Exosomal miR-378a-3p derived from keratinocytes inhibits transforming growth factor beta-induced fibroblast activation and attenuates bleomycin-induced skin fibrosis

Exosomal miR-378a-3p derived from keratinocytes inhibits transforming growth factor beta-induced fibroblast activation and attenuates bleomycin-induced skin fibrosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exosomal miR-378a-3p derived from keratinocytes inhibits transforming growth factor beta-induced fibroblast activation and attenuates bleomycin-induced skin fibrosis Lijun Yang, Guiping Tian, Baiting Liu, Chenxi Li, Yunyao Bo, Jianjun Si, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3829723/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The association between microRNAs (miRNAs) and the development of skin fibrosis has been established. Consequently, the investigation of miRNA is crucial for the management of cutaneous fibrotic diseases. Recent investigations have linked miR-378a to several fibrosis diseases. Here, we discovered that miR-378a-3p effectively inhibited the proliferative, migratory, and COL1A1 expression effects induced by TGF-β1 in fibroblasts. Additionally, we found that miR-378a-3p exerts its anti-fibrotic properties by directly binding to FSTL1, a downstream factor involved in TGF-β1 activation. We have also demonstrated that long non-coding RNA (lncRNA) NORAD functions as a competing endogenous RNA (ceRNA) for miR-378a-3p, thereby activating the expression of FTSL1. Additionally, miR-378a-3p can be encapsulated within exosomes derived from keratinocytes and transferred to fibroblasts, resulting in an antagonistic effect against TGF-β1. The subcutaneous injection of miR-378a-3p-overexpressing keratinocytes-derived exosomes significantly attenuated skin fibrosis induced by bleomycin (BLM), as evidenced by reduced dermal thickening and decreased expression of COL1A1. Consequently, exosome-mediated delivery of miR-378a-3p holds promise as a potential therapeutic strategy for the treatment of skin fibrosis. miR-378a-3p skin fibrosis exosomes TGF-β1 FSTL1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Skin fibrosis is a characteristic manifestation observed in multiple pathological conditions, such as scleroderma, keloids, hypertrophic scars, and graft versus host disease [ 1 – 4 ]. Current clinical interventions for skin fibrosis encompass local pharmaco-logical interventions, skin grafting, and surgical excision; nevertheless, their therapeutic effectiveness remains suboptimal [ 5 – 7 ]. Consequently, there exists a pressing necessity to acquire comprehensive insights that will enable the development of improved therapeutic approaches and furnish substantiation for clinical decision-making. MiRNAs exert substantial regulatory influence on gene expression by frequently targeting multiple genes within a specific gene network [ 8 , 9 ]. Consequently, the modulation of a single miRNA can have a significant impact, as it induces multiple changes in gene expression within the same pathway [ 10 , 11 ]. In recent times, a considerable number of miRNAs have been identified as playing crucial roles in the development of skin fibrosis. Previous research endeavors have made significant strides in understanding the impact of certain miRNAs on scar formation and investigating the potential underlying mechanisms [ 12 – 16 ]. The current study is specifically focused on miR-378a-3p, a miRNA that has been previously implicated in various biological functions and processes such as cell growth, cell cycle regulation, migration, differentiation, metabolism, and angiogenesis [ 17 – 21 ]. Recent investigations have also linked miR-378a to myocardial fibrosis, liver fibrosis, and interstitial fibrosis [ 22 , 23 ]. However, the precise role of miR-378a-3p in skin fibrosis remains unclear. Therefore, the objective of this study is to examine the potential involvement of miR-378a-3p in the development of skin fibrosis. As the discovery of disease-associated miRNAs and the advancement of in vivo miRNA manipulation techniques continue, a multitude of miRNA-based therapies have progressed to clinical trials, exhibiting promising therapeutic results. Recent scholarly investigations have provided evidence through multiple studies that miRNAs are enclosed within exosomes, thereby preserving their contents from degradation and facilitating the transfer of various small biomolecules, including proteins and RNAs, to adjacent cells [ 24 , 25 ]. Previous studies have demonstrated that exosomes derived from keratinocytes possess a robust and specific regulatory influence on fibro-blasts [ 26 ]. Expanding upon our previous investigations, we have observed that miR-16-5p can be enclosed within exosomes derived from keratinocytes and exert regulatory control over the functionality of fibroblasts. Therefore, we put forward the hypothesis that the inclusion of miR-378 within exosomes derived from keratinocytes may potentially demonstrate a synergistic effect, thereby offering a promising therapeutic strategy for the management of fibrosis. The primary objective of this study was to examine the role of miR-378a-3p in fibroblast activity and elucidate its regulatory mechanism. Additionally, we investigated the effects of miR-378a-3p-enriched exosomes on fibroblast activity and evaluated its potential therapeutic efficacy in a murine model of BLM-induced skin fibrosis. Materials and Methods Cell culture HaCaT cells were cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone, USA) containing 10% fetal bovine serum (Gibico, USA). Human skin samples were obtained from patients with consent forms. The dermal layers of the skin were dissected into small fragments and subsequently plated in DMEM supplemented with 10% fetal bovine serum. Fibroblasts from the third passage were employed for the study.. Plasmids, miRNAs, siRNA, lncRNA, and transfection Cells were seeded at a density of 1.2 × 10 4 cells per well in a 24-well plate and cultured for 24 hours. Transfection experiments of miR-378a-3p mimic/inhibitor (RiBoBio, Guangzhou, China), lncRNA NORAD, pcDNA3.1, or si-NORAD (RiBoBio, Guangzhou, China) were carried out according to Lipofectamine 2000 instructions (Thermo Fisher Scientific, CA, USA). EdU staining EdU (Beyotime, Shanghai, China) was added 48 hours after transfection for a further 4 hours of incubation, after which the cells were fixed with 4% paraformaldehyde for half an hour, washed with 3% bovine serum albumin (BSA), and permeabilized with 0.5% TritonX-100. Finally, Ultimately, cells were stained by BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, Shanghai, China) according to the manufacturer's instructions. Transwell assay The cells were digested and resuspended after being serum-free for 8 hours following transfection. The medium containing 5% fetal bovine serum (FBS) was added to the lower chamber of the transwell as an induction factor, and about 200 µl (1 × 10 4 cells) of serum-free cell suspension was added to the upper chamber. After culturing for 24 hours, the cells in the chamber were permeabilized with 4% paraformaldehyde. The cells in the chamber were stained with crystal violet solution for about 10–15 minutes. The upper chamber unpenetrated cells were wiped off, and photos of the cells that penetrated the chamber were taken using a Leica inverted microscope. Western blot analysis Cells and tissues were lysed with were lysed in ice-cold Radio-Immunoprecipitation Assay (RIPA, Sigma-Aldrich, St. Louis, MO, USA) after the initial treatment. The cells and tissues were thoroughly lysed and centrifuged for 15 minutes at 12,000 x g. After centrifugation, the supernatant was combined with 5× SDS Loading Buffer, and the protein was extracted after 10 minutes of boiling. Following that, the samples were separated on a 10% SDS-PAGE gel and transferred to PVDF membranes. The PVDF membrane was blocked with 5% nonfat milk powder and incubated with β-actin (1:1000, Proteintech, Wuhan, Hubei, China), FSTL1 (1:1000, Proteintech, Wuhan, Hubei, China) and COL1A1 (1:1000, Proteintech, Wuhan, Hubei, China) primary antibodies overnight at 4°C. The next day, incubated for 1 hours at room temperature with the corresponding secondary antibody (1:2000, Proteintech, Wuhan, Hubei, China) that can bind to the primary antibody, and visualized the protein bands by the ECL detection system. Luciferase assay First, the binding sites of miR-378a-3p in the 3'-UTR of FSTL1 and lncRNA NORAD were predicted, and then FSTL1 and lncRNA NORAD were amplified from cDNA by PCR. Then, the PCR fragment was inserted into the psiCHECK-2 vector. The successfully constructed plasmid was identified and co-transfected with miR-378a-3p mimic through Lipofectamine 2000 into HDFs. psiCHECK-2 and mimic negative control (NC) were used as negative controls for transfection according to the same method as above. Cells were collected 48 hours after transfection and were operated according to the Dual-Luciferase Reporter Gene Assay Kit (Beyotime, Shanghai, China) requirements. Quantitative real-time polymerase chain reaction (qRT-PCR) Cells/tissues were subjected to TRIzol reagent kit (Thermo Fisher Scientific, CA, USA) for the extraction of total RNA. The mRNA was then reverse transcribed for qRT-PCR using AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, Jiangsu, China). Subsequently, qRT-PCR analysis was conducted using AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, Jiangsu, China). The Ct values obtained were normalized using the GAPDH. For miRNA analysis, cDNA for miRNA was synthesized following the manufacturer's protocol of the miDETECT A TrackTM miRNA qRT-PCR Starter Kit. The U6 RNA level was utilized as an internal control for data normalization. The qRT-PCR reaction was conducted utilizing the miDETECT A TrackTM miRNA qPCR Kit, employing the miDETECT A TrackTM miR-378a-3p Forward Primer and miDETECT A TrackTM Uni-Reverse Primer (The above reagents about miRNA were from RiBoBio, Guangzhou, China). Isolation and characterization of exosomes HaCaT cells were cultured in serum-free DMEM for 48 hours. After collecting the conditioned medium, ExoEasy Maxi Kit (Qiagen, MD, USA) according to the manufacturer’s instructions. Then, the transmission electron microscope (JEOL, Japan) was used to observe the morphology of exosomes, and Nanosight LM 10 (Malvern Panalytical, UK) was used to identify the size, concentration, and distribution of exosomes. Cellular internalization First of all, exosomes were labeled using the PKH67 Green Fluorescent Cell Linker Mini Kit (Sigma-Aldrich, St. Louis, MO, USA). Then, PKH67-labeled exosomes were co-cultured with cells in FBS-free DMEM overnight. After fixing with 4% paraformaldehyde and staining with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA), cells were observed under a fluorescence microscope (Leica, Germany). BLM -induced skin fibrosis murine model Six-week-old male C57BL/6 mice were purchased from the Laboratory Animal Center of Southern Medical University. All animal protocols used in this study were approved by the Animal Care and Use Committee of Southern Medical University. For the BLM model, 100 ul of 0.5 mg/mL BLM (TargetMol, Shanghai, China) is administered by subcutaneous injection. Repeat injections every other day for 4 weeks. Mice are treated with PBS, Exo (NC), or Exo (miR-378a-3p) in parallel by intradermal injection every other day during the last two weeks. Histological analysis C57BL/6 mouse skin samples were firstly fixed with 4% paraformaldehyde for three days, then dehydrated in a gradient manner, transparent, and embedded in paraffin. They were then made into 5 um thick paraffin sections, and the tissue samples were stained according to the instructions for hematoxylin and eosin staining (H&E) and Masson’s trichrome staining Kit (both from Yeasen Biotechnology, Shanghai, China) and photographs were taken using a Leica upright microscope. Subsequent data processing was done using Image Pro Plus software. Immunohistochemical assay Paraffin section samples were dewaxed and hydrated, boiled with citrate solution (pH = 6.0) for antigen retrieval, and incubated with blocking solution (goat serum: 5% BSA: PBS = 1:2:7) for 1 hour. COL1A1 primary antibody (1:100, Proteintech, Wuhan, Hubei, China) was added dropwise and incubated overnight at 4°C. The next day after washing with PBS, an enzyme-labeled secondary antibody was added dropwise for 1 hour, and the DAB (ZSGB-BIO, Beijing, China) color was developed. Subsequently, hematoxylin staining was performed to stain the nuclei, and the cells were mounted by gradient dehydration. Photographs were taken with a Leica upright microscope and subsequent data processing was done with Image Pro Plus software. Statistical analysis All data are reported as the mean ± standard error (SEM) of at least three independent experiments (n ≥ 3). Data statistics were performed using GraphPad Prism 8 (GraphPad Software, Inc, La Jolla, CA) software, and comparisons between two groups were performed using independent samples t-test or one-way ANOVA between two groups for statistical analysis. Results miR-378a-3p inhibits the activity of TGF-β1-stimulated HDFs In this study, we examined the impact of miR-378a-3p on the activation of dermal fibroblasts. Our findings revealed that the overexpression of miR-378a-3p had a substantial effect on promoting the proliferation of dermal fibroblasts (Fig. 1 a). Additionally, we also investigated the influence of miR-378a-3p on the migration of these fibroblasts. The results obtained from the transwell assay, as depicted in Fig. 1 , demonstrated that the overexpression of miR-378a-3p significantly hindered the migration of dermal fibroblasts (Fig. 1 b). It is worth noting that excessive deposition of extracellular matrix (ECM) proteins in the dermis is a crucial characteristic of skin fibrosis. Therefore, we further detected the effect of miR-378a-3p on COL1A1 expression in dermal fibroblasts. The miR-378a-3p mimic was found to suppress the mRNA and protein expression of COL1A1 in HDFs when compared to the control group. Conversely, transfection with the miR-378a-3p inhibitor resulted in the opposite effect (Fig. 1 c, d). Furthermore, miR-378a-3p significantly inhibited the proliferative and migratory effects of TGF-β1 on HDFs (Fig. 1 e, f). The observed changes in COL1A1 mRNA and protein expression followed similar trends (Fig. 1 g, h). Based on these findings, it can be concluded that miR-378a-3p exhibits an antagonistic effect to TGF-β1 in the activation of dermal fibroblasts. miR-378a-3p played a role in dermal fibroblast via targeting FSTL1 Target Scan, miRanda, and miRDB predicted that the 3'-untranslated region (UTR) of FSTL1 has a miR-378a-3p binding site (Fig. 2 a). To validate this hypothesis, luciferase reporter gene constructs containing either the wild-type (WT) or mutant (MUT) versions of the putative miR-378a-3p binding site within the 3'-UTR of FSTL1 were prepared. Subsequent transfection of these reporter genes, along with a miR-378a-3p mimic, into HDFs confirmed that miR-378a-3p effectively inhibited the activity of the WT reporter construct, while having no impact on the MUT reporter construct (Fig. 2 b). Moreover, overexpression of miR-378a-3p reduced the mRNA and protein expression of FSTL1 in HDFs (Fig. 2 c, d). The further findings revealed that overexpression of miR-378a-3p in HDFs led to a reduction in proliferation and migration rates, which were subsequently rescued by the presence of 50 ng/ml FSTL1 (Fig. 2 e, f). Similar trends were observed in terms of mRNA transcription and COL1A1 protein expression (Fig. 2 g, h). Consequently, it can be inferred that miR-378a-3p exerts inhibitory effects on HDFs activation by targeting FSTL1. miR-378a-3p was regulated by lncRNA NORAD (NORAD) To study the upstream regulator of miR-378a-3p, we focused on long non-coding RNA (lncRNA) since accumulating evidence suggests that lncRNAs play significant roles in HS formation by regulating many processes, including ECM deposition and fibroblast proliferation. Analysis of data in the StarBase database revealed that lncRNA NORAD possibly binds miR-378a-3p (Fig. 3 a). To verify the interaction between NORAD and miR-378a-3p, constructed plasmids containing the wild-type sequence (NORAD-WT) and the mutant binding site sequence (NORAD-MUT). The results showed that overexpression of miR-378a-3p significantly reduced the luciferase activity of the NORAD -WT vector but did not reduce the luciferase activity of the empty vector or the NORAD-MUT vector (Fig. 3 b), confirming that NORAD-MUT can directly interact with miR-378a-3p. In addition, overexpression of NORAD significantly decreased the expression of miR-378a-3p, while the siRNA-NORAD inhibited the downregulated expression of miR-378a-3p (Fig. 3 c). Additionally, we also investigated the effect of NORAD on the expression of FSTL, the target of miR-378a-3p. The results showed that overexpression of NORAD significantly increased the mRNA and protein expression of FSTL1 (Fig. 3 d, e), which was inhibited by miR-378a-3p mimic. Therefore, we conclude that NORAD acts as an ceRNA of miR-378a-3p and further activates the expression of FTSL1. Preparation of keratinocytes-derived exosomes for miR-378a-3p delivery Exosomes derived from HaCaT cells infected with miR-378a-3p mimics or negative control mimics (NC) were isolated and subjected to morphological, numerical, and size analysis using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The findings revealed that both Exo (NC) and Exo (miR-378a-3p) exhibited a circular shape and were enveloped by double membranes within the size range of 30–150 nm (Fig. 4 a, b). Additionally, the integration capability of Exo (NC) and Exo (miR-378a-3p) was confirmed through PKH67 assay. Following a 10-hour incubation period of fibroblasts with PKH67-labeled exosomes, notable fluorescence levels were detected within the cytoplasm of fibroblasts, suggesting the internalization of Exo (NC) and Exo (miR-378a-3p) by fibroblasts (Fig. 4 c). Comparative analysis utilizing qRT-PCR demonstrated a significant increase in miR-378a-3p expression within Exo (miR-378a-3p)-treated fibroblasts when compared to those treated with Exo (NC) (Fig. 4 d). Exo (miR-378a-3p) inhibit activity of TGF-β1-stimulated dermal fibroblasts Initially, we investigated the impact of Exo (miR-378a-3p) on the proliferation of HDFs through the utilization of EdU staining. In the absence of TGF-β1 (10 ng/ml), the Exo (miR-378a-3p) group exhibited a substantial decrease in proliferation compared to both the Exo (NC) and PBS groups. Following treatment with TGF- β1, proliferation significantly increased in the Exo (NC) and PBS groups, whereas the Exo (miR-378a-3p) group effectively hindered the enhancement of TGF-β1 on cell proliferation (Fig. 5 a). Subsequently, the transwell assay demonstrated that treatment with Exo (NC) significantly enhanced the migration of HDFs, while the Exo (miR-378a-3p) group exhibited decreased migration compared to PBS. Furthermore, the migratory effect of TGF-β1 was also mitigated by Exo (miR-378a-3p) (Fig. 5 b). The mRNA expression of COL1A1 was reduced in both the Exo (NC) and Exo (miR-378a-3p) groups, with or without TGF-β1, when compared to the PBS group (Fig. 5 c). Additionally, the level of COL1A1 protein in the Exo (miR-378a-3p) groups was significantly lower than that in the Exo (NC) group (Fig. 5 d). Exo (miR-378a-3p) inhibits BLM-induced skin fibrosis We further investigated the involvement of miR-378a-3p in skin fibrosis induced by BLM through in vivo experimentation. The dermal thickness and collagen deposition were assessed using hematoxylin & eosin (H&E) and Masson staining techniques. Our findings revealed that the BLM group exhibited a significantly increased dermal thickness, elevated collagen deposition, and disorganized collagen arrangement in comparison to the WT group. Subsequent experiments demonstrated a significant reduction in skin tissue thickness and collagen deposition in BLM mice treated with Exo (miR-378a-3p) compared to those treated with PBS or Exo (NC) (Fig. 6 a). The immunohistochemical and qRT-PCR analysis further revealed higher expression of COL1A1 in the skin of BLM mice compared to WT mice. However, the expression of COL1A1 was decreased in the skin of Exo (miR-378a-3p)-treated BLM mice compared to those treated with PBS or Exo (NC) (Fig. 6 b, c). Western blotting analysis yielded consistent results with the immunohistochemical staining, further supporting these findings (Fig. 6 f). We also examined the expression of the FSTL1 gene, which is a target of miR-378a-3p. The results obtained from the Western blot and qRT-PCR analyses indicated that the expression of FSTL1 was significantly lower in BLM mice treated with Exo (miR-378a-3p) compared to those treated with PBS or Exo (NC) (Fig. 6 d, e). These findings are consistent with the in vitro experiments, which demonstrated that miR-378a-3p targets and inhibits the expression of FSTL1. Therefore, it can be concluded that miR-378a-3p plays a role in inhibiting skin fibrosis induced by BLM. Discussion This study aimed to investigate the potential anti-fibrotic role of miR-378a-3p in skin fibrosis. The findings demonstrated that miR-378a-3p effectively reduced proliferation, migration, and Collagen I expression in HDFs, both in the presence and absence of TGF-β1, by directly targeting FSTL1. Additionally, subsequent investigations revealed that exosomes derived from miR-378a-3p-overexpressing keratinocytes exhibited the ability to inhibit skin fibrosis, leading to a reduction in dermal thickening and collagen deposition. Multiple studies have provided evidence indicating that miR-378 possesses inhibitory properties against a range of fibrosis diseases [ 27 – 29 ]. Specifically, it has been observed that miR-378 plays a crucial role in suppressing cardiac fibrosis induced by pressure overload through the regulation of p38 MAPK signaling pathways [ 30 ]. Furthermore, a separate investigation has revealed that among the differentially expressed miRNAs in CCl4-induced liver fibrosis, the expression of miR-378 family members experienced the most significant reduction [ 22 ]. Additionally, miR-378 has demonstrated its ability to safeguard against renal interstitial fibrosis in mice subjected to ischemia-reperfusion injury (IRI) [ 29 ]. The current study provides evidence supporting the inhibitory regulatory role of miR-378a-3p in the activity of HDFs. Specifically, miR-378a-3p was found to significantly decrease the proliferation and migration of HDFs, as well as the expression of Collagen I. Conversely, suppressing the expression of miR-378a-3p resulted in increased HDFs activities. Additionally, as a profibrotic factor, transforming growth factor beta (TGF-β1) plays a crucial role in the development of organ fibrosis. Previous research has shown that miR-378 can attenuate the up-regulated expression of type 1 collagen, type IV collagen, and α-SMA induced by TGF-β1 in NRK52E cells [ 31 ]. Previous research has demonstrated that the upregulation of miR-378 in cardiac fibroblasts effectively impedes the expression of Col I, Col III, and GRB2 induced by TGF-β1 [ 32 ]. In our investigation, the introduction of miR-378a-3p mimics through transfection effectively suppressed the TGF-β1-mediated activity of HDFs. These findings strongly indicate that miR-378a-3p holds considerable promise as a pro-fibrosis factor. Our objective is to determine a specific target for miR-378a-3p using bioinformatics analysis and luciferase reporter assay. Both methodologies have provided evidence that FSTL1 is indeed a direct target of miR-378a-3p. Additionally, the expression of FSTL1 can be effectively modulated by the miR-378a-3p mimic or inhibitor. FSTL1, a glycoprotein presents in the extracellular matrix and induced by TGF-β, belongs to the SPARC family of matricellular proteins. FSTL1 exerts a substantial influence on the regulation of cellular interactions with the extracellular matrix through the integration of signaling networks that govern crucial cellular functions. Its involvement spans diverse biological processes such as development, tissue remodeling, repair, and inflammatory responses [ 33 , 34 ]. Furthermore, numerous studies have substantiated the therapeutic potential of targeting FSTL1 for the treatment of fibrosis. The administration of a specific antibody targeting FSTL1 in vivo experimentation effectively ameliorated lung fibrosis induced by bleomycin in mice [ 35 ]. This outcome closely resembled the effects observed in transgenic mice with FSTL1 genetic insufficiency resulting from heterozygosity for the FSTL1 knockout allele [ 36 ]. Further investigations utilizing this model revealed the impact of FSTL1 on multiple cellular processes associated with lung fibroblast deidentification, proliferation, migration, and invasion [ 37 ]. Research conducted on primary cultures of hematopoietic stem cells (HSCs) extracted from the liver of mice indicates that FSTL1 functions as a downstream factor of TGF-β1 activation and acts as an upstream regulator for the production of SMA and COL1A1 [ 38 ]. In line with these findings, our experimental results demonstrate that the inhibition of FSTL1 using siRNA in HDFs partially counteracted the activating effect of TGF-β1 on cellular activity. Furthermore, the observation of a substantial restoration in the diminished proliferation, migration, and COL1A1 expression in HDFs treated with TGF-β1, upon overexpression of miR-378a-3p, was further reinforced by the introduction of FSTL1. This finding provides additional evidence supporting the notion that miR-378a-3p may exert its influence on TGF-β-stimulated dermal fibroblasts by targeting FSTL1. In addition to identifying the downstream target, our study provides evidence that NORAD serves as a significant upstream regulator of miR-378a-3p expression. Recent research has extensively demonstrated NORAD's role as a competitive ceRNA in various cancers, where it modulates downstream mechanisms by sponging miRNAs such as miR-199a-3p, miR-202-5p, miR-125a-3p, miR-495-3p, and miR-590-3p [ 39 , 40 ]. Furthermore, NORAD's involvement in fibrosis has also been implicated. For example, the administration of NORAD lentivirus shRNA via intravenous injection resulted in a decrease in body weight and serum biochemical indexes, an improvement in cardiac function, and a reduction in inflammation and fibrosis in mice with Diabetic cardiomyopathy (DCM) [ 41 ]. Previous studies have indicated that NORAD may activate the TGF-β1/Smad3 pathway through miR-136-5p, thereby promoting tacrolimus-induced renal fibrosis [ 42 ]. Additionally, Jun et al. have demonstrated that NORAD plays a role in regulating the proliferation of hypertrophic scar fibroblasts by mediating the regulation of TGF-βR2/R1 through miR-26a [ 43 ]. In our study, it was initially discovered that miR-378a-3p serves as a downstream target of NORAD. Further investigation is required to determine whether NORAD modulates fibroblast activity by impeding the impact of miR-378a-3p on the suppression of FSTL1 expression. Exosomes, which are small extracellular vesicles released by diverse cells, possess the ability to modulate a range of cellular functions through the transfer of various intracellular components to target cells. Among these components, miRNAs are expressed and selectively packaged into exosomes, thereby regulating the functions of recipient cells. In recent times, exosomal miRNAs have emerged as promising candidates for novel therapeutic interventions. Research studies have provided evidence linking exosomes containing miR-378a-3p to several bio-functional processes, including wound healing and cancer progression [ 44 , 45 ]. Our previous research has indicated that miR-378a-3p exhibits high levels of expression within exosomes derived from induced pluripotent stem cell (iPSCs)-derived keratinocytes. The current study demonstrates the ability of keratinocyte-derived exosomes to transport miR-378a-3p into HDFs, resulting in the suppression of proliferation, migration, and Collagen I expression. Furthermore, in a mouse model of bleomycin-induced skin fibrosis, treatment with miR-378a-3p-overexpressing keratinocyte-derived exosomes led to a reduction in dermal fibrosis and collagen deposition within the skin. In the context of BLM-induced skin fibrosis, a reciprocal relationship was observed between miR-378a-3p expression and FSTL1, the gene targeted by miR-378a-3p, in the skin samples. This finding supports the notion that miR-378a-3p-overexpressing exosomes derived from keratinocytes could hold promise for clinical applications in the treatment of skin fibrosis. In summary, our study provides evidence for the anti-fibrotic properties of miR-378a-3p in HDFs. We have demonstrated that exosomes derived from keratinocytes overexpressing miR-378a-3p effectively mitigate skin fibrosis in a model of bleomycin-induced fibrosis. These findings suggest that utilizing exosomes derived from miR-378a-3p-overexpressing keratinocytes could potentially serve as a therapeutic approach for fibrotic conditions, including scleroderma and skin scarring. Declarations Author Contributions All authors contributed to the general study conception and design. Conceptualization and design were performed by YY and LZ. The research and data collection were performed by LY, GT, and BL. The data analysis and interpretation were finished by LY and CL. The first draft of the manuscript was written by YY, GT, YB, and JS. All authors have read and agreed to the published version of the manuscript. Funding This work was funded by the National Natural Science Foundation of China (No: 82072186, No: 82272265). Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Animal Care and Use Committee of Southern Medical University (protocol number SMUL2022044). Data Availability Statement The data presented in this study are available on reasonable request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. References Liu Y-X, Sun J-M, Ho C-K, Gao Y, Wen D-S, Liu Y-D, Huang L, Zhang Y-F (2023) Advancements in Adipose-Derived Stem Cell Therapy for Skin Fibrosis. 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Mol Med Rep 16:7119–7123. 10.3892/mmr.2017.7445 Zhang L, Wu H, Zhang Y, Xiao X, Chu F, Zhang L (2022) Induction of lncRNA NORAD Accounts for Hypoxia-Induced Chemoresistance and Vasculogenic Mimicry in Colorectal Cancer by Sponging the miR-495-3p/ Hypoxia-Inducible Factor-1α (HIF-1α). Bioengineered 13:950–962. 10.1080/21655979.2021.2015530 Shen J-G, Xu S-N, Yin L-G (2020) LncRNA NORAD/miR-202-5p Regulates the Drug Resistance of A549/DDP to Cisplatin by Targeting P-Gp. Gen Physiol Biophys 39:481–489. 10.4149/gpb_2020027 Liu Y, Zhu Y, Liu S, Liu J, Li XNORAD (2021) Lentivirus shRNA Mitigates Fibrosis and Inflammatory Responses in Diabetic Cardiomyopathy via the ceRNA Network of NORAD/miR-125a-3p/Fyn. Inflamm Res 70:1113–1127. 10.1007/s00011-021-01500-y Liu L, Guo J, Pang X-L, Shang W-J, Wang Z-G, Wang J-X, Yang X-L, Feng G-W (2023) Exploration of the Mechanism of NORAD Activation of TGF-Β1/Smad3 through miR-136-5p and Promotion of Tacrolimus-Induced Renal Fibrosis. Ren Fail 45:2147083. 10.1080/0886022X.2022.2147083 Qi J, Wu Y, Zhang H, Liu Y (2021) LncRNA NORAD Regulates Scar Hypertrophy via miRNA-26a Mediating the Regulation of TGFβR1/2. Adv Clin Exp Med 30:395–403. 10.17219/acem/133482 Yang Q, Zhao S, Shi Z, Cao L, Liu J, Pan T, Zhou D, Zhang J (2021) Chemotherapy-Elicited Exosomal miR-378a-3p and miR-378d Promote Breast Cancer Stemness and Chemoresistance via the Activation of EZH2/STAT3 Signaling. J Exp Clin Cancer Res 40:120. 10.1186/s13046-021-01901-1 Briand J, Garnier D, Nadaradjane A, Clément-Colmou K, Potiron V, Supiot S, Bougras-Cartron G, Frenel J-S, Heymann D, Vallette FM et al (2020) Radiotherapy-Induced Overexpression of Exosomal miRNA-378a-3p in Cancer Cells Limits Natural Killer Cells Cytotoxicity. Epigenomics 12:397–408. 10.2217/epi-2019-0193 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3829723","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":264997820,"identity":"8a67103d-5394-4889-ae94-d5ffa1f852d5","order_by":0,"name":"Lijun Yang","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lijun","middleName":"","lastName":"Yang","suffix":""},{"id":264997821,"identity":"d692aa09-dbe2-4d4f-9e36-7afda11e4903","order_by":1,"name":"Guiping Tian","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guiping","middleName":"","lastName":"Tian","suffix":""},{"id":264997822,"identity":"3fd3035f-bc09-4282-a43f-1f759eb281e7","order_by":2,"name":"Baiting Liu","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Baiting","middleName":"","lastName":"Liu","suffix":""},{"id":264997823,"identity":"b5ae81e4-01eb-4756-8f90-89f0c8b13f8c","order_by":3,"name":"Chenxi Li","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chenxi","middleName":"","lastName":"Li","suffix":""},{"id":264997824,"identity":"82445a5f-ac90-4ea1-9ae5-57cf5acc4ecf","order_by":4,"name":"Yunyao Bo","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yunyao","middleName":"","lastName":"Bo","suffix":""},{"id":264997825,"identity":"65975943-503f-40d3-977c-59b0d0ae09b5","order_by":5,"name":"Jianjun Si","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianjun","middleName":"","lastName":"Si","suffix":""},{"id":264997826,"identity":"c23c8f4a-1ea7-4ceb-8f4a-dd7fa9c0b299","order_by":6,"name":"Lin Zhang","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Zhang","suffix":""},{"id":264997827,"identity":"cad026ae-16b2-4761-8a8a-08ec1ec8054d","order_by":7,"name":"Yuan Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBADOQjFRoIWY9K1JDYQrcXgRo7h58Kcbenzp50xYPhQdpiBf3YDQS3G0jO33c7dcDvHgHHGucMMEncOENRiIM0L0iKdY8DM23aYwUAigbAtv4Fa0uVnA7X8JVKLGciWBAagw5gZidEieeZZmTVQi+GG22kFB3vOpfNI3CCghe948ubbQC3y8rOTNz74UWYtxz+DgBaFC0gKDgAxD371QCDff4CgmlEwCkbBKBjpAADbO0UbMd46CAAAAABJRU5ErkJggg==","orcid":"","institution":"Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Yan","suffix":""}],"badges":[],"createdAt":"2024-01-02 14:33:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3829723/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3829723/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49223241,"identity":"844f4ddd-24f2-4fba-ba93-ea74dac11ae1","added_by":"auto","created_at":"2024-01-05 13:21:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":302256,"visible":true,"origin":"","legend":"\u003cp\u003emiR-378a-3p inhibits activity of TGF-β1-stimulated HDFs. \u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eImaging of EdU staining of HDFs transfected with NC mimic, miR-378a-3p mimic, NC inhibitor, or miR-378a-3p inhibitor (left panel). Scale bar = 100 um. Quantification of the percentage of EdU-positive cells (right panel). \u003cstrong\u003eb\u003c/strong\u003e, Images of migrated HDFs per group. Scale bar = 50 um. Quantitative analysis of migrating cells (right panel). \u003cstrong\u003ec-d\u003c/strong\u003e, The mRNA and protein expressions of COL1A1 expression in HDFs per group using qRT-PCR and Western blot assays. \u003cstrong\u003ee\u003c/strong\u003e, EdU staining imaging of HDFs transfected with NC mimics or miR-378a-3p mimics with or without TGF-β1 treatment (left panel). Scale bar = 100 um. Quantitative analysis of the percentage of EdU-positive cells (right panel). \u003cstrong\u003ef\u003c/strong\u003e, Images of migrated HDFs per group. Scale bar = 50 um. Quantitative analysis of migrating cells (right panel). \u003cstrong\u003eg-h\u003c/strong\u003e, The mRNA and protein expressions of COL1A1 expression in HDFs per group using qRT-PCR and Western blot assays. All values are expressed as mean ± SD from three independent repeats, *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3829723/v1/c718e15e4b38507f6e87552f.png"},{"id":49223245,"identity":"27428ed0-fa1a-4791-a760-3abf41758d25","added_by":"auto","created_at":"2024-01-05 13:21:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":195132,"visible":true,"origin":"","legend":"\u003cp\u003emiR-378a-3p inhibits the proliferation and migration of HDFs by targeting FSTL1. \u003cstrong\u003ea\u003c/strong\u003e, Complementary binding sites between miR-378a-3p and the 3’UTR sequence of FSTL1 mRNA. \u003cstrong\u003eb\u003c/strong\u003e, Dual-luciferase reporter gene assays determined luciferase activity in HDFs co-transected with miR-378a-3p mimic and psiCHECK-FSTL1-wt-3'UTR (WT) or psiCHECK-FSTL1-mut-3'UTR (MUT). \u003cstrong\u003ec-d\u003c/strong\u003e, The mRNA and protein expressions of FSTL1 expression in HDFs transfected with NC mimic, miR-378a-3p mimic, NC inhibitor, or miR-378a-3p inhibitor using qRT-PCR and Western blot assays. \u003cstrong\u003ee\u003c/strong\u003e, EdU staining imaging of HDFs transfected with miR-378a-3p mimic with or without 50 ng/ml FSTL1 treatment (left panel). Scale bar = 100 um. Quantification of the percentage of EdU-positive cells (right panel). \u003cstrong\u003ef\u003c/strong\u003e, Images of migrated HDFs transfected with miR-378a-3p mimic with or without 50 ng/ml FSTL1 treatment (left panel). Scale bar = 50 um. quantitative analysis of migrating cells (right panel). \u003cstrong\u003eg-h\u003c/strong\u003e, The mRNA and protein expressions of COL1A1 expression in HDFs per group using qRT-PCR and Western blot assays. All values are expressed as mean ± SD from three independent repeats, *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3829723/v1/0f221663bef6ad3d12cde08b.png"},{"id":49223952,"identity":"8db7473d-b5fb-466a-bbb0-4bd6b92370fa","added_by":"auto","created_at":"2024-01-05 13:37:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73326,"visible":true,"origin":"","legend":"\u003cp\u003emiR-378a-3p was regulated by NORAD. \u003cstrong\u003ea\u003c/strong\u003e, Schematic representation of potential binding sites of miRNAs with NORAD. \u003cstrong\u003eb\u003c/strong\u003e, Luciferase reporter assay determined luciferase activity in 293T cells co-transfected with miR-378a-3p mimics and psiCHECK-NORAD-wt (WT) or psiCHECK-NORAD-mut (MUT). \u003cstrong\u003ec\u003c/strong\u003e, The expression of miR-378a-3p in HDFs transfected by pcDNA3.1-NORAD or si-NORAD using qRT-PCR.\u003cstrong\u003e d-e\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eThe mRNA and protein expression of FSTL1 in HDFs transfected with pcDNA3.1-NORAD in the presence or absence of miR-378a-3p mimics using qRT-PCR and Western blot assays. All values are expressed as mean ± SD from three independent repeats, *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3829723/v1/3867face86147fb6015f7089.png"},{"id":49223443,"identity":"e505af8b-14ce-4f6d-bbe4-763f4710d7ef","added_by":"auto","created_at":"2024-01-05 13:29:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116441,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of exosomes derived from modified Keratinocytes. \u003cstrong\u003ea\u003c/strong\u003e, The transmission electron microscope (TEM) images of Exo (NC) and Exo (miR-378a-3p). Scale bar = 100 nm. \u003cstrong\u003eb\u003c/strong\u003e, Nanoparticle tracking analysis (NTA) results of Exo (NC) and Exo (miR-378a-3p). \u003cstrong\u003ec\u003c/strong\u003e, Representative imaging of fibroblasts incubated with PKH67-labeled Exo (NC) or Exo (miR-378a-3p). Scale bar = 50 um. \u003cstrong\u003ed\u003c/strong\u003e, RT-qPCR analysis of miR-378a-3p expression of fibroblasts treated by PBS, Exo (NC), or Exo (miR-378a-3p). Results were normalized to U6 expression. All values are expressed as mean ± SD from three independent repeats, *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3829723/v1/8c9ea103a95a51c824e62a85.png"},{"id":49223243,"identity":"0fabd8b4-a590-4c46-9841-24a8576ce372","added_by":"auto","created_at":"2024-01-05 13:21:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":340539,"visible":true,"origin":"","legend":"\u003cp\u003eExo (miR-378a-3p) inhibits the activity of TGF-β1-stimulated dermal fibroblasts. \u003cstrong\u003ea\u003c/strong\u003e, Imaging of EdU staining of HDFs treated by PBS, Exo (NC), or Exo (miR-378a-3p) with or without TGF-β1 for 48 hours (left panel). Scale bar = 200 um. Quantitative analysis of the percentage of EdU-positive cells (right panel). \u003cstrong\u003eb\u003c/strong\u003e, Images of migrated HDFs per group. Scale bar = 200 um. Quantitative analysis of the migrated cells (right panel). \u003cstrong\u003ec-d\u003c/strong\u003e, The mRNA and protein expressions of COL1A1 expression of HDFs per group using qRT-PCR and Western blot assays. All values are expressed as mean ± SD from three independent repeats, *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3829723/v1/ef4640ad2265ea44649c5460.png"},{"id":49223247,"identity":"7239bc24-bd36-4a2c-8388-a69a6f2954f3","added_by":"auto","created_at":"2024-01-05 13:21:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":771890,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-fibrotic effects of Exo (miR-378a-3p) on skin fibrosis. \u003cstrong\u003ea\u003c/strong\u003e, Representative images of HE and Masson staining of skin tissues of wild-type (WT) mice and BLM mice treated by PBS, Exo (NC), or Exo (miR-378a-3p) (left panel). Scale bar = 100 um. Quantification of the dermal thickness of skin (right panel). \u003cstrong\u003eb\u003c/strong\u003e, Representative images of COL1A1 immunostaining of skin tissues in each group (left panel). Scale bar = 100 um. The areas stained with COL1A1 were determined using Image-Pro Plus 6.0 software (right panel). \u003cstrong\u003ec-d\u003c/strong\u003e, The mRNA expressions of COL1A1 and FSTL1 expression in skin tissues per group using qRT-PCR assays. \u003cstrong\u003ee-f\u003c/strong\u003e, The protein expressions of COL1A1 and FSTL1 in expression in skin tissues per group using Western blot assays. All values are expressed as mean ± SD from three independent repeats, *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3829723/v1/3a33fae7dd0360806eee1202.png"},{"id":49223246,"identity":"8da2a575-4f53-4fbe-8e3c-1a7e01d240ff","added_by":"auto","created_at":"2024-01-05 13:21:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":92900,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic model showing that exosomal miR-378a-3p derived from keratinocytes targeted FSTL1 and inhibited the TGF-β1 signaling pathways, further attenuating proliferation, migration, and COL1A1 expression of HDFs.\u003c/p\u003e","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3829723/v1/d93714ebe6ada9ccb07946a1.png"},{"id":49588454,"identity":"1c9a6843-c305-4a1c-818c-5bd00a8b89ed","added_by":"auto","created_at":"2024-01-14 16:37:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2968663,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3829723/v1/160e8e38-0f87-4e45-a9ed-d8b8948dd43d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exosomal miR-378a-3p derived from keratinocytes inhibits transforming growth factor beta-induced fibroblast activation and attenuates bleomycin-induced skin fibrosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSkin fibrosis is a characteristic manifestation observed in multiple pathological conditions, such as scleroderma, keloids, hypertrophic scars, and graft versus host disease [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Current clinical interventions for skin fibrosis encompass local pharmaco-logical interventions, skin grafting, and surgical excision; nevertheless, their therapeutic effectiveness remains suboptimal [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Consequently, there exists a pressing necessity to acquire comprehensive insights that will enable the development of improved therapeutic approaches and furnish substantiation for clinical decision-making.\u003c/p\u003e \u003cp\u003eMiRNAs exert substantial regulatory influence on gene expression by frequently targeting multiple genes within a specific gene network [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consequently, the modulation of a single miRNA can have a significant impact, as it induces multiple changes in gene expression within the same pathway [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In recent times, a considerable number of miRNAs have been identified as playing crucial roles in the development of skin fibrosis. Previous research endeavors have made significant strides in understanding the impact of certain miRNAs on scar formation and investigating the potential underlying mechanisms [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The current study is specifically focused on miR-378a-3p, a miRNA that has been previously implicated in various biological functions and processes such as cell growth, cell cycle regulation, migration, differentiation, metabolism, and angiogenesis [\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Recent investigations have also linked miR-378a to myocardial fibrosis, liver fibrosis, and interstitial fibrosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the precise role of miR-378a-3p in skin fibrosis remains unclear. Therefore, the objective of this study is to examine the potential involvement of miR-378a-3p in the development of skin fibrosis.\u003c/p\u003e \u003cp\u003eAs the discovery of disease-associated miRNAs and the advancement of in vivo miRNA manipulation techniques continue, a multitude of miRNA-based therapies have progressed to clinical trials, exhibiting promising therapeutic results. Recent scholarly investigations have provided evidence through multiple studies that miRNAs are enclosed within exosomes, thereby preserving their contents from degradation and facilitating the transfer of various small biomolecules, including proteins and RNAs, to adjacent cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Previous studies have demonstrated that exosomes derived from keratinocytes possess a robust and specific regulatory influence on fibro-blasts [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Expanding upon our previous investigations, we have observed that miR-16-5p can be enclosed within exosomes derived from keratinocytes and exert regulatory control over the functionality of fibroblasts. Therefore, we put forward the hypothesis that the inclusion of miR-378 within exosomes derived from keratinocytes may potentially demonstrate a synergistic effect, thereby offering a promising therapeutic strategy for the management of fibrosis.\u003c/p\u003e \u003cp\u003eThe primary objective of this study was to examine the role of miR-378a-3p in fibroblast activity and elucidate its regulatory mechanism. Additionally, we investigated the effects of miR-378a-3p-enriched exosomes on fibroblast activity and evaluated its potential therapeutic efficacy in a murine model of BLM-induced skin fibrosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHaCaT cells were cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone, USA) containing 10% fetal bovine serum (Gibico, USA). Human skin samples were obtained from patients with consent forms. The dermal layers of the skin were dissected into small fragments and subsequently plated in DMEM supplemented with 10% fetal bovine serum. Fibroblasts from the third passage were employed for the study..\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids, miRNAs, siRNA, lncRNA, and transfection\u003c/h2\u003e \u003cp\u003eCells were seeded at a density of 1.2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in a 24-well plate and cultured for 24 hours. Transfection experiments of miR-378a-3p mimic/inhibitor (RiBoBio, Guangzhou, China), lncRNA NORAD, pcDNA3.1, or si-NORAD (RiBoBio, Guangzhou, China) were carried out according to Lipofectamine 2000 instructions (Thermo Fisher Scientific, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEdU staining\u003c/h2\u003e \u003cp\u003eEdU (Beyotime, Shanghai, China) was added 48 hours after transfection for a further 4 hours of incubation, after which the cells were fixed with 4% paraformaldehyde for half an hour, washed with 3% bovine serum albumin (BSA), and permeabilized with 0.5% TritonX-100. Finally, Ultimately, cells were stained by BeyoClick\u0026trade; EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, Shanghai, China) according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTranswell assay\u003c/h2\u003e \u003cp\u003eThe cells were digested and resuspended after being serum-free for 8 hours following transfection. The medium containing 5% fetal bovine serum (FBS) was added to the lower chamber of the transwell as an induction factor, and about 200 \u0026micro;l (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells) of serum-free cell suspension was added to the upper chamber. After culturing for 24 hours, the cells in the chamber were permeabilized with 4% paraformaldehyde. The cells in the chamber were stained with crystal violet solution for about 10\u0026ndash;15 minutes. The upper chamber unpenetrated cells were wiped off, and photos of the cells that penetrated the chamber were taken using a Leica inverted microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eCells and tissues were lysed with were lysed in ice-cold Radio-Immunoprecipitation Assay (RIPA, Sigma-Aldrich, St. Louis, MO, USA) after the initial treatment. The cells and tissues were thoroughly lysed and centrifuged for 15 minutes at 12,000 x g. After centrifugation, the supernatant was combined with 5\u0026times; SDS Loading Buffer, and the protein was extracted after 10 minutes of boiling. Following that, the samples were separated on a 10% SDS-PAGE gel and transferred to PVDF membranes. The PVDF membrane was blocked with 5% nonfat milk powder and incubated with β-actin (1:1000, Proteintech, Wuhan, Hubei, China), FSTL1 (1:1000, Proteintech, Wuhan, Hubei, China) and COL1A1 (1:1000, Proteintech, Wuhan, Hubei, China) primary antibodies overnight at 4\u0026deg;C. The next day, incubated for 1 hours at room temperature with the corresponding secondary antibody (1:2000, Proteintech, Wuhan, Hubei, China) that can bind to the primary antibody, and visualized the protein bands by the ECL detection system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase assay\u003c/h2\u003e \u003cp\u003eFirst, the binding sites of miR-378a-3p in the 3'-UTR of FSTL1 and lncRNA NORAD were predicted, and then FSTL1 and lncRNA NORAD were amplified from cDNA by PCR. Then, the PCR fragment was inserted into the psiCHECK-2 vector. The successfully constructed plasmid was identified and co-transfected with miR-378a-3p mimic through Lipofectamine 2000 into HDFs. psiCHECK-2 and mimic negative control (NC) were used as negative controls for transfection according to the same method as above. Cells were collected 48 hours after transfection and were operated according to the Dual-Luciferase Reporter Gene Assay Kit (Beyotime, Shanghai, China) requirements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eCells/tissues were subjected to TRIzol reagent kit (Thermo Fisher Scientific, CA, USA) for the extraction of total RNA. The mRNA was then reverse transcribed for qRT-PCR using AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, Jiangsu, China). Subsequently, qRT-PCR analysis was conducted using AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, Jiangsu, China). The Ct values obtained were normalized using the GAPDH. For miRNA analysis, cDNA for miRNA was synthesized following the manufacturer's protocol of the miDETECT A TrackTM miRNA qRT-PCR Starter Kit. The U6 RNA level was utilized as an internal control for data normalization. The qRT-PCR reaction was conducted utilizing the miDETECT A TrackTM miRNA qPCR Kit, employing the miDETECT A TrackTM miR-378a-3p Forward Primer and miDETECT A TrackTM Uni-Reverse Primer (The above reagents about miRNA were from RiBoBio, Guangzhou, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and characterization of exosomes\u003c/h2\u003e \u003cp\u003eHaCaT cells were cultured in serum-free DMEM for 48 hours. After collecting the conditioned medium, ExoEasy Maxi Kit (Qiagen, MD, USA) according to the manufacturer\u0026rsquo;s instructions. Then, the transmission electron microscope (JEOL, Japan) was used to observe the morphology of exosomes, and Nanosight LM 10 (Malvern Panalytical, UK) was used to identify the size, concentration, and distribution of exosomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCellular internalization\u003c/h2\u003e \u003cp\u003eFirst of all, exosomes were labeled using the PKH67 Green Fluorescent Cell Linker Mini Kit (Sigma-Aldrich, St. Louis, MO, USA). Then, PKH67-labeled exosomes were co-cultured with cells in FBS-free DMEM overnight. After fixing with 4% paraformaldehyde and staining with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA), cells were observed under a fluorescence microscope (Leica, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBLM -induced skin fibrosis murine model\u003c/h2\u003e \u003cp\u003eSix-week-old male C57BL/6 mice were purchased from the Laboratory Animal Center of Southern Medical University. All animal protocols used in this study were approved by the Animal Care and Use Committee of Southern Medical University. For the BLM model, 100 ul of 0.5 mg/mL BLM (TargetMol, Shanghai, China) is administered by subcutaneous injection. Repeat injections every other day for 4 weeks. Mice are treated with PBS, Exo (NC), or Exo (miR-378a-3p) in parallel by intradermal injection every other day during the last two weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eC57BL/6 mouse skin samples were firstly fixed with 4% paraformaldehyde for three days, then dehydrated in a gradient manner, transparent, and embedded in paraffin. They were then made into 5 um thick paraffin sections, and the tissue samples were stained according to the instructions for hematoxylin and eosin staining (H\u0026amp;E) and Masson\u0026rsquo;s trichrome staining Kit (both from Yeasen Biotechnology, Shanghai, China) and photographs were taken using a Leica upright microscope. Subsequent data processing was done using Image Pro Plus software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical assay\u003c/h2\u003e \u003cp\u003eParaffin section samples were dewaxed and hydrated, boiled with citrate solution (pH\u0026thinsp;=\u0026thinsp;6.0) for antigen retrieval, and incubated with blocking solution (goat serum: 5% BSA: PBS\u0026thinsp;=\u0026thinsp;1:2:7) for 1 hour. COL1A1 primary antibody (1:100, Proteintech, Wuhan, Hubei, China) was added dropwise and incubated overnight at 4\u0026deg;C. The next day after washing with PBS, an enzyme-labeled secondary antibody was added dropwise for 1 hour, and the DAB (ZSGB-BIO, Beijing, China) color was developed. Subsequently, hematoxylin staining was performed to stain the nuclei, and the cells were mounted by gradient dehydration. Photographs were taken with a Leica upright microscope and subsequent data processing was done with Image Pro Plus software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SEM) of at least three independent experiments (n\u0026thinsp;\u0026ge;\u0026thinsp;3). Data statistics were performed using GraphPad Prism 8 (GraphPad Software, Inc, La Jolla, CA) software, and comparisons between two groups were performed using independent samples t-test or one-way ANOVA between two groups for statistical analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003emiR-378a-3p inhibits the activity of TGF-β1-stimulated HDFs\u003c/h2\u003e \u003cp\u003eIn this study, we examined the impact of miR-378a-3p on the activation of dermal fibroblasts. Our findings revealed that the overexpression of miR-378a-3p had a substantial effect on promoting the proliferation of dermal fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Additionally, we also investigated the influence of miR-378a-3p on the migration of these fibroblasts. The results obtained from the transwell assay, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, demonstrated that the overexpression of miR-378a-3p significantly hindered the migration of dermal fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). It is worth noting that excessive deposition of extracellular matrix (ECM) proteins in the dermis is a crucial characteristic of skin fibrosis. Therefore, we further detected the effect of miR-378a-3p on COL1A1 expression in dermal fibroblasts. The miR-378a-3p mimic was found to suppress the mRNA and protein expression of COL1A1 in HDFs when compared to the control group. Conversely, transfection with the miR-378a-3p inhibitor resulted in the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). Furthermore, miR-378a-3p significantly inhibited the proliferative and migratory effects of TGF-β1 on HDFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). The observed changes in COL1A1 mRNA and protein expression followed similar trends (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, h). Based on these findings, it can be concluded that miR-378a-3p exhibits an antagonistic effect to TGF-β1 in the activation of dermal fibroblasts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003emiR-378a-3p played a role in dermal fibroblast via targeting FSTL1\u003c/h2\u003e \u003cp\u003eTarget Scan, miRanda, and miRDB predicted that the 3'-untranslated region (UTR) of FSTL1 has a miR-378a-3p binding site (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). To validate this hypothesis, luciferase reporter gene constructs containing either the wild-type (WT) or mutant (MUT) versions of the putative miR-378a-3p binding site within the 3'-UTR of FSTL1 were prepared. Subsequent transfection of these reporter genes, along with a miR-378a-3p mimic, into HDFs confirmed that miR-378a-3p effectively inhibited the activity of the WT reporter construct, while having no impact on the MUT reporter construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Moreover, overexpression of miR-378a-3p reduced the mRNA and protein expression of FSTL1 in HDFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). The further findings revealed that overexpression of miR-378a-3p in HDFs led to a reduction in proliferation and migration rates, which were subsequently rescued by the presence of 50 ng/ml FSTL1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). Similar trends were observed in terms of mRNA transcription and COL1A1 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, h). Consequently, it can be inferred that miR-378a-3p exerts inhibitory effects on HDFs activation by targeting FSTL1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003emiR-378a-3p was regulated by lncRNA NORAD (NORAD)\u003c/h2\u003e \u003cp\u003eTo study the upstream regulator of miR-378a-3p, we focused on long non-coding RNA (lncRNA) since accumulating evidence suggests that lncRNAs play significant roles in HS formation by regulating many processes, including ECM deposition and fibroblast proliferation. Analysis of data in the StarBase database revealed that lncRNA NORAD possibly binds miR-378a-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). To verify the interaction between NORAD and miR-378a-3p, constructed plasmids containing the wild-type sequence (NORAD-WT) and the mutant binding site sequence (NORAD-MUT). The results showed that overexpression of miR-378a-3p significantly reduced the luciferase activity of the NORAD -WT vector but did not reduce the luciferase activity of the empty vector or the NORAD-MUT vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), confirming that NORAD-MUT can directly interact with miR-378a-3p. In addition, overexpression of NORAD significantly decreased the expression of miR-378a-3p, while the siRNA-NORAD inhibited the downregulated expression of miR-378a-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Additionally, we also investigated the effect of NORAD on the expression of FSTL, the target of miR-378a-3p. The results showed that overexpression of NORAD significantly increased the mRNA and protein expression of FSTL1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e), which was inhibited by miR-378a-3p mimic. Therefore, we conclude that NORAD acts as an ceRNA of miR-378a-3p and further activates the expression of FTSL1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of keratinocytes-derived exosomes for miR-378a-3p delivery\u003c/h2\u003e \u003cp\u003eExosomes derived from HaCaT cells infected with miR-378a-3p mimics or negative control mimics (NC) were isolated and subjected to morphological, numerical, and size analysis using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The findings revealed that both Exo (NC) and Exo (miR-378a-3p) exhibited a circular shape and were enveloped by double membranes within the size range of 30\u0026ndash;150 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Additionally, the integration capability of Exo (NC) and Exo (miR-378a-3p) was confirmed through PKH67 assay. Following a 10-hour incubation period of fibroblasts with PKH67-labeled exosomes, notable fluorescence levels were detected within the cytoplasm of fibroblasts, suggesting the internalization of Exo (NC) and Exo (miR-378a-3p) by fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Comparative analysis utilizing qRT-PCR demonstrated a significant increase in miR-378a-3p expression within Exo (miR-378a-3p)-treated fibroblasts when compared to those treated with Exo (NC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eExo (miR-378a-3p) inhibit activity of TGF-β1-stimulated dermal fibroblasts\u003c/h2\u003e \u003cp\u003eInitially, we investigated the impact of Exo (miR-378a-3p) on the proliferation of HDFs through the utilization of EdU staining. In the absence of TGF-β1 (10 ng/ml), the Exo (miR-378a-3p) group exhibited a substantial decrease in proliferation compared to both the Exo (NC) and PBS groups. Following treatment with TGF- β1, proliferation significantly increased in the Exo (NC) and PBS groups, whereas the Exo (miR-378a-3p) group effectively hindered the enhancement of TGF-β1 on cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Subsequently, the transwell assay demonstrated that treatment with Exo (NC) significantly enhanced the migration of HDFs, while the Exo (miR-378a-3p) group exhibited decreased migration compared to PBS. Furthermore, the migratory effect of TGF-β1 was also mitigated by Exo (miR-378a-3p) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The mRNA expression of COL1A1 was reduced in both the Exo (NC) and Exo (miR-378a-3p) groups, with or without TGF-β1, when compared to the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Additionally, the level of COL1A1 protein in the Exo (miR-378a-3p) groups was significantly lower than that in the Exo (NC) group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eExo (miR-378a-3p) inhibits BLM-induced skin fibrosis\u003c/h2\u003e \u003cp\u003eWe further investigated the involvement of miR-378a-3p in skin fibrosis induced by BLM through in vivo experimentation. The dermal thickness and collagen deposition were assessed using hematoxylin \u0026amp; eosin (H\u0026amp;E) and Masson staining techniques. Our findings revealed that the BLM group exhibited a significantly increased dermal thickness, elevated collagen deposition, and disorganized collagen arrangement in comparison to the WT group. Subsequent experiments demonstrated a significant reduction in skin tissue thickness and collagen deposition in BLM mice treated with Exo (miR-378a-3p) compared to those treated with PBS or Exo (NC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The immunohistochemical and qRT-PCR analysis further revealed higher expression of COL1A1 in the skin of BLM mice compared to WT mice. However, the expression of COL1A1 was decreased in the skin of Exo (miR-378a-3p)-treated BLM mice compared to those treated with PBS or Exo (NC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c). Western blotting analysis yielded consistent results with the immunohistochemical staining, further supporting these findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). We also examined the expression of the FSTL1 gene, which is a target of miR-378a-3p. The results obtained from the Western blot and qRT-PCR analyses indicated that the expression of FSTL1 was significantly lower in BLM mice treated with Exo (miR-378a-3p) compared to those treated with PBS or Exo (NC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, e). These findings are consistent with the in vitro experiments, which demonstrated that miR-378a-3p targets and inhibits the expression of FSTL1. Therefore, it can be concluded that miR-378a-3p plays a role in inhibiting skin fibrosis induced by BLM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study aimed to investigate the potential anti-fibrotic role of miR-378a-3p in skin fibrosis. The findings demonstrated that miR-378a-3p effectively reduced proliferation, migration, and Collagen I expression in HDFs, both in the presence and absence of TGF-β1, by directly targeting FSTL1. Additionally, subsequent investigations revealed that exosomes derived from miR-378a-3p-overexpressing keratinocytes exhibited the ability to inhibit skin fibrosis, leading to a reduction in dermal thickening and collagen deposition.\u003c/p\u003e \u003cp\u003eMultiple studies have provided evidence indicating that miR-378 possesses inhibitory properties against a range of fibrosis diseases [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Specifically, it has been observed that miR-378 plays a crucial role in suppressing cardiac fibrosis induced by pressure overload through the regulation of p38 MAPK signaling pathways [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Furthermore, a separate investigation has revealed that among the differentially expressed miRNAs in CCl4-induced liver fibrosis, the expression of miR-378 family members experienced the most significant reduction [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Additionally, miR-378 has demonstrated its ability to safeguard against renal interstitial fibrosis in mice subjected to ischemia-reperfusion injury (IRI) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The current study provides evidence supporting the inhibitory regulatory role of miR-378a-3p in the activity of HDFs. Specifically, miR-378a-3p was found to significantly decrease the proliferation and migration of HDFs, as well as the expression of Collagen I. Conversely, suppressing the expression of miR-378a-3p resulted in increased HDFs activities. Additionally, as a profibrotic factor, transforming growth factor beta (TGF-β1) plays a crucial role in the development of organ fibrosis. Previous research has shown that miR-378 can attenuate the up-regulated expression of type 1 collagen, type IV collagen, and α-SMA induced by TGF-β1 in NRK52E cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Previous research has demonstrated that the upregulation of miR-378 in cardiac fibroblasts effectively impedes the expression of Col I, Col III, and GRB2 induced by TGF-β1 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In our investigation, the introduction of miR-378a-3p mimics through transfection effectively suppressed the TGF-β1-mediated activity of HDFs. These findings strongly indicate that miR-378a-3p holds considerable promise as a pro-fibrosis factor.\u003c/p\u003e \u003cp\u003eOur objective is to determine a specific target for miR-378a-3p using bioinformatics analysis and luciferase reporter assay. Both methodologies have provided evidence that FSTL1 is indeed a direct target of miR-378a-3p. Additionally, the expression of FSTL1 can be effectively modulated by the miR-378a-3p mimic or inhibitor. FSTL1, a glycoprotein presents in the extracellular matrix and induced by TGF-β, belongs to the SPARC family of matricellular proteins. FSTL1 exerts a substantial influence on the regulation of cellular interactions with the extracellular matrix through the integration of signaling networks that govern crucial cellular functions. Its involvement spans diverse biological processes such as development, tissue remodeling, repair, and inflammatory responses [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, numerous studies have substantiated the therapeutic potential of targeting FSTL1 for the treatment of fibrosis. The administration of a specific antibody targeting FSTL1 in vivo experimentation effectively ameliorated lung fibrosis induced by bleomycin in mice [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This outcome closely resembled the effects observed in transgenic mice with FSTL1 genetic insufficiency resulting from heterozygosity for the FSTL1 knockout allele [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Further investigations utilizing this model revealed the impact of FSTL1 on multiple cellular processes associated with lung fibroblast deidentification, proliferation, migration, and invasion [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Research conducted on primary cultures of hematopoietic stem cells (HSCs) extracted from the liver of mice indicates that FSTL1 functions as a downstream factor of TGF-β1 activation and acts as an upstream regulator for the production of SMA and COL1A1 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In line with these findings, our experimental results demonstrate that the inhibition of FSTL1 using siRNA in HDFs partially counteracted the activating effect of TGF-β1 on cellular activity. Furthermore, the observation of a substantial restoration in the diminished proliferation, migration, and COL1A1 expression in HDFs treated with TGF-β1, upon overexpression of miR-378a-3p, was further reinforced by the introduction of FSTL1. This finding provides additional evidence supporting the notion that miR-378a-3p may exert its influence on TGF-β-stimulated dermal fibroblasts by targeting FSTL1.\u003c/p\u003e \u003cp\u003eIn addition to identifying the downstream target, our study provides evidence that NORAD serves as a significant upstream regulator of miR-378a-3p expression. Recent research has extensively demonstrated NORAD's role as a competitive ceRNA in various cancers, where it modulates downstream mechanisms by sponging miRNAs such as miR-199a-3p, miR-202-5p, miR-125a-3p, miR-495-3p, and miR-590-3p [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Furthermore, NORAD's involvement in fibrosis has also been implicated. For example, the administration of NORAD lentivirus shRNA via intravenous injection resulted in a decrease in body weight and serum biochemical indexes, an improvement in cardiac function, and a reduction in inflammation and fibrosis in mice with Diabetic cardiomyopathy (DCM) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Previous studies have indicated that NORAD may activate the TGF-β1/Smad3 pathway through miR-136-5p, thereby promoting tacrolimus-induced renal fibrosis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, Jun et al. have demonstrated that NORAD plays a role in regulating the proliferation of hypertrophic scar fibroblasts by mediating the regulation of TGF-βR2/R1 through miR-26a [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In our study, it was initially discovered that miR-378a-3p serves as a downstream target of NORAD. Further investigation is required to determine whether NORAD modulates fibroblast activity by impeding the impact of miR-378a-3p on the suppression of FSTL1 expression.\u003c/p\u003e \u003cp\u003eExosomes, which are small extracellular vesicles released by diverse cells, possess the ability to modulate a range of cellular functions through the transfer of various intracellular components to target cells. Among these components, miRNAs are expressed and selectively packaged into exosomes, thereby regulating the functions of recipient cells. In recent times, exosomal miRNAs have emerged as promising candidates for novel therapeutic interventions. Research studies have provided evidence linking exosomes containing miR-378a-3p to several bio-functional processes, including wound healing and cancer progression [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our previous research has indicated that miR-378a-3p exhibits high levels of expression within exosomes derived from induced pluripotent stem cell (iPSCs)-derived keratinocytes. The current study demonstrates the ability of keratinocyte-derived exosomes to transport miR-378a-3p into HDFs, resulting in the suppression of proliferation, migration, and Collagen I expression. Furthermore, in a mouse model of bleomycin-induced skin fibrosis, treatment with miR-378a-3p-overexpressing keratinocyte-derived exosomes led to a reduction in dermal fibrosis and collagen deposition within the skin. In the context of BLM-induced skin fibrosis, a reciprocal relationship was observed between miR-378a-3p expression and FSTL1, the gene targeted by miR-378a-3p, in the skin samples. This finding supports the notion that miR-378a-3p-overexpressing exosomes derived from keratinocytes could hold promise for clinical applications in the treatment of skin fibrosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, our study provides evidence for the anti-fibrotic properties of miR-378a-3p in HDFs. We have demonstrated that exosomes derived from keratinocytes overexpressing miR-378a-3p effectively mitigate skin fibrosis in a model of bleomycin-induced fibrosis. These findings suggest that utilizing exosomes derived from miR-378a-3p-overexpressing keratinocytes could potentially serve as a therapeutic approach for fibrotic conditions, including scleroderma and skin scarring.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the general study conception and design. Conceptualization and design were performed by YY and LZ. The research and data collection were performed by LY, GT, and BL. The data analysis and interpretation were finished by LY and CL. The first draft of the manuscript was written by YY, GT, YB, and JS. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was funded by the National Natural Science Foundation of China (No: 82072186, No: 82272265).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Animal Care and Use Committee of Southern Medical University (protocol number SMUL2022044).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are available on reasonable request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiu Y-X, Sun J-M, Ho C-K, Gao Y, Wen D-S, Liu Y-D, Huang L, Zhang Y-F (2023) Advancements in Adipose-Derived Stem Cell Therapy for Skin Fibrosis. 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Epigenomics 12:397\u0026ndash;408. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2217/epi-2019-0193\u003c/span\u003e\u003cspan address=\"10.2217/epi-2019-0193\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"miR-378a-3p, skin fibrosis, exosomes, TGF-β1, FSTL1","lastPublishedDoi":"10.21203/rs.3.rs-3829723/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3829723/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe association between microRNAs (miRNAs) and the development of skin fibrosis has been established. Consequently, the investigation of miRNA is crucial for the management of cutaneous fibrotic diseases. Recent investigations have linked miR-378a to several fibrosis diseases. Here, we discovered that miR-378a-3p effectively inhibited the proliferative, migratory, and COL1A1 expression effects induced by TGF-β1 in fibroblasts. Additionally, we found that miR-378a-3p exerts its anti-fibrotic properties by directly binding to FSTL1, a downstream factor involved in TGF-β1 activation. We have also demonstrated that long non-coding RNA (lncRNA) NORAD functions as a competing endogenous RNA (ceRNA) for miR-378a-3p, thereby activating the expression of FTSL1. Additionally, miR-378a-3p can be encapsulated within exosomes derived from keratinocytes and transferred to fibroblasts, resulting in an antagonistic effect against TGF-β1. The subcutaneous injection of miR-378a-3p-overexpressing keratinocytes-derived exosomes significantly attenuated skin fibrosis induced by bleomycin (BLM), as evidenced by reduced dermal thickening and decreased expression of COL1A1. Consequently, exosome-mediated delivery of miR-378a-3p holds promise as a potential therapeutic strategy for the treatment of skin fibrosis.\u003c/p\u003e","manuscriptTitle":"Exosomal miR-378a-3p derived from keratinocytes inhibits transforming growth factor beta-induced fibroblast activation and attenuates bleomycin-induced skin fibrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-05 13:20:57","doi":"10.21203/rs.3.rs-3829723/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"73329bf0-0c83-4a5a-b5be-9437c31cae5a","owner":[],"postedDate":"January 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-14T16:29:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-05 13:20:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3829723","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3829723","identity":"rs-3829723","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}