expression. A KEGG pathway enrichment analysis of miR-214 using the mirPath v.3 tool. B RT-qPCR analysis of the expression of ECM-related genes. C Immunoblotting analysis of the expression of ECM-related proteins. *, p < 0.05; **, p < 0.01 MECP2 is a potential functional target of miR-214 The cellular distribution of miRNA determines its regulatory mechanism. Therefore, cell fractionation assay was performed and we found that miR-214 predominantly localized at the cytoplasm of LX2 cells (Fig. [123]8A), indicating a classical regulatory function of miR-214 in HSCs. Then, potential targeted genes of miR-214 were predicted by using the online tools TarBase, miRDB, TargetScan, and ENCORI (Fig. [124]8B). Among the 20 overlapped targets, MECP2 was selected as the candidate gene, as solid evidences have validated the antifibrotic role of MeCP2 (Fig. S5) [[125]18, [126]37–[127]40]. Moreover, miR-214 overexpression significantly inhibited MECP2 expression both in LX2 cells and fibrotic livers (Fig. [128]8C, D). Overexpression of MeCP2 significantly promoted cell proliferation which was inhibited by miR-214 (Fig. [129]8E). And results from the luciferase reporter assay showed that miR-214 could target a CDS sequence of MECP2 and inhibit the luciferase activity (Fig. [130]8F, G). Together, our data indicated that miR-214 might exert its anti-fibrosis function at least in part by suppressing MECP2 expression. Fig. 8. [131]Fig. 8 [132]Open in a new tab MECP2 is a potential target of miR-214. A Nuclear-cytoplasmic fractionation assay showing the cellular localization of miR-214 in LX2 cell. B Prediction of potential target genes of miR-214 using the TarBase, miRDB, TargetScan, and ENCORI online tools. C The expression levels of MECP2 in miR-214 overexpressing LX2 cells. D RT-qPCR analysis of the expression of Mecp2 in liver-wide and HSC-specific miR-214 overexpression group. E Cell ability analysis of MeCP2 overexpression in miR-214 OE LX2 cells. F Targeted binding site of miR-214 to MECP2 mRNA in human and mouse. G Luciferase reporter gene assay showing the direct binding of miR-214 to MECP2 mRNA. *, p < 0.05; **, p < 0.01 Discussion In the present study, we provide evidence supporting a regulatory role for miR-214 in HSC activation and liver fibrosis. We observed that miR-214 was downregulated during HSC activation and that its expression negatively correlated with the levels of key fibrogenic genes, Acta2 and Fn1. Functionally, whole-liver overexpression of miR-214 alleviated liver fibrosis but led to pronounced lipid accumulation and inflammatory responses. In contrast, HSC-specific expression of miR-214 effectively attenuated hepatic fibrosis without inducing adverse effects such as lipid accumulation or inflammation. Mechanistically, miR-214 might inhibit HSC activation by negatively regulating MeCP2, a critical fibrogenic regulator. Taken together, we uncover that HSC-specific delivery of miR-214 represents a promising therapeutic approach for liver fibrosis, capable of minimizing adverse metabolic effects. miR-214 is a highly conserved miRNA which play various roles in human diseases such as diabetic kidney disease [[133]41], hypertension [[134]27], preeclampsia-like symptoms [[135]42], and heart failure [[136]23]. However, the functional role of miR-214 in liver fibrogenesis remains unclear due to conflicting findings from previous studies [[137]29–[138]31, [139]43, [140]44]. Here, we provide evidence that downregulation of miR-214 promoted HSC differentiation while overexpression of miR-214 inhibited HSC activation in vitro. To date, clinical miRNA expression is typically achieved by using chemically modified oligonucleotides, such as LNA-antimiRs and miRNA mimics, and recombinant expression vectors carrying miRNA encoding sequences [[141]45]. We observed an intriguing phenomenon: studies using synthetic miRNA oligonucleotides in liver fibrosis intervention suggested pro-fibrotic role of miR-214 [[142]31, [143]44], whereas our research, based on AAV-mediated miR-214 delivery, supported an anti-fibrotic role for miR-214. Although synthetic miRNA oligo-based therapies have acquired great progress in relevant clinical trials, however, many trials were stopped/terminated and no therapy reach Phase III trials [[144]46]. In contrast, AAV-based gene therapies have shown greater translational success, with approved applications for diseases such as hemophilia B and Duchenne muscular dystrophy. These distinctions highlight that, compared to studies using synthetic miRNA oligonucleotides, research based on AAV-mediated gene delivery produces more physiologically relevant and reproducible outcomes. In other words, our study based on miR-214 overexpression for liver fibrosis intervention using AAV delivery methods provides more robust and trustworthy results. AAV8 is particularly efficient at targeting the liver, as it has a strong tropism for hepatocytes. In this study, we found that liver-wide miR-214 overexpression driven by CMV promoter could alleviate liver fibrosis but boost severer steatosis and inflammation. Interestingly, HSC-specific expression of miR-214 driven by GFAP promoter not only ameliorated hepatic fibrosis but also avoided adverse effects like lipid accumulation and immune infiltration. This phenomenon might be partly due to the cell-specific roles of miR-214 in the liver. Liver-wide expression of miR-214 probably caused increased expression of miR-214 in hepatocytes, which may contribute to miR-214-induced adverse effects. For example, our recent study reported that miR-214 could sensitize hepatocytes to ferroptosis [[145]28], and one other study suggested a role of miR-214 in promoting lipid accumulation in hepatocytes [[146]47]. Previous literatures have also reported that the same biomolecules perform different or even opposite functions in different cells. For example, Zhang etc. reported the double face of miR-320 in heart failure, that miR-320 overexpression in cardiomyocytes exacerbated cardiac dysfunction, whereas overexpression of miR-320 in cardiac fibroblasts alleviated cardiac fibrosis and hypertrophy [[147]48]. Thus, both our findings and previous findings emphasize the importance of cell-type-specific targeting when designing miRNA-based therapies. Mechanistically, MeCP2 is a well-established regulator of liver fibrosis by epigenetically repressing PPARγ, a transcription factor that represses HSC differentiation [[148]18, [149]37]. MeCP2 phosphorylation has also been linked to HSC proliferation and induction of fibrosis [[150]39]. Here, we identified that miR-214 could suppress MECP2 expression, and overexpression of MeCP2 significantly recovered the cell proliferation inhibited by miR-214. Given that one miRNA can target many mRNAs, and genes, such as CCN2 [[151]29], ATF4 [[152]24], PTEN [[153]49], GPX4 [[154]50], PPARG [[155]23], and Tlr4 [[156]51], have been identified as the targets of miR-214 in published researches. Thus, further efforts are needed to identified more targets of miR-214 in the context of liver fibrosis. Despite the promising findings, our study also had some limitations. First, while HSC-specific expression of miR-214 was inferred from GFAP promoter-driven AAV delivery, direct confirmation in purified primary HSCs was lacking. Second, the upstream mechanism for the down-regulation of miR-214 in the early stage of HSC activation remains to be explored. Finally, HSC-specific overexpression of miR-214 decreased the liver TC and TG levels, but the mechanism underlining this effect remains unclear. Conclusion In conclusion, our study demonstrated that AAV-mediated HSC-specific expression of miR-214 effectively ameliorated liver fibrosis without boosting steatosis and inflammation. These findings provide a rationale for developing AAV-based strategies to deliver miR-214 specifically to HSCs as a precision medicine approach for liver fibrosis. Supplementary Information [157]Supplementary Material 1^ (2.3MB, docx) Abbreviations AAV Adeno-associated virus serotype ALF Alcohol-related fibrosis CTGF Connective tissue growth factor ECM Extracellular matrix HSCs Hepatic stellate cells MeCP2 Methyl-CpG-binding domain protein miRNA MicroRNA miR-214 MiR-214-3p MPO Myeloperoxidase TC Total cholesterol TG Triglyceride Author contributions Fangqing Zhao and Xuan Niu: investigation, data curation and visualization; Ge Song: investigation and writing-original draft preparation; Lijie Wang: investigation; Yisheng Fu: investigation; Qingkun Wang: investigation; Shuwen Li: software; Xinxin Gu: software; Jiao Luo: conceptualization, project administration, writing-original draft preparation, funding acquirement. Funding This work was supported by the National Natural Science Foundation of China (Nos. 82273670). Data availability The data used to support the findings of this study are available from the corresponding author upon reasonable request. Declarations Ethics approval and consent to participate All animal experiments were approved by the Institutional Animal Care and Use Committee of Qingdao University (No. 20230901C577220240121151). All experimental procedures followed the approved guidelines. Consent for publication All authors consent this manuscript for publication. Competing interests The authors have no conflicts of interest to report. Footnotes Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Fangqing Zhao, Xuan Niu and Ge Song contributed equally to this work. References