{"paper_id":"02d26158-4e6f-4fa3-a288-4d8be4dcada4","body_text":"Epithelial cell-derived exosomes carry NamiRNA-143-5p and promote ASPN expression in fibroblasts to drive Helicobacter pylori infected gastritis progression | 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 Epithelial cell-derived exosomes carry NamiRNA-143-5p and promote ASPN expression in fibroblasts to drive Helicobacter pylori infected gastritis progression Zheng Zhang, Shuyue Yang, Mengran Zhao, Wenjing Sun, Rui Xu, Anni Zhou, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7945968/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Feb, 2026 Read the published version in Gut Pathogens → Version 1 posted 11 You are reading this latest preprint version Abstract In Helicobacter pylori (H. pylori) associated gastritis, fibroblasts are recruited to inflammatory sites and secrete multiple pro-inflammatory cytokines, but the underlying mechanism remains unclear. Here, immunohistochemical staining revealed that ASPN expression was significantly upregulated in fibroblasts from H. pylori positive gastritis tissues, whereas its level remained unchanged in fibroblasts directly infected with H. pylori. Co-culture assays demonstrated that exosomes released from H. pylori infected epithelial cells induced the upregulation of ASPN and its downstream cytokines (IL-4, IL-6, and TGF-β) in fibroblasts. MicroRNA sequencing and correlation analyses identified miR-143-5p as an exosomal miRNA enriched after H. pylori infection that potentially regulates ASPN. Immunofluorescence confirmed that exosomes derived from epithelial cells carrying miR-143-5p were internalized by fibroblasts. Further immunofluorescence and immunohistochemical analyses showed nuclear accumulation of miR-143-5p in fibroblasts in both the H. pylori infected epithelial cell co-culture system and H. pylori positive gastritis tissues. Mechanistic studies demonstrated that miR-143-5p functions as a nuclear activating microRNA (NamiRNA-143-5p), binding to the super-enhancer region of ASPN, increasing H3K27ac enrichment, and promoting its transcription. In vivo, antagomir-143-5p reduced H. pylori induced ASPN and cytokine overexpression, thereby alleviating gastric inflammation. Thus, we conclude that exosomal NamiRNA-143-5p from H. pylori infected epithelial cells upregulate pro-inflammatory cytokines in fibroblasts by activating ASPN expression through a super-enhancer-dependent pathway, thereby positioning this axis as a potential therapeutic target for H. pylori associated gastric disease. Helicobacter pylori Exosome NamiRNA ASPN Fibroblast Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction H. pylori is a Group I carcinogen that drives the Correa cascade and contributes to peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma [ 1 , 2 , 3 ]. Activated stromal fibroblasts are emerging as key amplifiers of chronic gastric inflammation: they secrete IL-6, IL-4, IL-13, and TGF-β, facilitate neutrophil infiltration via ICAM-1, and undergo autocrine differentiation toward myofibroblasts [ 4 , 5 , 6 ]. Yet H. pylori typically colonizes the epithelial surface without invading the lamina propria, making direct microbe–fibroblast contact uncommon [ 7 , 8 ]. This creates a central unresolved question: how are fibroblasts activated and maintained in a pro-inflammatory state during H. pylori–associated gastritis? Asporin (ASPN), a small leucine-rich proteoglycan expressed in fibroblasts, modulates extracellular signaling and matrix biology in inflammatory disorders such as osteoarthritis and pulpitis [ 9 , 10 ]. Our prior work showed that ASPN is upregulated in gastric cancer and precursor lesions; intracellular ASPN binds LEF1 and selectively enhances transcription of inflammation-related genes (e.g., PTGS2, IL6, WISP1) and components of the IL-4/TGF-β axis [ 11 , 12 ]. These data nominate ASPN as a candidate driver of fibroblast-dependent inflammation in H. pylori gastritis. Whether ASPN is indeed induced in gastric fibroblasts during infection and functionally promotes disease progression remains unknown. Exosomes (30–150 nm) are efficient long-range messengers that transport nucleic acids, proteins, and lipids to reprogram recipient cells through membrane fusion, cargo release, or receptor interaction [ 13 , 14 ]. During H. pylori infection, gastric epithelial cells actively secrete exosomes enriched with small RNAs that function as key mediators of epithelial–recipient cell communication [ 15 , 16 , 17 ]. For instance, epithelial exosomal miR-25 targets KLF2 in endothelial cells, thereby activating vascular inflammatory signaling, while p-MET–bearing vesicles educate macrophages toward tumor-promoting and pro-inflammatory phenotypes [ 18 ]. Collectively, these findings support that H. pylori infection stimulates epithelial exosome production, enabling the transfer of internal signaling molecules to distant recipient cells and promoting downstream inflammatory mediator expression. Although most studies of exosomal miRNAs have focused on their traditional role as cytoplasmic repressors of translation, increasing evidence indicates that miRNA function varies considerably depending on their subcellular localization [ 19 , 20 , 21 ]. Recent research has revealed that several mature miRNAs translocate into the nucleus and directly modulate transcription [ 22 , 23 ]. For example, miR-195-5p targets the FOXO3 promoter through AGO2–TATA-box interaction to activate gene transcription [ 24 ], while miR-26a-1 and miR-24-1 act on enhancer regions, promoting H3K27ac enrichment and eRNA-driven activation of neighboring gene [ 25 ]. Building on ChIP-seq evidence, Xiao et al. defined nuclear activating miRNAs (NamiRNAs) that associate with H3K27ac-marked enhancers and nearby super-enhancers to stimulate gene expression on the same chromosome; NamiR-143 is among these candidates [ 23 , 26 ]. Subsequent studies demonstrate that NamiRNAs can transcriptionally activate disease-relevant genes in breast cancer [ 27 ]. Whether such enhancer-activating miRNAs operate in H. pylori gastritis, and specifically drive ASPN in fibroblasts, has not been addressed. Here, we report that both ASPN and NamiRNA-143-5p accumulate in gastric fibroblasts during H. pylori. H. pylori exposure increases the release of epithelial exosomes carrying NamiRNA-143-5p; these vesicles are internalized by fibroblasts, where nuclear NamiRNA-143-5p directly engages an ASPN enhancer to elevate its transcription. Elevated ASPN, in turn, augments downstream inflammatory mediators including IL-6, IL-4 and TGF-β. Together, these findings delineate a mechanistic epithelial-to-stromal route that reprograms fibroblasts via enhancer-targeting NamiRNAs, positioning ASPN as a transcriptionally controlled hub with biomarker and therapeutic potential. Materials and Methods Patients and tissue specimens A total of 150 biopsy specimens with complete clinicopathologic characteristics were used for IHC staining, including 50 cases of H. pylori negative gastric epithelial tissue, 50 cases of H. pylori positive superficial gastritis tissue, and 50 cases of H. pylori positive atrophic gastritis tissue. All of these tissues come from our own biobank. This study was conducted in compliance with the principles of the Declaration of Helsinki. Informed consent was obtained from all the subjects. Ethics approval for human subjects was provided by Ethics Committee of the Beijing Friendship Hospital, Capital Medical University. Immunohistochemistry Formalin-fixed paraffin-embedded sections were deparaffinized, rehydrated, and subjected to high-pressure antigen retrieval. Endogenous peroxidase was blocked with 3% H₂O₂ for 20 min, followed by serum blocking for 1 h. Slides were incubated overnight at 4°C with antibodies including anti-ASPN, anti-Vimentin, anti-IL4, anti-IL6(detailed in Table S1 : Information of primary antibodies used in WB and IHC), then with an HRP-conjugated anti-rabbit secondary antibody for 2 h at room temperature, and visualized using a DAB kit (ZSGB-Bio, Beijing, China). IHC staining was independently evaluated by two blinded pathologists. Intensity was scored as 0 (none), 1 (weak), 2 (moderate), or 3 (strong), and the percentage of positive cells as 1 (0–25%), 2 (26–50%), 3 (51–75%), or 4 (> 75%). The total score (0–12) was classified as negative (0), weak (1–4), moderate (5–8), or strong (9–12); scores ≥ 5 indicated high expression. Western Blotting Western Blotting Protein concentrations were determined using the Bicinchoninic Acid Assay Kit (Thermo Fisher, Massachu-setts). Equal amounts of denatured protein (50 µg per lane) were resolved by SDS–PAGE and transferred onto PVDF membranes. After blocking with 5% nonfat milk, membranes were incubated overnight at 4°C with primary an-ti-bodies against ASPN, IL6, IL4, TGF-β, GAPDH, GM130, CD63, TSG101, CD9 (de-tailed in Table S1 : Information of primary antibodies used in WB and IHC). The following day, membranes were exposed to HRP-conjugated secondary antibodies for 1 h at room temperature. Protein sig-nals were visualized using an enhanced chemiluminescence system (Bio-Rad, California). Quantitative real-time PCR (qRT-PCR) RNA extraction. Total RNA was isolated with TRIzol (DNase I treated as needed). Concentration/purity were checked spectrophotometrically; integrity by agarose gel. For EV samples, RNase-free procedures were used. mRNA qRT-PCR: cDNA was synthesized from 0.5–1.0 µg RNA using oligo(dT) ± random hexamers. SYBR Green qPCR (10–20 µL) was run on an ABI 7500 with 80–150 bp amplicons designed across exon–exon junctions. Cycling: 95°C 2 min; 40× (95°C 15 s, 60°C 30 s). GAPDH (or RPLP0; stability verified) was the ref-erence. Technical triplicates were used. miRNA qRT-PCR: Mature miRNAs (e.g., miR-143-5p) were quantified by stem-loop RT followed by SYBR qPCR using a miRNA-specific forward and universal reverse primer (amplicon ~ 60–80 bp). Cycling: 95°C 2 min; 40× (95°C 15 s, 60°C 30–60 s). Normalization: U6 for cells/tissues (stability verified); Controls & analysis. No-template and No-RT controls were included. Single-peak melt curves confirmed specificity. Relative expression was calculated by 2^-ΔΔCt with effi-ciency 90–110% (standard curves). All the primer sequences of mRNA and miRNA are provided in Table S2 : Information of primers used in RT-qPCR. Cell culture and co-culture system Two cell lines (GES-1 and primary fibroblasts) were used in the study. GES-1 was purchased from the National Infrastructure of Cell Line Resource of China. The human primary gastric fibroblast Cells (HUM-iCELL-d006) was purchased from Cellverse. GES-1 was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO2. The gastric fibroblasts were cultured in the special medium of PriMed-iCELL-003, which was produced by Cellverse. The cell lines ap-plied in the experiments underwent fewer than 5 passages and were authenticated using short tandem repeat analysis. Cells were cultured in permeable membrane in-serts (Transwell, Corning; 0.4-µm pores, polycarbonate, 12-mm) placed in companion plates to establish a two-compartment co-culture. GES-1 cells were seeded in the upper Transwell insert and gastric fibroblasts in the lower well; both compartments contained PriMed-iCELL-003 medium during co-culture. H. pylori strasins and infection with H. pylori H.pylori strains 26695 were inoculated in Columbia blood agar (OXOID, CM0331b) plates supplemented with 10% off fiber sheep blood and mixed antibiotics (vancomy-cin 10mg/L, amphotericin 5mg/L, trimethoprim 5mg/L, and polymyxin 5mg/L) under 37℃ microaerobic atmosphere by anaeropack (MGC, C-2). A total of 3×105 GES1 cells were seeded into 10cm culture dish with 10% exosome-free FBS (Wenren, EXO-FBS-50A-1). H.pylori strain 26695 was used for co-culture with GES1 cell line at 5 MOI for 96 h. We collect 48h and 96h mediun after the infection of H.pylori. We cultured gastric fibroblasts in the conditioned medium treated with a 0.22µm filter for 48h. sEV isolation To isolate vesicles, the conditioned medium was subjected to centrifugation at 8,500g 30min to remove cell debris, H.pylori and other impurities. Then the conditioned me-dium was subjected to ultracentrifugation at 120,000 × g for 70 min at 4℃, followed by a single wash with phosphate-buffered saline (PBS), and the pellet was resus-pended in 200 µL PBS. EVs were stored at -80℃ before use. Nanoparticle Tracking Analysis of exosome Nanoparticle tracking analysis (NTA) was performed on supernatants after 10,000 × g centrifugation using a NanoSight LM10 (Nanosight, UK). About 0.3 mL sample was loaded, and three 30–60 s videos were recorded. Data were analyzed with NTA soft-ware (v1.1/2.1) to calculate particle size and concentration via the Stokes–Einstein equation. Instrument settings were optimized with 100–400 nm calibration beads (Duke Scientific, USA). Biological samples were measured at 15–30 ms shutter speed and camera gain 280–560. Results are presented as mean ± SD of triplicates, with reli-able quantification between 2 × 10^8 and 2 × 10^9 particles/mL; more concentrated samples were diluted accordingly. Immunofluorescence To assess the subcellular localization of PKH67, Cy3 and β-actin, gastric fibroblasts were cultured on sterile coverslips placed in 6-well plates. After three washes with PBS, cells were fixed in 4% paraformaldehyde for 15 min and permea-bilized with 0.25% Triton X-100 in PBS. Non-specific binding was blocked using 5% BSA in PBST for 1 h. Samples were then incubated overnight at 4°C with primary antibodies against β-actin diluted in PBS, followed by exposure to Alexa Fluor 488–conjugated anti-mouse IgG (Life Technolo-gies, MA) for 2 h in the dark. Nuclei were counter-stained with DAPI, and images were acquired using a confocal microscope (IX83, FLUOVIEW FV1200; Olympus, Japan). Exosomes miRNA sequencing Following the isolation of total RNA (containing the small RNA fraction) from purified exosomes of H. pylori-infected and control GES-1 cells, high-throughput sequencing was performed by Majorbio (Shanghai, China). This process included RNA quality assessment, library preparation, and sequencing on HiSeq/MiSeq platforms, all conducted as per the manufacturer's instructions. In situ hybridization ISH was performed on 5 µm paraffin sections using a Tecan Genepaint system as de-scribed [ 28 ]. Sections were treated with proteinase K (25 µg/mL, 37°C, 8 min) and hy-bridized in Exiqon buffer with double-FAM–labeled LNA probes for miR-143, or scramble controls at 55–57°C. After SSC washes, blocking was carried out with PBS con-taining 0.1% Tween, 4% sheep serum, and 1% BSA. Probes were detected with AP-conjugated anti-FAM Fab frag-ments (1:800, Roche), followed by NBT/BCIP sub-strate with 0.25 mM levamisole for 60 min. Slides were counter-stained with Nuclear Fast Red, dehydrated, and mounted. After multiple sections of the same part of the specimen, the in situ hybridization results of miR-143 were compared with the IHC staining results of VIM. Luciferase reporter assay PGL3 reporter plasmids containing the DNA fragments (about 1000bp), which is the miR-143-5p genome locus in the upstream 20 kb of ASPN, as well as a luciferase re-porter gene, named as pGL3-miR143, were obtained from YouBio (China). gastric fibroblasts (4 × 10^4 per well) were seeded into 24-well plates and cotransfected with 800 ng of pro-mot-er-reporter plasmid, 200 ng of pRL-TK plasmid, using Lipofectamine 3000 (Invi-trogen), following the manufactur-er’s protocol. After 36 h of incubation at 37°C, firefly and Renilla luciferase activities were measured with the Du-al-Luciferase Reporter Assay System (Thermo Fisher, MA). Firefly signals were normalized to Renilla activity for each sample. Chromatin immunoprecipitation assay (ChIP) ChIP was performed using the Pierce™ Magnetic ChIP Kit (Thermo Fisher) according to the manufacturer’s protocol. After 3 days transfected with miR-143-5p mimic or in-hibitor, gastric fibroblasts were cultured to ~ 80% confluence and crosslinked with 1% formaldehyde for 10 min. Genomic DNA was fragmented into 300–1000 bp fragments using a Bio-ruptor® Pico sonicator, and chromatin supernatants were incubated overnight at 4°C with either anti-H3K27ac or control IgG antibody under rotation. DNA recovered from magnetic beads was analyzed by qPCR with special primer pairs (listed in Table S3 : Information of primers used in ChIP-PCR). Animal care and treatment All mouse studies were performed with approval by Institutional Animal Care and Use Committee (IACUC) of Tsinghua University, Beijing, China. Six- to eight-week-old mice were modeled for infection or infected with H. pylori via oral gavage. Food was withdrawn the night before the procedure, and water was removed at 8:00 AM the next morning. The oral gavage experiment commenced at 12:00 PM, and after con-firming no abnormalities at 4:00 PM, food and water were restored. The mock-infected group received 500 µL of sterile Brucella broth, while the H. pylori infected group was inoculated with 500 µL of Brucella broth containing 1×10⁹ CFU of H. pylori. The in-ocu-lations were administered twice, with a 24-hour interval between doses. Both male and female mice were included in the H. pylori infection study. Antagomir-143-5p and Antagomir-NC were purchased in GenePharma and 50nmol/100ul per mice were in-jected though tail vein. Statistical Analysis Data represent the mean ± SD (standard deviation); all statistical analyses were performed using GraphPad Prism and R Software. Statistical tests were one-sided or two-sided, and Mann-Whitney U-test and Wilcoxon matched-pairs test were conducted to evaluate the differences between two groups, while ANOVA was used to compare multiple groups. P < 0.05 was considered significant. Results H. pylori infection elevated ASPN levels in gastric fibroblasts Building on the aberrant elevation of ASPN in gastric cancer observed in our previous research and its emerging role as a crucial fibroblast-derived modulator in matrix-associated inflammation, we first examined its expression in H. pylori related pre-neoplastic lesions. Immunohistochemistry (IHC) was performed on 50 cases each of H. pylori negative normal gastric mucosa (NT), H. pylori positive non-atrophic gastritis (NAG), and H. pylori positive atrophic gastritis (AG). ASPN expression was significantly higher in both H. pylori positive NAG and AG compared with NT (p < 0.01), whereas no difference was observed between NAG and AG. Importantly, ASPN staining was predominantly localized to mesenchymal cells, such as fibroblasts, rather than epithelial compartments (Fig. 1 A, B). To further confirm fibroblast localization, IHC for the stromal marker Vimentin (VIM) revealed a marked accumulation of fibroblasts in H. pylori infected tissues compared with H. pylori negative controls (p < 0.01, Fig. 1 C, D). Strong staining for both ASPN and VIM in H. pylori associated gastritis specimens supports fibroblasts as the primary source of ASPN. Western blotting (WB) analysis of tissue lysates corroborated the IHC findings, showing elevated ASPN protein levels in H. pylori positive NAG and AG relative to NT (Fig. 1 E). Consistently, qRT-PCR demonstrated increased ASPN mRNA levels in H. pylori positive states (Fig. 1 F). However, when primary gastric fibroblasts were directly infected with H. pylori, neither WB nor qRT-PCR showed changes in ASPN expression (Fig. 1 G, H). Together, these results indicates that ASPN upregulation in H. pylori associated gastritis occurs within fibroblasts in vivo, but is not directly induced by bacterial infection of fibroblasts. Exosomes from H. pylori infected GES-1 cells could induce upregulation of ASPN signaling in fibroblasts To further detect the mechanism of the overexpression of ASPN in gastric fibroblasts, we first conducted immunofluorescence (IF) staining of VIM to confirm cell purity of the primary fibroblasts above 90% (Fig. 2 A, left). Then, we established a Transwell co-culture system with a 0.4 µm pore membrane to prevent cell migration (Fig. 2 A, right). H. pylori infected GES-1 cells (upper chamber) were co-cultured with fibroblasts (lower chamber) for 48 h. Compared with controls using H. pylori negative GES-1 cells, WB analysis showed that fibroblasts co-cultured with H. pylori infected epithelial cells exhibited increased ASPN protein expression, accompanied by upregulation of IL-6, IL-4, and TGF-β (Fig. 2 B). Consistent with our prior GSEA analysis linking ASPN with IL-4 and TGF-β signaling pathways (Fig. 2 C), overexpression of ASPN in fibroblasts enhanced the expression of these inflammatory cytokines (Fig. 2 D). Together, these results demonstrate that H. pylori infected epithelial cells can modulate ASPN and downstream inflammatory mediators in fibroblasts through indirect mechanisms. To identify the mediator of this epithelial–fibroblast crosstalk, exosomes were isolated from the supernatant of H. pylori infected GES-1 cells using ultracentrifugation. Transmission electron microscopy revealed vesicles of ~ 100 nm with characteristic cup-shaped, double-membrane morphology (Fig. 2 E). Western blot confirmed enrichment of exosome markers CD63, TSG101, and CD9 in the EV fractions, while the Golgi marker GM130 was absent, validating exosome purity (Fig. 2 F). Equal amounts of EVs from H. pylori positive or H. pylori negative GES-1 cells were added to fibroblast cultures. After 24–36 h, qRT-PCR analysis revealed increased ASPN, IL-6, IL-4, and TGF-β mRNA levels in fibroblasts treated with H. pylori positive EVs compared with controls (Fig. 2 H). Collectively, these findings indicate that exosomes derived from H. pylori infected epithelial cells mediate the upregulation of ASPN and its downstream inflammatory genes in gastric fibroblasts at both transcriptional and protein levels. H. pylori infection promoted exosomes transferring into fibroblasts and the overexpression of exosomal miR-143-5p To determine the effect of H. pylori infection on exosome secretion, we performed nanoparticle tracking analysis (NTA) of exosomes isolated from GES-1 cells. Both the average particle count and the total protein content were significantly higher in the H. pylori infected group than in H. pylori negative controls (Fig. 3 A). Exosomes derived from equal numbers of H. pylori positive or H. pylori negative GES-1 cells were then labeled with PKH67 and co-cultured with gastric fibroblasts. IF assays revealed exosome uptake by gastric fibroblasts, with significantly stronger fluorescence intensity observed in the H. pylori positive group (Fig. 3 B, C). These data indicate that H. pylori infection enhances exosome production by epithelial cells and promotes their uptake by fibroblasts. To identify the cargo responsible for ASPN induction, exosomes from H. pylori positive and H. pylori negative GES-1 cells were analyzed by small RNA sequencing. Approximately 400 miRNAs were detected, among which 102 were differentially expressed (50 upregulated, 52 downregulated; Fig. 3 D, screening criteria: FDR < 0.14, p < 0.05). A heatmap of the top 60 differentially expressed miRNAs highlighted miR-143-5p (Fig. 3 E). Correlation analysis across multiple cancer datasets (miRactDB, https://ccsm.uth.edu/miRactDB/index.html ) showed a strong positive association between miR-143 and ASPN expression in more than ten cancer types, including esophageal, gastric, colorectal, and liver cancers (Fig. 3 F). These results suggest that miR-143-5p is enriched in exosomes from H. pylori infected epithelial cells and may regulate ASPN expression in fibroblasts. To validate the sequencing data, qRT-PCR was performed on GES-1 cells infected with H. pylori for 24 or 48 h. miR-143-5p expression was significantly higher at 48 h compared with H. pylori negative controls (Fig. S1 A). Consistently, qRT-PCR analysis of exosomes secreted by H. pylori infected cells confirmed elevated miR-143-5p levels (Fig. 3 G). In co-culture assays, gastric fibroblasts exposed to H. pylori positive GES-1 cells exhibited higher intracellular miR-143-5p levels than those co-cultured with H. pylori negative cells (Fig. 3 H). Furthermore, Cy3-labeled miR-143-5p transfected into GES-1 cells was detected in gastric fibroblasts nuclei using a Transwell system, confirming direct uptake of miR-143-5p by fibroblasts (Fig. S1 B). Collectively, these findings demonstrate that H. pylori infection increases both the release of exosomes from epithelial cells into fibroblasts and the level of exosomal miR-143-5p, which may contribute to ASPN upregulation. H. pylori infection enhanced exosomal miR-143-5p transferring into nuclei of fibroblasts To further elucidate whether miR-143-5p could be carried by exosomes from H. pylori infected GES-1 cells entered gastric fibroblasts and promotes downstream inflammatory factor expression, we conducted a series of experiments. H. pylori positive GES-1 cells were transfected with Cy3-labeled miR-143-5p and co-cultured with gastric fibroblasts. Control conditions included H. pylori negative GES-1 cells transfected with Cy3-miR-143-5p and Cy3 alone. After 48h co-culture, IF assays in gastric fibroblasts revealed the strongest intracellular Cy3 signal in group of Cy3-labeled miR-143-5p, with a portion of the fluorescence localizing to nuclei, whereas transfer efficiency was reduced under H. pylori negative conditions (Fig. 4 A, B). Following co-culture with H. pylori infected GES-1 cells, nuclear–cytoplasmic fractionation was performed in gastric fibroblasts. Stem-loop qRT-PCR showed a significant increase of miR-143-5p in the nuclear fraction under H. pylori infection (p < 0.01; Fig. 4 C). In patient tissues, in situ hybridization (ISH) showed elevated nuclear miR-143-5p in H. pylori positive specimens compared with H. pylori negative controls (Fig. 4 D, E). Stratified analysis further indicated a more pronounced increase in the stromal compartment than in the epithelium under H. pylori infection, suggesting that H. pylori not only increased miR-143-5p levels but also promotes its transfer into stromal regions (Fig. 4 F). All above results prove that upon H. pylori infection, exosomal miR-143-5p derived from GES-1 cells is delivered to fibroblasts and ultimately localizes to the nucleus. Nuclear miR-143-5p (NamiRNA-143-5p) promoted ASPN expression though binding to super-enhancer sites For further exploring the function and mechanism of nuclear miR-143-5p, a miR-143-5p inhibitor was added to EV-conditioned medium from H. pylori positive GES-1 cells prior to co-culture with gastric fibroblasts, whereas synthetic miR-143-5p was added to medium from H. pylori negative cells (with matched controls). Gastric fibroblasts lysates were subsequently analyzed by WB analysis. Exogenous miR-143-5p enhanced ASPN and downstream inflammatory mediators in the H. pylori negative EV condition, whereas the miR-143-5p inhibitor attenuated the promotive effect of H. pylori positive EVs on ASPN and downstream proteins in gastric fibroblasts (Fig. 5 A). Given the nuclear localization of miR-143-5p in gastric fibroblasts, we hypothesized a NamiRNA mechanism. Sequence alignment between miR-143-5p and the ASPN SE region in fibroblasts (SEdb v3.0 entry SE_02_214900539) predicted strong binding potential (Fig. 5 B), suggesting that miR-143-5p acts as a NamiRNA (NamiRNA-143-5p) to enhance transcription. To test this, a 700-bp ASPN enhancer fragment harboring the predicted miR-143-5p site was cloned into pGL3 (pGL3-miR143). Compared with empty pGL3, transfection into gastric fibroblasts significantly increased the Firefly/Renilla luciferase activity ratio (Luc+/Rluc+) (Fig. 5 C). Furthermore, chromatin immunoprecipitation (ChIP) with an H3K27ac antibody in gastric fibroblasts overexpressing NamiRNA-143-5p showed enriched H3K27ac at primers flanking the predicted binding site, relative to empty-vector controls (Fig. 5 D). We then generated dual-reporter constructs carrying either the WT ASPN enhancer (wt-pGL3-miR143) or a mutated site (mut-pGL3-miR143), and pSUPER vectors expressing either WT NamiRNA-143-5p (wt-pSR-miR143) or a binding-defective mutant (mut-pSR-miR143). Mutations in mut-pGL3-miR143 and mut-pSR-miR143 were designed as complementary pairs to allow interaction rescue. Pairwise co-transfections in HEK293T cells yielded four conditions for Luc+/Rluc + comparison. As shown in Fig. 5 E: (a) wt-pGL3-miR143 + wt-pSR-miR143 significantly increased the level of Luc+/Rluc + versus control (wt-pGL3-miR143 + empty pSUPER); (b–d) mutating either the enhancer (mut-pGL3-miR143 + wt-pSR-miR143) or the miRNA seed (wt-pGL3-miR143 + mut-pSR-miR143) reduced the activity of luciferease; (e) co-transfection of complementary mutants (mut-pGL3-miR143 + mut-pSR-miR143) restored the activity. Collectively, these data demonstrate that EV-delivered NamiRNA-143-5p enters gastric fibroblasts nuclei, binds the ASPN SE, and activates ASPN transcription, thereby driving downstream inflammatory factor expression. Antagomir of NamiRNA-143-5p alleviated H. pylori induced gastric inflammation in vivo To assess the in vivo expression pattern and pro-inflammatory function of NamiRNA-143-5p, we established a mouse H. pylori infection model. Infection status was confirmed by Steiner silver staining (Fig. 6 A). ISH revealed markedly increased nuclear NamiRNA-143-5p in H. pylori positive stomachs versus H. pylori negative controls, with a more pronounced elevation in the stromal compartment than in the epithelium (Fig. 6 B). In addition, ASPN also was significantly elevated in H. pylori infected gastric stromal (Fig. 6 C). To test functional relevance, NamiRNA-143-5p antagomir was administered via tail vein to H. pylori positive mice. IHC showed higher IL-6 and IL-4 in H. pylori positive mucosa relative to H. pylori negative tissue, whereas antagomir-143 reduced these cytokines, particularly within stromal regions (Fig. 6 D, Fig. S2 A). Consistently, WB of gastric lysates across four groups (H. pylori−, H. pylori+, H. pylori+/antagomir-NC, H. pylori+/antagomir-143) demonstrated that miR-143-5p antagomir attenuated H. pylori induced upregulation of ASPN, IL-4, IL-6, and TGF-β (Fig. 6 E). As shown in the schematic figure (Fig. 6 F), all our data suggested that H. pylori infection could promote the secretion of exosomal NamiRNA-143-5p from gastric epithelium transferring to fibroblasts and then activating ASPN expression by binding to its SE site to drive gastritis progression. Discussion This study defines an epithelial–stromal communication axis whereby H. pylori indirectly program a pro-inflammatory gastric fibroblasts phenotype. H. pylori infected epithelial cells release EVs enriched for NamiRNA-143-5p, which accumulates in gastric fibroblasts nuclei, engages an ASPN SE, elevates H3K27ac, and activates ASPN transcription, in turn upregulating IL-6, IL-4, and TGF-β. ASPN is increased in H. pylori positive gastritis and localizes to the stroma, while direct H. pylori exposure does not induce ASPN in primary gastric fibroblasts, indicating an indirect mechanism. Together, these findings illuminate how epithelial infection is transduced to stromal activation during H. pylori associated inflammation. Our data position gastric fibroblasts as active amplifiers of chronic gastric inflammation. Prior reports implicated fibroblast activation and aggregation in H. pylori associated gastritis and tissue remodeling [ 29 , 30 ]. We extend this by identifying ASPN as a fibroblast-centered effector whose upregulation is driven by EV-mediated NamiRNA transfer rather than direct bacterial contact. Although a gastric fibroblasts-specific marker was not available, VIM IHC supported stromal-including gastric fibroblasts-ASPN elevation [ 31 , 32 ], and co-culture experiments verified increased ASPN and downstream cytokines when GES-1 cells are H. pylori infected. Beyond EV quantity, cargo matters: NamiRNA-143-5p acts within recipient nuclei, consistent with NamiRNAs that activate transcription via enhancer/SE binding [ 27 ]. Concordant spatial signals (VIM/ASPN by IHC and miR-143-5p by ISH), ChIP evidence of H3K27ac enrichment, and complementary-mutation luciferase rescue collectively support direct engagement of the ASPN SE despite modest predicted base-pairing compared with other contexts. Functionally, ASPN links H. pylori colonization to stromal remodeling and inflammatory circuitry. Together with our prior work showing intracellular ASPN cooperates with LEF1 to enhance transcription of PTGS2, IL6, and WISP1 [ 23 ], the present findings suggest the NamiRNA-143-5p–ASPN axis may help drive the transition from acute to chronic/atrophic gastritis and ultimately a tumor-permissive microenvironment. Translationally, tissue or circulating EV levels of NamiRNA-143-5p and ASPN merit evaluation as biomarkers to stratify H. pylori positive individuals at risk for progression. Therapeutically, disrupting EV biogenesis/uptake, NamiRNA cargo loading, SE engagement, or downstream ASPN signaling are plausible strategies[ 33 ]. The in vivo attenuation of ASPN and cytokines by antagomir-143 provides preliminary proof-of-concept, which is particularly relevant given the high H. pylori burden in East Asia and limitations of eradication therapy[ 34 ]. Limitations include the potential contribution of other EV miRNAs/non-coding RNAs; incomplete delineation of nuclear cofactors and chromatin partners that mediate NamiRNA-143-5p–SE activation; and unresolved determinants of EV tropism and uptake by gastric fibroblasts. Larger human cohorts-deally integrating biofluid EV profiling and longitudinal outcomes-are needed to validate biomarker utility and therapeutic targeting. In summary, H. pylori infection stimulates gastric epithelial cells to secrete exosomes enriched with nuclear-activating miR-143-5p (NamiR-143-5p). These vesicles are internalized by fibroblasts, where NamiR-143-5p binds an ASPN super-enhancer, elevating H3K27ac enrichment and driving transcription of ASPN and its downstream cytokines (Fig. 6 F). The resulting ASPN–IL-6/IL-4/TGF-β axis amplifies stromal inflammation, contributing to gastritis progression. Targeting this exosome-NamiR-143-5p-ASPN pathway may offer a promising therapeutic strategy against H. pylori associated gastric disease. Declarations Author information Zheng Zhang and Shuyue Yang have contributed equally to this work. Authors and Affiliations Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, State Key Laboratory of Digestive Health, National Clinical Research Center for Digestive Disease, Beijing Key Laboratory of Early Gastrointestinal Cancer Medicine and Medical Devices Zheng Zhang, Mengran Zhao, Wenjing Sun, Anni Zhou, Sifan Liu, Mingyang Ma, Peng Li Department of Gastroenterology, Tianjin Union Medical Center, the First Affiliated Hospital of Nankai University Shuyue Yang Department of Pathology, Beijing Friendship Hospital, Capital Medical University Rui Xu Contributions Zheng Zhang, Shuyue Yang, Wenjing Sun, Mingyang Ma, and Sifan Liu performed the research and collected the data. Zheng Zhang and Shuyue Yang prepared the manuscript. Rui Xu reviewed and interpreted the pathology data. Zheng Zhang, Mengran Zhao, Anni Zhou, and Peng Li designed the study and supervised the study. Peng Li supervised the research and help to revised the manuscript. All authors have read and agreed to the published version of the manuscript. Corresponding author Correspondence to Peng Li or Zheng Zhang. Ethics Statement This study was conducted in compliance with the principles of the Declaration of Helsinki. Ethics approval for human subjects was provided by Ethics Committee of the Beijing Friendship Hospital, Capital Medical University. (DR20210109). All mouse studies were performed with approval by Institutional Animal Care and Use Committee (IACUC) of Tsinghua University(22-ZB1), Beijing, China. Competing interests The authors declare no conflicts of interest. Funding This study was supported by National Natural Science Foundation of China (No. 82200622) and Beijing Natural Science Foundation (No. 2332028) Author Contribution Zheng Zhang, Shuyue Yang, Wenjing Sun, Mingyang Ma, and Sifan Liu performed the research and collected the data. Zheng Zhang and Shuyue Yang prepared the manuscript. Rui Xu reviewed and interpreted the pathology data. Zheng Zhang, Mengran Zhao, Anni Zhou, and Peng Li designed the study and supervised the study. Peng Li supervised the research and help to revised the manuscript. All authors have read and agreed to the published version of the manuscript. Data Availability All data supporting the findings of this study are available within the paper and its Supplementary Information. References Amieva M, Peek RM. Jr. Pathobiology of Helicobacter pylori-Induced Gastric Cancer. Gastroenterology. 2016;150(1):64–78. Noto JM, Peek RM Jr. The gastric microbiome, its interaction with Helicobacter pylori, and its potential role in the progression to stomach cancer. PLoS Pathog. 2017;13(10):e1006573. LeBleu VS, Neilson EG. Origin and functional heterogeneity of fibroblasts. FASEB J. 2020;34(3):3519–36. Kendall RT, Feghali-Bostwick CA. Fibroblasts in fibrosis: novel roles and mediators. Front Pharmacol. 2014;5:123. Croft AP, Campos J, Jansen K, Turner JD, Marshall J, Attar M, et al. Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature. 2019;570(7760):246–51. Mescher AL. Macrophages and fibroblasts during inflammation and tissue repair in models of organ regeneration. Regeneration (Oxf). 2017;4(2):39–53. Chen X, Chen W, Zhao Y, Wang Q, Wang W, Xiang Y, et al. Interplay of Helicobacter pylori, fibroblasts, and cancer cells induces fibroblast activation and serpin E1 expression by cancer cells to promote gastric tumorigenesis. J Transl Med. 2022;20(1):322. Wang YK, Li C, Zhou YM, Zeng L, Li YY, Huang SL, et al. Histopathological Features of Helicobacter pylori Infection in Gastric Mucosa. J Inflamm Res. 2022;15:6231–43. Zhao Z, Wijerathne H, Godwin AK, Soper SA. Isolation and analysis methods of extracellular vesicles (EVs). Extracell Vesicles Circ Nucl Acids. 2021;2(1):80–103. Xu Y, Yu Y, Yang B, Hui J, Zhang C, Fang H, et al. Extracellular Mitochondrial Components and Effects on Cardiovascular Disease. DNA Cell Biol. 2021;40(9):1131–43. Liu YJ, Wang C. A review of the regulatory mechanisms of extracellular vesicles-mediated intercellular communication. Cell Commun Signal. 2023;21(1):77. Wang C, Li W, Shao L, Zhou A, Zhao M, Li P, et al. Both extracellular vesicles from helicobacter pylori-infected cells and helicobacter pylori outer membrane vesicles are involved in gastric/extragastric diseases. Eur J Med Res. 2023;28(1):484. Xia X, Zhang L, Chi J, Li H, Liu X, Hu T, et al. Helicobacter pylori Infection Impairs Endothelial Function Through an Exosome-Mediated Mechanism. J Am Heart Assoc. 2020;9(6):e014120. Li N, Liu SF, Dong K, Zhang GC, Huang J, Wang ZH, et al. Exosome-Transmitted miR-25 Induced by H. pylori Promotes Vascular Endothelial Cell Injury by Targeting KLF2. Front Cell Infect Microbiol. 2019;9:366. Zou Q, Liang Y, Luo H, Yu W. miRNA-Mediated RNAa by Targeting Enhancers. Adv Exp Med Biol. 2017;983:113–25. Xiao M, Li J, Li W, Wang Y, Wu F, Xi Y, et al. MicroRNAs activate gene transcription epigenetically as an enhancer trigger. RNA Biol. 2017;14(10):1326–34. Liang Y, Xu P, Zou Q, Luo H, Yu W. An epigenetic perspective on tumorigenesis: Loss of cell identity, enhancer switching, and NamiRNA network. Semin Cancer Biol. 2019;57:1–9. Liang Y, Lu Q, Li W, Zhang D, Zhang F, Zou Q, et al. Reactivation of tumour suppressor in breast cancer by enhancer switching through NamiRNA network. Nucleic Acids Res. 2021;49(15):8556–72. Li W, Yang S, Xu P, Zhang D, Tong Y, Chen L, et al. SARS-CoV-2 RNA elements share human sequence identity and upregulate hyaluronan via NamiRNA-enhancer network. EBioMedicine. 2022;76:103861. Ikegawa S. Expression, regulation and function of asporin, a susceptibility gene in common bone and joint diseases. Curr Med Chem. 2008;15(7):724–8. Fan R, Yan X, Zhang W. Relationship between asporin and extracellular matrix behavior: A literature review. Med (Baltim). 2022;101(51):e32490. Li H, Zhang Z, Chen L, Sun X, Zhao Y, Guo Q, et al. Cytoplasmic Asporin promotes cell migration by regulating TGF-beta/Smad2/3 pathway and indicates a poor prognosis in colorectal cancer. Cell Death Dis. 2019;10(2):109. Zhang Z, Min L, Li H, Chen L, Zhao Y, Liu S, et al. Asporin represses gastric cancer apoptosis via activating LEF1-mediated gene transcription independent of beta-catenin. Oncogene. 2021;40(27):4552–66. Xu R, Yang L, Zhang Z, Liao Y, Yu Y, Zhou D, et al. Cancer-associated fibroblast related gene signature in Helicobacter pylori-based subtypes of gastric carcinoma for prognosis and tumor microenvironment estimation in silico analysis. Front Med (Lausanne). 2023;10:1079470. Ren X, Liu G, Zhou J. Nuclear-activating miRNAs: unveiling the intricacies of subcellular miRNA function and regulation in cancer and immunity disease. Cancer Cell Int. 2025;25(1):147. Kuo YT, Liou JM, El-Omar EM, Wu JY, Leow AHR, Goh KL, et al. Primary antibiotic resistance in Helicobacter pylori in the Asia-Pacific region: a systematic review and meta-analysis. Lancet Gastroenterol Hepatol. 2017;2(10):707–15. Wei H, Chen Q, Lin L, Sha C, Li T, Liu Y, et al. Regulation of exosome production and cargo sorting. Int J Biol Sci. 2021;17(1):163. Xie S, Zhang Q, Jiang L. Current knowledge on exosome biogenesis, cargo-sorting mechanism and therapeutic implications. Membranes. 2022;12(5):498. Zhao L, Liu W, Xiao J, Cao B. The role of exosomes and exosomal shuttle microRNA in tumorigenesis and drug resistance. Cancer Lett. 2015;356(2):339–46. Liu Q, Wang J, Zhao Y, Li C-I, Stengel KR, Acharya P, et al. Identification of active miRNA promoters from nuclear run-on RNA sequencing. Nucleic Acids Res. 2017;45(13):e121–e. Billi M, De Marinis E, Gentile M, Nervi C, Grignani F. Nuclear miRNAs: gene regulation activities. Int J Mol Sci. 2024;25(11):6066. Bai Y, Pan B, Zhan X, Silver H, Li J. MicroRNA 195-5p targets Foxo3 promoter region to regulate its expression in granulosa cells. Int J Mol Sci. 2021;22(13):6721. Anderson CM, Zhang B, Miller M, Butko E, Wu X, Laver T, et al. Fully automated RNAscope in situ hybridization assays for formalin-fixed paraffin‐embedded cells and tissues. J Cell Biochem. 2016;117(10):2201–8. Gonciarz W, Krupa A, Hinc K, Obuchowski M, Moran AP, Gajewski A, et al. The effect of Helicobacter pylori infection and different H. pylori components on the proliferation and apoptosis of gastric epithelial cells and fibroblasts. PLoS ONE. 2019;14(8):e0220636. Additional Declarations No competing interests reported. Supplementary Files supplementarytables.docx Supplemetaryfigures.pdf miRNAseqdata.xlsx fulluncroppedGelsandBlotsimages.pdf Cite Share Download PDF Status: Published Journal Publication published 21 Feb, 2026 Read the published version in Gut Pathogens → Version 1 posted Editorial decision: Revision requested 05 Jan, 2026 Reviews received at journal 02 Jan, 2026 Reviews received at journal 22 Dec, 2025 Reviews received at journal 08 Dec, 2025 Reviewers agreed at journal 08 Dec, 2025 Reviewers agreed at journal 07 Dec, 2025 Reviewers agreed at journal 04 Dec, 2025 Reviewers invited by journal 12 Nov, 2025 Editor assigned by journal 27 Oct, 2025 Submission checks completed at journal 27 Oct, 2025 First submitted to journal 25 Oct, 2025 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. 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14:56:15\",\"extension\":\"xml\",\"order_by\":19,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"acdc-reference\",\"size\":106465,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"537285101cd44196b912dd647c1679eb1structuring.xml\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/05d3cea3ba95ab1211740ef7.xml\"},{\"id\":96709268,\"identity\":\"2d0949da-3f07-42f2-a4a7-78742b14d8a7\",\"added_by\":\"auto\",\"created_at\":\"2025-11-25 10:08:33\",\"extension\":\"html\",\"order_by\":20,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"acdc-reference\",\"size\":117070,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"earlyproof.html\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/c87ef1861fe3bdfa92a166ad.html\"},{\"id\":96642726,\"identity\":\"5df1de42-0403-42b9-8541-b2416042b1d3\",\"added_by\":\"auto\",\"created_at\":\"2025-11-24 14:56:14\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":533543,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eH. pylori infection elevated ASPN levels in gastric fibroblasts. (\\u003cstrong\\u003eA\\u003c/strong\\u003e) IHC staining of ASPN in H. pylori negative normal gastric tissues (NT), H. pylori positive non-atrophic gastritis (NAG) tissues, and H. pylori positive atrophic gastritis (AG) tissues. (\\u003cstrong\\u003eB\\u003c/strong\\u003e) Analysis of the IHC scores of ASPN (50cases per group). (\\u003cstrong\\u003eC\\u003c/strong\\u003e) IHC staining of Vimentin (VIM) in H. pylori- NT, H. pylori+ NAG and H. pylori+ AG. (\\u003cstrong\\u003eD\\u003c/strong\\u003e) Analysis of the IHC scores of VIM (50cases per group). (\\u003cstrong\\u003eE\\u003c/strong\\u003e) WB analysis of ASPN in two NTs and two H. pylori+ NAGs (top); two NTs and two H. pylori+ AGs (bottom); and the relative gray value (right). (\\u003cstrong\\u003eF\\u003c/strong\\u003e) RT-qPCR analysis of ASPN in the three groups of H. pylori- NT, H. pylori+ NAG and H. pylori+ AG. (\\u003cstrong\\u003eG\\u003c/strong\\u003e) WB analysis of ASPN in H. pylori negative gastric fibroblasts (GF) and H. pylori infected GF (MOI 25-50-100). (\\u003cstrong\\u003eH\\u003c/strong\\u003e) RT-qPCR analysis of ASPN in the above cells. Values are presented as the mean±SD. **P\\u0026lt;0.01, ***P\\u0026lt;0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/2d38a35e6a3449d32085a1e9.png\"},{\"id\":96642727,\"identity\":\"e98fc13e-95fb-47e7-93e0-fdf6201d4e64\",\"added_by\":\"auto\",\"created_at\":\"2025-11-24 14:56:15\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":398626,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eExosomes from H. pylori infected epithelial cells induced ASPN signaling in fibroblasts. \\u003cstrong\\u003e(A) \\u003c/strong\\u003eIF staining of VIM in GF (left). A schematic diagram of the GES-1 and GF co-culture system using a Transwell chamber with a pore size of 0.4 μm (right). (\\u003cstrong\\u003eB) \\u003c/strong\\u003eWB analysis of ASPN, IL6, IL4 and TGF-β using the above co-culture system. (\\u003cstrong\\u003eC) \\u003c/strong\\u003eGSEA analysis indicated positive associations between ASPN and IL4 signaling (top), TGF-β signaling (bottom). (\\u003cstrong\\u003eD) \\u003c/strong\\u003eWB analysis of ASPN, IL6, IL4 and TGF-β in GF with overexpression of ASPN. (\\u003cstrong\\u003eE) \\u003c/strong\\u003eElectron micrographs of exosomes secreted by H. pylori- and H. pylori+ GES-1 cells\\u003cstrong\\u003e.\\u003c/strong\\u003e(\\u003cstrong\\u003eF) \\u003c/strong\\u003eWB analysis of GM130, CD63, TSG101, and CD9 to validate exosome isolation technique. (\\u003cstrong\\u003eG) \\u003c/strong\\u003eRT-qPCR analysis of ASPN, IL6, IL4 and TGF-β in GF with H. pylori- and H. pylori+ EVs of GF. Values are presented as the mean±SD. *P\\u0026lt;0.05, **P\\u0026lt;0.01, ***P\\u0026lt;0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/22753531d970b9c3959a3e6a.png\"},{\"id\":96642731,\"identity\":\"cad0afe3-fa8f-4ae9-b5a5-b18159590747\",\"added_by\":\"auto\",\"created_at\":\"2025-11-24 14:56:15\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":393169,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eH. pylori promoted exosomes transfer and increased exosomal miR-143-5p expression. (\\u003cstrong\\u003eA) \\u003c/strong\\u003eNanoparticle tracking analysis (NTA) of exosomes from H. pylori- and H. pylori+ GES-1. (\\u003cstrong\\u003eB) \\u003c/strong\\u003eIF analysis of PKH67 in GF cells transfected with the PKH67 marked exosomes of H. pylori- and H. pylori+ GES-1. (\\u003cstrong\\u003eC) \\u003c/strong\\u003eComparison of mean gray value of PKH67 staining between the above two groups. (\\u003cstrong\\u003eD, E) \\u003c/strong\\u003eMicroRNA sequencing analysis of H. pylori- and H. pylori+ GES-1 exosomes were conducted. Volcano chart and heatmap were presented to show the differentially expressed miRNAs. (\\u003cstrong\\u003eF) \\u003c/strong\\u003eCorrelation analysis verified the positive association between ASPN and has-miR-143 in 9 cancers. (\\u003cstrong\\u003eG) \\u003c/strong\\u003ePCR analysis of miR-143-5p in H. pylori- and H. pylori+ GES-1 exsomes. (\\u003cstrong\\u003eH)\\u003c/strong\\u003e PCR analysis of miR-143-5p in GF co-cultured with H. pylori-, H. pylori+ GES-1 and H. pylori+ GES-1+GW4689. Values are presented as the mean±SD. **P\\u0026lt;0.01.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/0cbf0cfeb7b87397b1be16ff.png\"},{\"id\":96642733,\"identity\":\"f7e70e3b-3577-449a-a378-e17642a2afbc\",\"added_by\":\"auto\",\"created_at\":\"2025-11-24 14:56:15\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":435081,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eH. pylori infection enhanced nuclear transfer of exosomal miR-143-5p in fibroblasts. (\\u003cstrong\\u003eA) \\u003c/strong\\u003eCy3-labeled miR-143-5p was transfected into GES-1 cells and then co-cultured with GF, three group included H. pylori /Cy3, H. pylori /Cy3-miR-143, H. pylori/Cy3-miR-143. Then, IF staining of GF were conducted (Blue: DAPI, Green: Actin Tracker, Red: Cy3-labeled miR-143-5p). (\\u003cstrong\\u003eB) \\u003c/strong\\u003eIF analysis (mean gray value) of Cy3(Red) in GF cells of the above three groups. (\\u003cstrong\\u003eC) \\u003c/strong\\u003eFollowing co-culture of GF with H. pylori infected/noninfected GES-1 cells, nuclear and cytoplasmic fractions were separated\\u003cstrong\\u003e. \\u003c/strong\\u003eRT-PCR analysis of relative miR-143-5p expression between H. pylori- and H. pylori+ groups. (\\u003cstrong\\u003eD) \\u003c/strong\\u003eISH staining of miR-143-5p in H. pylori- and H. pylori+ tissues. (\\u003cstrong\\u003eE) \\u003c/strong\\u003eAnalysis of the ISH scores of totally miR-143-5p (50cases per group). (\\u003cstrong\\u003eF)\\u003c/strong\\u003eMiR-143-5p ISH signals were evaluated in epithelium and stroma separately. Values are presented as the mean±SD. *P\\u0026lt;0.05, **P\\u0026lt;0.01, ***P\\u0026lt;0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/b700eef22d82893ae561895b.png\"},{\"id\":96710170,\"identity\":\"41324100-e759-4f62-a0ba-ac94b30c2386\",\"added_by\":\"auto\",\"created_at\":\"2025-11-25 10:10:15\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":243238,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNuclear miR-143-5p (NamiRNA-143-5p) activated ASPN transcription through super-enhancer binding. (\\u003cstrong\\u003eA) \\u003c/strong\\u003eWB analysis of ASPN, IL6, IL4 and TGF-β in GF when co-cultured with H. pylori- GES-1 EVs, H. pylori+ GES-1 EVs, H. pylori- GES-1 EVs combined with miR-143-5p, H. pylori+ GES-1 EVs combined with miR-143-5p inhibitor. (\\u003cstrong\\u003eB) \\u003c/strong\\u003eSchematic diagram showed the binding site of NamiRNA-143-5p on the super-enhancer of ASPN. (\\u003cstrong\\u003eC)\\u003c/strong\\u003e Luciferase reporter assays in GF cells transfected with pGL3-miR143 or pGL3-con. (\\u003cstrong\\u003eD) \\u003c/strong\\u003eChIP-qPCR assays were performed in GF cells transfected with miR-143-5p mimics or miR-143-5p inhibitor, by using an H3K27ac antibody. (\\u003cstrong\\u003eE)\\u003c/strong\\u003e Luciferase reporter assays in GF cells with five conditions (wt-pGL3-miR143 + wt-pSR-con, wt-pGL3-miR143 + wt-pSR-miR143, wt-pGL3-miR143 + mut-pSR-miR143, mut-pGL3-miR143 + wt-pSR-miR143, mut-pGL3-miR143 + mut-pSR-miR143).\\u003cstrong\\u003e \\u003c/strong\\u003eValues are presented as the mean±SD. *P\\u0026lt;0.05, **P\\u0026lt;0.01, ***P\\u0026lt;0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/1b2a3d14838cdc1c4e358d26.png\"},{\"id\":96709237,\"identity\":\"7bad7ff5-07a0-4916-8f15-c095d02ee8b6\",\"added_by\":\"auto\",\"created_at\":\"2025-11-25 10:08:20\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":566994,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAntagomir of NamiRNA-143-5p (Antagomir-143) alleviated H. pylori induced gastric inflammation in vivo. (\\u003cstrong\\u003eA\\u003c/strong\\u003e) H. pylori IHC staining of gastric specimens from H. pylori negative (H. pylori−) and H. pylori positive (H. pylori+) mice, with \\u003cem\\u003eH. pylori\\u003c/em\\u003e organisms visualized as black structures. (\\u003cstrong\\u003eB\\u003c/strong\\u003e) ISH staining showing NamiRNA-143-5p expression in stomach tissues from H. pylori- and H. pylori+ mice. (\\u003cstrong\\u003eC\\u003c/strong\\u003e) IHC staining of ASPN in the stomach from H. pylori and H. pylori+mice. (\\u003cstrong\\u003eD\\u003c/strong\\u003e) IHC staining of IL-6 in gastric mucosa across three groups: H. pylori−, H. pylori+, and H. pylori+/Antagomir-143. (\\u003cstrong\\u003eE\\u003c/strong\\u003e) Western blot analysis of ASPN, IL-4, IL-6, and TGF-β protein levels in four groups: H. pylori-, H. pylori+, H. pylori+/Antagomir-NC, and H. pylori+/Antagomir-143. (\\u003cstrong\\u003eF\\u003c/strong\\u003e) Schematic figure showed NamiRNA-143-5p from H. pylori–infected gastric exosomes activate ASPN expression by binding to the SE site in fibroblasts to drive gastritis progression. Data are presented as mean ± SD. *P \\u0026lt; 0.05, **P \\u0026lt; 0.01, ***P \\u0026lt; 0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/f74b7f94457c6a3000a29677.png\"},{\"id\":103251120,\"identity\":\"eced45c5-d275-4083-ae21-f09684089faa\",\"added_by\":\"auto\",\"created_at\":\"2026-02-23 16:04:17\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3754760,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/d742d1a7-7027-470f-99f3-4d1ce0556d70.pdf\"},{\"id\":96709525,\"identity\":\"e776027a-8f4c-4d06-ba65-83e91a9756c7\",\"added_by\":\"auto\",\"created_at\":\"2025-11-25 10:09:12\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":21544,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"supplementarytables.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/78d5a1ea251789e268f04420.docx\"},{\"id\":96709255,\"identity\":\"707cc437-893b-433a-b28e-4de45acc89de\",\"added_by\":\"auto\",\"created_at\":\"2025-11-25 10:08:29\",\"extension\":\"pdf\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":852540,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplemetaryfigures.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/d558ffcabbebdf842faf4cea.pdf\"},{\"id\":96709359,\"identity\":\"9302bf7b-fb9b-4a3e-b5d4-92c8fa3b8713\",\"added_by\":\"auto\",\"created_at\":\"2025-11-25 10:08:48\",\"extension\":\"xlsx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":16050,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"miRNAseqdata.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/78ad5036b9e42aa7afa16a0a.xlsx\"},{\"id\":96709934,\"identity\":\"b871a186-7bf5-4ee8-ad52-b112841eb9b3\",\"added_by\":\"auto\",\"created_at\":\"2025-11-25 10:09:48\",\"extension\":\"pdf\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":344579,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"fulluncroppedGelsandBlotsimages.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7945968/v1/4b9f343e7b3462496656a76d.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Epithelial cell-derived exosomes carry NamiRNA-143-5p and promote ASPN expression in fibroblasts to drive Helicobacter pylori infected gastritis progression\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eH. pylori is a Group I carcinogen that drives the Correa cascade and contributes to peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Activated stromal fibroblasts are emerging as key amplifiers of chronic gastric inflammation: they secrete IL-6, IL-4, IL-13, and TGF-β, facilitate neutrophil infiltration via ICAM-1, and undergo autocrine differentiation toward myofibroblasts [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. Yet H. pylori typically colonizes the epithelial surface without invading the lamina propria, making direct microbe\\u0026ndash;fibroblast contact uncommon [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. This creates a central unresolved question: how are fibroblasts activated and maintained in a pro-inflammatory state during H. pylori\\u0026ndash;associated gastritis?\\u003c/p\\u003e\\u003cp\\u003eAsporin (ASPN), a small leucine-rich proteoglycan expressed in fibroblasts, modulates extracellular signaling and matrix biology in inflammatory disorders such as osteoarthritis and pulpitis [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. Our prior work showed that ASPN is upregulated in gastric cancer and precursor lesions; intracellular ASPN binds LEF1 and selectively enhances transcription of inflammation-related genes (e.g., PTGS2, IL6, WISP1) and components of the IL-4/TGF-β axis [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. These data nominate ASPN as a candidate driver of fibroblast-dependent inflammation in H. pylori gastritis. Whether ASPN is indeed induced in gastric fibroblasts during infection and functionally promotes disease progression remains unknown.\\u003c/p\\u003e\\u003cp\\u003eExosomes (30\\u0026ndash;150 nm) are efficient long-range messengers that transport nucleic acids, proteins, and lipids to reprogram recipient cells through membrane fusion, cargo release, or receptor interaction [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. During \\u003cem\\u003eH. pylori\\u003c/em\\u003e infection, gastric epithelial cells actively secrete exosomes enriched with small RNAs that function as key mediators of epithelial\\u0026ndash;recipient cell communication [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. For instance, epithelial exosomal miR-25 targets KLF2 in endothelial cells, thereby activating vascular inflammatory signaling, while p-MET\\u0026ndash;bearing vesicles educate macrophages toward tumor-promoting and pro-inflammatory phenotypes [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. Collectively, these findings support that \\u003cem\\u003eH. pylori\\u003c/em\\u003e infection stimulates epithelial exosome production, enabling the transfer of internal signaling molecules to distant recipient cells and promoting downstream inflammatory mediator expression.\\u003c/p\\u003e\\u003cp\\u003eAlthough most studies of exosomal miRNAs have focused on their traditional role as cytoplasmic repressors of translation, increasing evidence indicates that miRNA function varies considerably depending on their subcellular localization [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Recent research has revealed that several mature miRNAs translocate into the nucleus and directly modulate transcription [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. For example, miR-195-5p targets the FOXO3 promoter through AGO2\\u0026ndash;TATA-box interaction to activate gene transcription [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e], while miR-26a-1 and miR-24-1 act on enhancer regions, promoting H3K27ac enrichment and eRNA-driven activation of neighboring gene [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. Building on ChIP-seq evidence, Xiao et al. defined nuclear activating miRNAs (NamiRNAs) that associate with H3K27ac-marked enhancers and nearby super-enhancers to stimulate gene expression on the same chromosome; NamiR-143 is among these candidates [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Subsequent studies demonstrate that NamiRNAs can transcriptionally activate disease-relevant genes in breast cancer [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Whether such enhancer-activating miRNAs operate in H. pylori gastritis, and specifically drive ASPN in fibroblasts, has not been addressed.\\u003c/p\\u003e\\u003cp\\u003eHere, we report that both ASPN and NamiRNA-143-5p accumulate in gastric fibroblasts during H. pylori. H. pylori exposure increases the release of epithelial exosomes carrying NamiRNA-143-5p; these vesicles are internalized by fibroblasts, where nuclear NamiRNA-143-5p directly engages an ASPN enhancer to elevate its transcription. Elevated ASPN, in turn, augments downstream inflammatory mediators including IL-6, IL-4 and TGF-β. Together, these findings delineate a mechanistic epithelial-to-stromal route that reprograms fibroblasts via enhancer-targeting NamiRNAs, positioning ASPN as a transcriptionally controlled hub with biomarker and therapeutic potential.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003ePatients and tissue specimens\\u003c/h2\\u003e\\u003cp\\u003eA total of 150 biopsy specimens with complete clinicopathologic characteristics were used for IHC staining, including 50 cases of H. pylori negative gastric epithelial tissue, 50 cases of H. pylori positive superficial gastritis tissue, and 50 cases of H. pylori positive atrophic gastritis tissue. All of these tissues come from our own biobank. This study was conducted in compliance with the principles of the Declaration of Helsinki. Informed consent was obtained from all the subjects. Ethics approval for human subjects was provided by Ethics Committee of the Beijing Friendship Hospital, Capital Medical University.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eImmunohistochemistry\\u003c/h3\\u003e\\n\\u003cp\\u003eFormalin-fixed paraffin-embedded sections were deparaffinized, rehydrated, and subjected to high-pressure antigen retrieval. Endogenous peroxidase was blocked with 3% H₂O₂ for 20 min, followed by serum blocking for 1 h. Slides were incubated overnight at 4\\u0026deg;C with antibodies including anti-ASPN, anti-Vimentin, anti-IL4, anti-IL6(detailed in Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e: Information of primary antibodies used in WB and IHC), then with an HRP-conjugated anti-rabbit secondary antibody for 2 h at room temperature, and visualized using a DAB kit (ZSGB-Bio, Beijing, China). IHC staining was independently evaluated by two blinded pathologists. Intensity was scored as 0 (none), 1 (weak), 2 (moderate), or 3 (strong), and the percentage of positive cells as 1 (0\\u0026ndash;25%), 2 (26\\u0026ndash;50%), 3 (51\\u0026ndash;75%), or 4 (\\u0026gt;\\u0026thinsp;75%). The total score (0\\u0026ndash;12) was classified as negative (0), weak (1\\u0026ndash;4), moderate (5\\u0026ndash;8), or strong (9\\u0026ndash;12); scores\\u0026thinsp;\\u0026ge;\\u0026thinsp;5 indicated high expression.\\u003c/p\\u003e\\n\\u003ch3\\u003eWestern Blotting\\u003c/h3\\u003e\\n\\u003cdiv class=\\\"Heading\\\"\\u003eWestern Blotting\\u003c/div\\u003e\\u003cp\\u003eProtein concentrations were determined using the Bicinchoninic Acid Assay Kit (Thermo Fisher, Massachu-setts). Equal amounts of denatured protein (50 \\u0026micro;g per lane) were resolved by SDS\\u0026ndash;PAGE and transferred onto PVDF membranes. After blocking with 5% nonfat milk, membranes were incubated overnight at 4\\u0026deg;C with primary an-ti-bodies against ASPN, IL6, IL4, TGF-β, GAPDH, GM130, CD63, TSG101, CD9 (de-tailed in Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e: Information of primary antibodies used in WB and IHC). The following day, membranes were exposed to HRP-conjugated secondary antibodies for 1 h at room temperature. Protein sig-nals were visualized using an enhanced chemiluminescence system (Bio-Rad, California).\\u003c/p\\u003e\\n\\u003ch3\\u003eQuantitative real-time PCR (qRT-PCR)\\u003c/h3\\u003e\\n\\u003cp\\u003eRNA extraction. Total RNA was isolated with TRIzol (DNase I treated as needed). Concentration/purity were checked spectrophotometrically; integrity by agarose gel. For EV samples, RNase-free procedures were used.\\u003c/p\\u003e\\u003cp\\u003emRNA qRT-PCR: cDNA was synthesized from 0.5\\u0026ndash;1.0 \\u0026micro;g RNA using oligo(dT)\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;random hexamers. SYBR Green qPCR (10\\u0026ndash;20 \\u0026micro;L) was run on an ABI 7500 with 80\\u0026ndash;150 bp amplicons designed across exon\\u0026ndash;exon junctions. Cycling: 95\\u0026deg;C 2 min; 40\\u0026times; (95\\u0026deg;C 15 s, 60\\u0026deg;C 30 s). GAPDH (or RPLP0; stability verified) was the ref-erence. Technical triplicates were used.\\u003c/p\\u003e\\u003cp\\u003emiRNA qRT-PCR: Mature miRNAs (e.g., miR-143-5p) were quantified by stem-loop RT followed by SYBR qPCR using a miRNA-specific forward and universal reverse primer (amplicon\\u0026thinsp;~\\u0026thinsp;60\\u0026ndash;80 bp). Cycling: 95\\u0026deg;C 2 min; 40\\u0026times; (95\\u0026deg;C 15 s, 60\\u0026deg;C 30\\u0026ndash;60 s). Normalization: U6 for cells/tissues (stability verified);\\u003c/p\\u003e\\u003cp\\u003eControls \\u0026amp; analysis. No-template and No-RT controls were included. Single-peak melt curves confirmed specificity. Relative expression was calculated by 2^-ΔΔCt with effi-ciency 90\\u0026ndash;110% (standard curves). All the primer sequences of mRNA and miRNA are provided in Table \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e: Information of primers used in RT-qPCR.\\u003c/p\\u003e\\n\\u003ch3\\u003eCell culture and co-culture system\\u003c/h3\\u003e\\n\\u003cp\\u003eTwo cell lines (GES-1 and primary fibroblasts) were used in the study. GES-1 was purchased from the National Infrastructure of Cell Line Resource of China. The human primary gastric fibroblast Cells (HUM-iCELL-d006) was purchased from Cellverse. GES-1 was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37\\u0026deg;C in a humidified incubator with 5% CO2. The gastric fibroblasts were cultured in the special medium of PriMed-iCELL-003, which was produced by Cellverse. The cell lines ap-plied in the experiments underwent fewer than 5 passages and were authenticated using short tandem repeat analysis. Cells were cultured in permeable membrane in-serts (Transwell, Corning; 0.4-\\u0026micro;m pores, polycarbonate, 12-mm) placed in companion plates to establish a two-compartment co-culture. GES-1 cells were seeded in the upper Transwell insert and gastric fibroblasts in the lower well; both compartments contained PriMed-iCELL-003 medium during co-culture.\\u003c/p\\u003e\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eH. pylori strasins and infection with H. pylori\\u003c/h2\\u003e\\u003cp\\u003eH.pylori strains 26695 were inoculated in Columbia blood agar (OXOID, CM0331b) plates supplemented with 10% off fiber sheep blood and mixed antibiotics (vancomy-cin 10mg/L, amphotericin 5mg/L, trimethoprim 5mg/L, and polymyxin 5mg/L) under 37℃ microaerobic atmosphere by anaeropack (MGC, C-2).\\u003c/p\\u003e\\u003cp\\u003eA total of 3\\u0026times;105 GES1 cells were seeded into 10cm culture dish with 10% exosome-free FBS (Wenren, EXO-FBS-50A-1). H.pylori strain 26695 was used for co-culture with GES1 cell line at 5 MOI for 96 h. We collect 48h and 96h mediun after the infection of H.pylori. We cultured gastric fibroblasts in the conditioned medium treated with a 0.22\\u0026micro;m filter for 48h.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003esEV isolation\\u003c/h3\\u003e\\n\\u003cp\\u003eTo isolate vesicles, the conditioned medium was subjected to centrifugation at 8,500g 30min to remove cell debris, H.pylori and other impurities. Then the conditioned me-dium was subjected to ultracentrifugation at 120,000 \\u0026times; g for 70 min at 4℃, followed by a single wash with phosphate-buffered saline (PBS), and the pellet was resus-pended in 200 \\u0026micro;L PBS. EVs were stored at -80℃ before use.\\u003c/p\\u003e\\n\\u003ch3\\u003eNanoparticle Tracking Analysis of exosome\\u003c/h3\\u003e\\n\\u003cp\\u003eNanoparticle tracking analysis (NTA) was performed on supernatants after 10,000 \\u0026times; g centrifugation using a NanoSight LM10 (Nanosight, UK). About 0.3 mL sample was loaded, and three 30\\u0026ndash;60 s videos were recorded. Data were analyzed with NTA soft-ware (v1.1/2.1) to calculate particle size and concentration via the Stokes\\u0026ndash;Einstein equation. Instrument settings were optimized with 100\\u0026ndash;400 nm calibration beads (Duke Scientific, USA). Biological samples were measured at 15\\u0026ndash;30 ms shutter speed and camera gain 280\\u0026ndash;560. Results are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD of triplicates, with reli-able quantification between 2 \\u0026times; 10^8 and 2 \\u0026times; 10^9 particles/mL; more concentrated samples were diluted accordingly.\\u003c/p\\u003e\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eImmunofluorescence\\u003c/h2\\u003e\\u003cp\\u003eTo assess the subcellular localization of PKH67, Cy3 and β-actin, gastric fibroblasts were cultured on sterile coverslips placed in 6-well plates. After three washes with PBS, cells were fixed in 4% paraformaldehyde for 15 min and permea-bilized with 0.25% Triton X-100 in PBS. Non-specific binding was blocked using 5% BSA in PBST for 1 h. Samples were then incubated overnight at 4\\u0026deg;C with primary antibodies against β-actin diluted in PBS, followed by exposure to Alexa Fluor 488\\u0026ndash;conjugated anti-mouse IgG (Life Technolo-gies, MA) for 2 h in the dark. Nuclei were counter-stained with DAPI, and images were acquired using a confocal microscope (IX83, FLUOVIEW FV1200; Olympus, Japan).\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e\\u003cb\\u003eExosomes miRNA sequencing\\u003c/b\\u003e\\u003c/h2\\u003e\\u003cp\\u003eFollowing the isolation of total RNA (containing the small RNA fraction) from purified exosomes of H. pylori-infected and control GES-1 cells, high-throughput sequencing was performed by Majorbio (Shanghai, China). This process included RNA quality assessment, library preparation, and sequencing on HiSeq/MiSeq platforms, all conducted as per the manufacturer's instructions.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eIn situ hybridization\\u003c/h2\\u003e\\u003cp\\u003eISH was performed on 5 \\u0026micro;m paraffin sections using a Tecan Genepaint system as de-scribed [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. Sections were treated with proteinase K (25 \\u0026micro;g/mL, 37\\u0026deg;C, 8 min) and hy-bridized in Exiqon buffer with double-FAM\\u0026ndash;labeled LNA probes for miR-143, or scramble controls at 55\\u0026ndash;57\\u0026deg;C. After SSC washes, blocking was carried out with PBS con-taining 0.1% Tween, 4% sheep serum, and 1% BSA. Probes were detected with AP-conjugated anti-FAM Fab frag-ments (1:800, Roche), followed by NBT/BCIP sub-strate with 0.25 mM levamisole for 60 min. Slides were counter-stained with Nuclear Fast Red, dehydrated, and mounted. After multiple sections of the same part of the specimen, the in situ hybridization results of miR-143 were compared with the IHC staining results of VIM.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eLuciferase reporter assay\\u003c/h2\\u003e\\u003cp\\u003ePGL3 reporter plasmids containing the DNA fragments (about 1000bp), which is the miR-143-5p genome locus in the upstream 20 kb of ASPN, as well as a luciferase re-porter gene, named as pGL3-miR143, were obtained from YouBio (China). gastric fibroblasts (4 \\u0026times; 10^4 per well) were seeded into 24-well plates and cotransfected with 800 ng of pro-mot-er-reporter plasmid, 200 ng of pRL-TK plasmid, using Lipofectamine 3000 (Invi-trogen), following the manufactur-er\\u0026rsquo;s protocol. After 36 h of incubation at 37\\u0026deg;C, firefly and Renilla luciferase activities were measured with the Du-al-Luciferase Reporter Assay System (Thermo Fisher, MA). Firefly signals were normalized to Renilla activity for each sample.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eChromatin immunoprecipitation assay (ChIP)\\u003c/h2\\u003e\\u003cp\\u003eChIP was performed using the Pierce\\u0026trade; Magnetic ChIP Kit (Thermo Fisher) according to the manufacturer\\u0026rsquo;s protocol. After 3 days transfected with miR-143-5p mimic or in-hibitor, gastric fibroblasts were cultured to ~\\u0026thinsp;80% confluence and crosslinked with 1% formaldehyde for 10 min. Genomic DNA was fragmented into 300\\u0026ndash;1000 bp fragments using a Bio-ruptor\\u0026reg; Pico sonicator, and chromatin supernatants were incubated overnight at 4\\u0026deg;C with either anti-H3K27ac or control IgG antibody under rotation. DNA recovered from magnetic beads was analyzed by qPCR with special primer pairs (listed in Table \\u003cspan refid=\\\"MOESM3\\\" class=\\\"InternalRef\\\"\\u003eS3\\u003c/span\\u003e: Information of primers used in ChIP-PCR).\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eAnimal care and treatment\\u003c/h2\\u003e\\u003cp\\u003eAll mouse studies were performed with approval by Institutional Animal Care and Use Committee (IACUC) of Tsinghua University, Beijing, China. Six- to eight-week-old mice were modeled for infection or infected with H. pylori via oral gavage. Food was withdrawn the night before the procedure, and water was removed at 8:00 AM the next morning. The oral gavage experiment commenced at 12:00 PM, and after con-firming no abnormalities at 4:00 PM, food and water were restored. The mock-infected group received 500 \\u0026micro;L of sterile Brucella broth, while the H. pylori infected group was inoculated with 500 \\u0026micro;L of Brucella broth containing 1\\u0026times;10⁹ CFU of H. pylori. The in-ocu-lations were administered twice, with a 24-hour interval between doses. Both male and female mice were included in the H. pylori infection study. Antagomir-143-5p and Antagomir-NC were purchased in GenePharma and 50nmol/100ul per mice were in-jected though tail vein.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eStatistical Analysis\\u003c/h2\\u003e\\u003cp\\u003eData represent the mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD (standard deviation); all statistical analyses were performed using GraphPad Prism and R Software. Statistical tests were one-sided or two-sided, and Mann-Whitney U-test and Wilcoxon matched-pairs test were conducted to evaluate the differences between two groups, while ANOVA was used to compare multiple groups. P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was considered significant.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eH. pylori infection elevated ASPN levels in gastric fibroblasts\\u003c/h2\\u003e\\u003cp\\u003eBuilding on the aberrant elevation of ASPN in gastric cancer observed in our previous research and its emerging role as a crucial fibroblast-derived modulator in matrix-associated inflammation, we first examined its expression in H. pylori related pre-neoplastic lesions. Immunohistochemistry (IHC) was performed on 50 cases each of H. pylori negative normal gastric mucosa (NT), H. pylori positive non-atrophic gastritis (NAG), and H. pylori positive atrophic gastritis (AG). ASPN expression was significantly higher in both H. pylori positive NAG and AG compared with NT (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01), whereas no difference was observed between NAG and AG. Importantly, ASPN staining was predominantly localized to mesenchymal cells, such as fibroblasts, rather than epithelial compartments (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA, B). To further confirm fibroblast localization, IHC for the stromal marker Vimentin (VIM) revealed a marked accumulation of fibroblasts in H. pylori infected tissues compared with H. pylori negative controls (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC, D). Strong staining for both ASPN and VIM in H. pylori associated gastritis specimens supports fibroblasts as the primary source of ASPN.\\u003c/p\\u003e\\u003cp\\u003eWestern blotting (WB) analysis of tissue lysates corroborated the IHC findings, showing elevated ASPN protein levels in H. pylori positive NAG and AG relative to NT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE). Consistently, qRT-PCR demonstrated increased ASPN mRNA levels in H. pylori positive states (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eF). However, when primary gastric fibroblasts were directly infected with H. pylori, neither WB nor qRT-PCR showed changes in ASPN expression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eG, H). Together, these results indicates that ASPN upregulation in H. pylori associated gastritis occurs within fibroblasts in vivo, but is not directly induced by bacterial infection of fibroblasts.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eExosomes from H. pylori infected GES-1 cells could induce upregulation of ASPN signaling in fibroblasts\\u003c/h2\\u003e\\u003cp\\u003eTo further detect the mechanism of the overexpression of ASPN in gastric fibroblasts, we first conducted immunofluorescence (IF) staining of VIM to confirm cell purity of the primary fibroblasts above 90% (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, left). Then, we established a Transwell co-culture system with a 0.4 \\u0026micro;m pore membrane to prevent cell migration (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, right). H. pylori infected GES-1 cells (upper chamber) were co-cultured with fibroblasts (lower chamber) for 48 h. Compared with controls using H. pylori negative GES-1 cells, WB analysis showed that fibroblasts co-cultured with H. pylori infected epithelial cells exhibited increased ASPN protein expression, accompanied by upregulation of IL-6, IL-4, and TGF-β (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). Consistent with our prior GSEA analysis linking ASPN with IL-4 and TGF-β signaling pathways (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC), overexpression of ASPN in fibroblasts enhanced the expression of these inflammatory cytokines (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). Together, these results demonstrate that H. pylori infected epithelial cells can modulate ASPN and downstream inflammatory mediators in fibroblasts through indirect mechanisms.\\u003c/p\\u003e\\u003cp\\u003eTo identify the mediator of this epithelial\\u0026ndash;fibroblast crosstalk, exosomes were isolated from the supernatant of H. pylori infected GES-1 cells using ultracentrifugation. Transmission electron microscopy revealed vesicles of ~\\u0026thinsp;100 nm with characteristic cup-shaped, double-membrane morphology (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE). Western blot confirmed enrichment of exosome markers CD63, TSG101, and CD9 in the EV fractions, while the Golgi marker GM130 was absent, validating exosome purity (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eF). Equal amounts of EVs from H. pylori positive or H. pylori negative GES-1 cells were added to fibroblast cultures. After 24\\u0026ndash;36 h, qRT-PCR analysis revealed increased ASPN, IL-6, IL-4, and TGF-β mRNA levels in fibroblasts treated with H. pylori positive EVs compared with controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eH).\\u003c/p\\u003e\\u003cp\\u003eCollectively, these findings indicate that exosomes derived from H. pylori infected epithelial cells mediate the upregulation of ASPN and its downstream inflammatory genes in gastric fibroblasts at both transcriptional and protein levels.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eH. pylori infection promoted exosomes transferring into fibroblasts and the overexpression of exosomal miR-143-5p\\u003c/h2\\u003e\\u003cp\\u003eTo determine the effect of H. pylori infection on exosome secretion, we performed nanoparticle tracking analysis (NTA) of exosomes isolated from GES-1 cells. Both the average particle count and the total protein content were significantly higher in the H. pylori infected group than in H. pylori negative controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). Exosomes derived from equal numbers of H. pylori positive or H. pylori negative GES-1 cells were then labeled with PKH67 and co-cultured with gastric fibroblasts. IF assays revealed exosome uptake by gastric fibroblasts, with significantly stronger fluorescence intensity observed in the H. pylori positive group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB, C). These data indicate that H. pylori infection enhances exosome production by epithelial cells and promotes their uptake by fibroblasts.\\u003c/p\\u003e\\u003cp\\u003eTo identify the cargo responsible for ASPN induction, exosomes from H. pylori positive and H. pylori negative GES-1 cells were analyzed by small RNA sequencing. Approximately 400 miRNAs were detected, among which 102 were differentially expressed (50 upregulated, 52 downregulated; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD, screening criteria: FDR\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.14, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). A heatmap of the top 60 differentially expressed miRNAs highlighted miR-143-5p (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE). Correlation analysis across multiple cancer datasets (miRactDB, \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://ccsm.uth.edu/miRactDB/index.html\\u003c/span\\u003e\\u003cspan address=\\\"https://ccsm.uth.edu/miRactDB/index.html\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) showed a strong positive association between miR-143 and ASPN expression in more than ten cancer types, including esophageal, gastric, colorectal, and liver cancers (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF). These results suggest that miR-143-5p is enriched in exosomes from H. pylori infected epithelial cells and may regulate ASPN expression in fibroblasts.\\u003c/p\\u003e\\u003cp\\u003eTo validate the sequencing data, qRT-PCR was performed on GES-1 cells infected with H. pylori for 24 or 48 h. miR-143-5p expression was significantly higher at 48 h compared with H. pylori negative controls (Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eA). Consistently, qRT-PCR analysis of exosomes secreted by H. pylori infected cells confirmed elevated miR-143-5p levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eG). In co-culture assays, gastric fibroblasts exposed to H. pylori positive GES-1 cells exhibited higher intracellular miR-143-5p levels than those co-cultured with H. pylori negative cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eH). Furthermore, Cy3-labeled miR-143-5p transfected into GES-1 cells was detected in gastric fibroblasts nuclei using a Transwell system, confirming direct uptake of miR-143-5p by fibroblasts (Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eB).\\u003c/p\\u003e\\u003cp\\u003eCollectively, these findings demonstrate that H. pylori infection increases both the release of exosomes from epithelial cells into fibroblasts and the level of exosomal miR-143-5p, which may contribute to ASPN upregulation.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eH. pylori infection enhanced exosomal miR-143-5p transferring into nuclei of fibroblasts\\u003c/h2\\u003e\\u003cp\\u003eTo further elucidate whether miR-143-5p could be carried by exosomes from H. pylori infected GES-1 cells entered gastric fibroblasts and promotes downstream inflammatory factor expression, we conducted a series of experiments. H. pylori positive GES-1 cells were transfected with Cy3-labeled miR-143-5p and co-cultured with gastric fibroblasts. Control conditions included H. pylori negative GES-1 cells transfected with Cy3-miR-143-5p and Cy3 alone. After 48h co-culture, IF assays in gastric fibroblasts revealed the strongest intracellular Cy3 signal in group of Cy3-labeled miR-143-5p, with a portion of the fluorescence localizing to nuclei, whereas transfer efficiency was reduced under H. pylori negative conditions (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA, B). Following co-culture with H. pylori infected GES-1 cells, nuclear\\u0026ndash;cytoplasmic fractionation was performed in gastric fibroblasts. Stem-loop qRT-PCR showed a significant increase of miR-143-5p in the nuclear fraction under H. pylori infection (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC).\\u003c/p\\u003e\\u003cp\\u003eIn patient tissues, in situ hybridization (ISH) showed elevated nuclear miR-143-5p in H. pylori positive specimens compared with H. pylori negative controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD, E). Stratified analysis further indicated a more pronounced increase in the stromal compartment than in the epithelium under H. pylori infection, suggesting that H. pylori not only increased miR-143-5p levels but also promotes its transfer into stromal regions (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eF). All above results prove that upon H. pylori infection, exosomal miR-143-5p derived from GES-1 cells is delivered to fibroblasts and ultimately localizes to the nucleus.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cdiv id=\\\"Sec23\\\" class=\\\"Section3\\\"\\u003e\\u003ch2\\u003eNuclear miR-143-5p (NamiRNA-143-5p) promoted ASPN expression though binding to super-enhancer sites\\u003c/h2\\u003e\\u003cp\\u003eFor further exploring the function and mechanism of nuclear miR-143-5p, a miR-143-5p inhibitor was added to EV-conditioned medium from H. pylori positive GES-1 cells prior to co-culture with gastric fibroblasts, whereas synthetic miR-143-5p was added to medium from H. pylori negative cells (with matched controls). Gastric fibroblasts lysates were subsequently analyzed by WB analysis. Exogenous miR-143-5p enhanced ASPN and downstream inflammatory mediators in the H. pylori negative EV condition, whereas the miR-143-5p inhibitor attenuated the promotive effect of H. pylori positive EVs on ASPN and downstream proteins in gastric fibroblasts (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA).\\u003c/p\\u003e\\u003cp\\u003eGiven the nuclear localization of miR-143-5p in gastric fibroblasts, we hypothesized a NamiRNA mechanism. Sequence alignment between miR-143-5p and the ASPN SE region in fibroblasts (SEdb v3.0 entry SE_02_214900539) predicted strong binding potential (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB), suggesting that miR-143-5p acts as a NamiRNA (NamiRNA-143-5p) to enhance transcription. To test this, a 700-bp ASPN enhancer fragment harboring the predicted miR-143-5p site was cloned into pGL3 (pGL3-miR143). Compared with empty pGL3, transfection into gastric fibroblasts significantly increased the Firefly/Renilla luciferase activity ratio (Luc+/Rluc+) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC). Furthermore, chromatin immunoprecipitation (ChIP) with an H3K27ac antibody in gastric fibroblasts overexpressing NamiRNA-143-5p showed enriched H3K27ac at primers flanking the predicted binding site, relative to empty-vector controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD).\\u003c/p\\u003e\\u003cp\\u003eWe then generated dual-reporter constructs carrying either the WT ASPN enhancer (wt-pGL3-miR143) or a mutated site (mut-pGL3-miR143), and pSUPER vectors expressing either WT NamiRNA-143-5p (wt-pSR-miR143) or a binding-defective mutant (mut-pSR-miR143). Mutations in mut-pGL3-miR143 and mut-pSR-miR143 were designed as complementary pairs to allow interaction rescue. Pairwise co-transfections in HEK293T cells yielded four conditions for Luc+/Rluc\\u0026thinsp;+\\u0026thinsp;comparison. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE: (a) wt-pGL3-miR143\\u0026thinsp;+\\u0026thinsp;wt-pSR-miR143 significantly increased the level of Luc+/Rluc\\u0026thinsp;+\\u0026thinsp;versus control (wt-pGL3-miR143\\u0026thinsp;+\\u0026thinsp;empty pSUPER); (b\\u0026ndash;d) mutating either the enhancer (mut-pGL3-miR143\\u0026thinsp;+\\u0026thinsp;wt-pSR-miR143) or the miRNA seed (wt-pGL3-miR143\\u0026thinsp;+\\u0026thinsp;mut-pSR-miR143) reduced the activity of luciferease; (e) co-transfection of complementary mutants (mut-pGL3-miR143\\u0026thinsp;+\\u0026thinsp;mut-pSR-miR143) restored the activity. Collectively, these data demonstrate that EV-delivered NamiRNA-143-5p enters gastric fibroblasts nuclei, binds the ASPN SE, and activates ASPN transcription, thereby driving downstream inflammatory factor expression.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec24\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eAntagomir of NamiRNA-143-5p alleviated H. pylori induced gastric inflammation in vivo\\u003c/h2\\u003e\\u003cp\\u003eTo assess the in vivo expression pattern and pro-inflammatory function of NamiRNA-143-5p, we established a mouse H. pylori infection model. Infection status was confirmed by Steiner silver staining (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA). ISH revealed markedly increased nuclear NamiRNA-143-5p in H. pylori positive stomachs versus H. pylori negative controls, with a more pronounced elevation in the stromal compartment than in the epithelium (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB). In addition, ASPN also was significantly elevated in H. pylori infected gastric stromal (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eC). To test functional relevance, NamiRNA-143-5p antagomir was administered via tail vein to H. pylori positive mice. IHC showed higher IL-6 and IL-4 in H. pylori positive mucosa relative to H. pylori negative tissue, whereas antagomir-143 reduced these cytokines, particularly within stromal regions (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eD, Fig. \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003eA). Consistently, WB of gastric lysates across four groups (H. pylori\\u0026minus;, H. pylori+, H. pylori+/antagomir-NC, H. pylori+/antagomir-143) demonstrated that miR-143-5p antagomir attenuated H. pylori induced upregulation of ASPN, IL-4, IL-6, and TGF-β (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eE). As shown in the schematic figure (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eF), all our data suggested that H. pylori infection could promote the secretion of exosomal NamiRNA-143-5p from gastric epithelium transferring to fibroblasts and then activating ASPN expression by binding to its SE site to drive gastritis progression.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThis study defines an epithelial\\u0026ndash;stromal communication axis whereby H. pylori indirectly program a pro-inflammatory gastric fibroblasts phenotype. H. pylori infected epithelial cells release EVs enriched for NamiRNA-143-5p, which accumulates in gastric fibroblasts nuclei, engages an ASPN SE, elevates H3K27ac, and activates ASPN transcription, in turn upregulating IL-6, IL-4, and TGF-β. ASPN is increased in H. pylori positive gastritis and localizes to the stroma, while direct H. pylori exposure does not induce ASPN in primary gastric fibroblasts, indicating an indirect mechanism. Together, these findings illuminate how epithelial infection is transduced to stromal activation during H. pylori associated inflammation.\\u003c/p\\u003e\\u003cp\\u003eOur data position gastric fibroblasts as active amplifiers of chronic gastric inflammation. Prior reports implicated fibroblast activation and aggregation in H. pylori associated gastritis and tissue remodeling [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. We extend this by identifying ASPN as a fibroblast-centered effector whose upregulation is driven by EV-mediated NamiRNA transfer rather than direct bacterial contact. Although a gastric fibroblasts-specific marker was not available, VIM IHC supported stromal-including gastric fibroblasts-ASPN elevation [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e], and co-culture experiments verified increased ASPN and downstream cytokines when GES-1 cells are H. pylori infected. Beyond EV quantity, cargo matters: NamiRNA-143-5p acts within recipient nuclei, consistent with NamiRNAs that activate transcription via enhancer/SE binding [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Concordant spatial signals (VIM/ASPN by IHC and miR-143-5p by ISH), ChIP evidence of H3K27ac enrichment, and complementary-mutation luciferase rescue collectively support direct engagement of the ASPN SE despite modest predicted base-pairing compared with other contexts.\\u003c/p\\u003e\\u003cp\\u003eFunctionally, ASPN links H. pylori colonization to stromal remodeling and inflammatory circuitry. Together with our prior work showing intracellular ASPN cooperates with LEF1 to enhance transcription of PTGS2, IL6, and WISP1 [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e], the present findings suggest the NamiRNA-143-5p\\u0026ndash;ASPN axis may help drive the transition from acute to chronic/atrophic gastritis and ultimately a tumor-permissive microenvironment.\\u003c/p\\u003e\\u003cp\\u003eTranslationally, tissue or circulating EV levels of NamiRNA-143-5p and ASPN merit evaluation as biomarkers to stratify H. pylori positive individuals at risk for progression. Therapeutically, disrupting EV biogenesis/uptake, NamiRNA cargo loading, SE engagement, or downstream ASPN signaling are plausible strategies[\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. The in vivo attenuation of ASPN and cytokines by antagomir-143 provides preliminary proof-of-concept, which is particularly relevant given the high H. pylori burden in East Asia and limitations of eradication therapy[\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eLimitations include the potential contribution of other EV miRNAs/non-coding RNAs; incomplete delineation of nuclear cofactors and chromatin partners that mediate NamiRNA-143-5p\\u0026ndash;SE activation; and unresolved determinants of EV tropism and uptake by gastric fibroblasts. Larger human cohorts-deally integrating biofluid EV profiling and longitudinal outcomes-are needed to validate biomarker utility and therapeutic targeting.\\u003c/p\\u003e\\u003cp\\u003eIn summary, H. pylori infection stimulates gastric epithelial cells to secrete exosomes enriched with nuclear-activating miR-143-5p (NamiR-143-5p). These vesicles are internalized by fibroblasts, where NamiR-143-5p binds an ASPN super-enhancer, elevating H3K27ac enrichment and driving transcription of ASPN and its downstream cytokines (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eF). The resulting ASPN\\u0026ndash;IL-6/IL-4/TGF-β axis amplifies stromal inflammation, contributing to gastritis progression. Targeting this exosome-NamiR-143-5p-ASPN pathway may offer a promising therapeutic strategy against H. pylori associated gastric disease.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003ch2\\u003eAuthor information\\u003c/h2\\u003e\\u003cp\\u003eZheng Zhang and Shuyue Yang have contributed equally to this work.\\u003c/p\\u003e\\u003c/p\\u003e\\u003ch2\\u003e\\u003cb\\u003eAuthors and Affiliations\\u003c/b\\u003e\\u003c/h2\\u003e\\u003cp\\u003e\\u003cstrong\\u003eDepartment of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, State Key Laboratory of Digestive Health, National Clinical Research Center for Digestive Disease, Beijing Key Laboratory of Early Gastrointestinal Cancer Medicine and Medical Devices\\u003c/strong\\u003e\\u003cp\\u003eZheng Zhang, Mengran Zhao, Wenjing Sun, Anni Zhou, Sifan Liu, Mingyang Ma, Peng Li\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eDepartment of Gastroenterology, Tianjin Union Medical Center, the First Affiliated Hospital of Nankai University\\u003c/strong\\u003e\\u003cp\\u003eShuyue Yang\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eDepartment of Pathology, Beijing Friendship Hospital, Capital Medical University\\u003c/strong\\u003e\\u003cp\\u003eRui Xu\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eContributions\\u003c/strong\\u003e\\u003cp\\u003eZheng Zhang, Shuyue Yang, Wenjing Sun, Mingyang Ma, and Sifan Liu performed the research and collected the data. Zheng Zhang and Shuyue Yang prepared the manuscript. Rui Xu reviewed and interpreted the pathology data. Zheng Zhang, Mengran Zhao, Anni Zhou, and Peng Li designed the study and supervised the study. Peng Li supervised the research and help to revised the manuscript. All authors have read and agreed to the published version of the manuscript.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eCorresponding author\\u003c/strong\\u003e\\u003cp\\u003eCorrespondence to Peng Li or Zheng Zhang.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eEthics Statement\\u003c/strong\\u003e\\u003cp\\u003eThis study was conducted in compliance with the principles of the Declaration of Helsinki. Ethics approval for human subjects was provided by Ethics Committee of the Beijing Friendship Hospital, Capital Medical University. (DR20210109). All mouse studies were performed with approval by Institutional Animal Care and Use Committee (IACUC) of Tsinghua University(22-ZB1), Beijing, China.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003ch2\\u003eCompeting interests\\u003c/h2\\u003e\\u003cp\\u003eThe authors declare no conflicts of interest.\\u003c/p\\u003e\\u003c/p\\u003e\\u003ch2\\u003eFunding\\u003c/h2\\u003e\\u003cp\\u003eThis study was supported by National Natural Science Foundation of China (No. 82200622) and Beijing Natural Science Foundation (No. 2332028)\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eZheng Zhang, Shuyue Yang, Wenjing Sun, Mingyang Ma, and Sifan Liu performed the research and collected the data. Zheng Zhang and Shuyue Yang prepared the manuscript. Rui Xu reviewed and interpreted the pathology data. Zheng Zhang, Mengran Zhao, Anni Zhou, and Peng Li designed the study and supervised the study. Peng Li supervised the research and help to revised the manuscript. All authors have read and agreed to the published version of the manuscript.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAmieva M, Peek RM. Jr. Pathobiology of Helicobacter pylori-Induced Gastric Cancer. Gastroenterology. 2016;150(1):64\\u0026ndash;78.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eNoto JM, Peek RM Jr. The gastric microbiome, its interaction with Helicobacter pylori, and its potential role in the progression to stomach cancer. PLoS Pathog. 2017;13(10):e1006573.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLeBleu VS, Neilson EG. Origin and functional heterogeneity of fibroblasts. FASEB J. 2020;34(3):3519\\u0026ndash;36.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eKendall RT, Feghali-Bostwick CA. Fibroblasts in fibrosis: novel roles and mediators. Front Pharmacol. 2014;5:123.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eCroft AP, Campos J, Jansen K, Turner JD, Marshall J, Attar M, et al. Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature. 2019;570(7760):246\\u0026ndash;51.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMescher AL. Macrophages and fibroblasts during inflammation and tissue repair in models of organ regeneration. Regeneration (Oxf). 2017;4(2):39\\u0026ndash;53.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eChen X, Chen W, Zhao Y, Wang Q, Wang W, Xiang Y, et al. Interplay of Helicobacter pylori, fibroblasts, and cancer cells induces fibroblast activation and serpin E1 expression by cancer cells to promote gastric tumorigenesis. J Transl Med. 2022;20(1):322.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eWang YK, Li C, Zhou YM, Zeng L, Li YY, Huang SL, et al. Histopathological Features of Helicobacter pylori Infection in Gastric Mucosa. J Inflamm Res. 2022;15:6231\\u0026ndash;43.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eZhao Z, Wijerathne H, Godwin AK, Soper SA. Isolation and analysis methods of extracellular vesicles (EVs). Extracell Vesicles Circ Nucl Acids. 2021;2(1):80\\u0026ndash;103.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eXu Y, Yu Y, Yang B, Hui J, Zhang C, Fang H, et al. Extracellular Mitochondrial Components and Effects on Cardiovascular Disease. DNA Cell Biol. 2021;40(9):1131\\u0026ndash;43.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLiu YJ, Wang C. A review of the regulatory mechanisms of extracellular vesicles-mediated intercellular communication. Cell Commun Signal. 2023;21(1):77.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eWang C, Li W, Shao L, Zhou A, Zhao M, Li P, et al. Both extracellular vesicles from helicobacter pylori-infected cells and helicobacter pylori outer membrane vesicles are involved in gastric/extragastric diseases. Eur J Med Res. 2023;28(1):484.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eXia X, Zhang L, Chi J, Li H, Liu X, Hu T, et al. Helicobacter pylori Infection Impairs Endothelial Function Through an Exosome-Mediated Mechanism. J Am Heart Assoc. 2020;9(6):e014120.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLi N, Liu SF, Dong K, Zhang GC, Huang J, Wang ZH, et al. Exosome-Transmitted miR-25 Induced by H. pylori Promotes Vascular Endothelial Cell Injury by Targeting KLF2. Front Cell Infect Microbiol. 2019;9:366.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eZou Q, Liang Y, Luo H, Yu W. miRNA-Mediated RNAa by Targeting Enhancers. Adv Exp Med Biol. 2017;983:113\\u0026ndash;25.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eXiao M, Li J, Li W, Wang Y, Wu F, Xi Y, et al. MicroRNAs activate gene transcription epigenetically as an enhancer trigger. RNA Biol. 2017;14(10):1326\\u0026ndash;34.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLiang Y, Xu P, Zou Q, Luo H, Yu W. An epigenetic perspective on tumorigenesis: Loss of cell identity, enhancer switching, and NamiRNA network. Semin Cancer Biol. 2019;57:1\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLiang Y, Lu Q, Li W, Zhang D, Zhang F, Zou Q, et al. Reactivation of tumour suppressor in breast cancer by enhancer switching through NamiRNA network. Nucleic Acids Res. 2021;49(15):8556\\u0026ndash;72.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLi W, Yang S, Xu P, Zhang D, Tong Y, Chen L, et al. SARS-CoV-2 RNA elements share human sequence identity and upregulate hyaluronan via NamiRNA-enhancer network. EBioMedicine. 2022;76:103861.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eIkegawa S. Expression, regulation and function of asporin, a susceptibility gene in common bone and joint diseases. Curr Med Chem. 2008;15(7):724\\u0026ndash;8.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eFan R, Yan X, Zhang W. Relationship between asporin and extracellular matrix behavior: A literature review. Med (Baltim). 2022;101(51):e32490.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLi H, Zhang Z, Chen L, Sun X, Zhao Y, Guo Q, et al. Cytoplasmic Asporin promotes cell migration by regulating TGF-beta/Smad2/3 pathway and indicates a poor prognosis in colorectal cancer. Cell Death Dis. 2019;10(2):109.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eZhang Z, Min L, Li H, Chen L, Zhao Y, Liu S, et al. Asporin represses gastric cancer apoptosis via activating LEF1-mediated gene transcription independent of beta-catenin. Oncogene. 2021;40(27):4552\\u0026ndash;66.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eXu R, Yang L, Zhang Z, Liao Y, Yu Y, Zhou D, et al. Cancer-associated fibroblast related gene signature in Helicobacter pylori-based subtypes of gastric carcinoma for prognosis and tumor microenvironment estimation in silico analysis. Front Med (Lausanne). 2023;10:1079470.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eRen X, Liu G, Zhou J. Nuclear-activating miRNAs: unveiling the intricacies of subcellular miRNA function and regulation in cancer and immunity disease. Cancer Cell Int. 2025;25(1):147.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eKuo YT, Liou JM, El-Omar EM, Wu JY, Leow AHR, Goh KL, et al. Primary antibiotic resistance in Helicobacter pylori in the Asia-Pacific region: a systematic review and meta-analysis. Lancet Gastroenterol Hepatol. 2017;2(10):707\\u0026ndash;15.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eWei H, Chen Q, Lin L, Sha C, Li T, Liu Y, et al. Regulation of exosome production and cargo sorting. Int J Biol Sci. 2021;17(1):163.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eXie S, Zhang Q, Jiang L. Current knowledge on exosome biogenesis, cargo-sorting mechanism and therapeutic implications. Membranes. 2022;12(5):498.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eZhao L, Liu W, Xiao J, Cao B. The role of exosomes and exosomal shuttle microRNA in tumorigenesis and drug resistance. Cancer Lett. 2015;356(2):339\\u0026ndash;46.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLiu Q, Wang J, Zhao Y, Li C-I, Stengel KR, Acharya P, et al. Identification of active miRNA promoters from nuclear run-on RNA sequencing. Nucleic Acids Res. 2017;45(13):e121\\u0026ndash;e.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBilli M, De Marinis E, Gentile M, Nervi C, Grignani F. Nuclear miRNAs: gene regulation activities. Int J Mol Sci. 2024;25(11):6066.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBai Y, Pan B, Zhan X, Silver H, Li J. MicroRNA 195-5p targets Foxo3 promoter region to regulate its expression in granulosa cells. Int J Mol Sci. 2021;22(13):6721.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAnderson CM, Zhang B, Miller M, Butko E, Wu X, Laver T, et al. Fully automated RNAscope in situ hybridization assays for formalin-fixed paraffin‐embedded cells and tissues. J Cell Biochem. 2016;117(10):2201\\u0026ndash;8.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eGonciarz W, Krupa A, Hinc K, Obuchowski M, Moran AP, Gajewski A, et al. The effect of Helicobacter pylori infection and different H. pylori components on the proliferation and apoptosis of gastric epithelial cells and fibroblasts. PLoS ONE. 2019;14(8):e0220636.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"gut-pathogens\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"gutp\",\"sideBox\":\"Learn more about [Gut Pathogens](http://gutpathogens.biomedcentral.com/)\",\"snPcode\":\"13099\",\"submissionUrl\":\"https://submission.nature.com/new-submission/13099/3\",\"title\":\"Gut Pathogens\",\"twitterHandle\":\"@GutPathogens\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Helicobacter pylori, Exosome, NamiRNA, ASPN, Fibroblast\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7945968/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7945968/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eIn Helicobacter pylori (H. pylori) associated gastritis, fibroblasts are recruited to inflammatory sites and secrete multiple pro-inflammatory cytokines, but the underlying mechanism remains unclear. Here, immunohistochemical staining revealed that ASPN expression was significantly upregulated in fibroblasts from H. pylori positive gastritis tissues, whereas its level remained unchanged in fibroblasts directly infected with H. pylori. Co-culture assays demonstrated that exosomes released from H. pylori infected epithelial cells induced the upregulation of ASPN and its downstream cytokines (IL-4, IL-6, and TGF-β) in fibroblasts. MicroRNA sequencing and correlation analyses identified miR-143-5p as an exosomal miRNA enriched after H. pylori infection that potentially regulates ASPN. Immunofluorescence confirmed that exosomes derived from epithelial cells carrying miR-143-5p were internalized by fibroblasts. Further immunofluorescence and immunohistochemical analyses showed nuclear accumulation of miR-143-5p in fibroblasts in both the H. pylori infected epithelial cell co-culture system and H. pylori positive gastritis tissues. Mechanistic studies demonstrated that miR-143-5p functions as a nuclear activating microRNA (NamiRNA-143-5p), binding to the super-enhancer region of ASPN, increasing H3K27ac enrichment, and promoting its transcription. In vivo, antagomir-143-5p reduced H. pylori induced ASPN and cytokine overexpression, thereby alleviating gastric inflammation. Thus, we conclude that exosomal NamiRNA-143-5p from H. pylori infected epithelial cells upregulate pro-inflammatory cytokines in fibroblasts by activating ASPN expression through a super-enhancer-dependent pathway, thereby positioning this axis as a potential therapeutic target for H. pylori associated gastric disease.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Epithelial cell-derived exosomes carry NamiRNA-143-5p and promote ASPN expression in fibroblasts to drive Helicobacter pylori infected gastritis progression\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-11-24 14:56:10\",\"doi\":\"10.21203/rs.3.rs-7945968/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2026-01-05T19:09:32+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-01-03T04:10:59+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-12-22T17:00:00+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-12-09T00:11:12+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"176694026227528782574119120749694766463\",\"date\":\"2025-12-09T00:08:04+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"54492002486672614746824311080757927014\",\"date\":\"2025-12-07T06:53:49+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"333883772578766553375704909011523984682\",\"date\":\"2025-12-04T14:39:26+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-11-12T17:45:13+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-10-27T07:56:44+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-10-27T07:55:29+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Gut Pathogens\",\"date\":\"2025-10-25T09:45:50+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"gut-pathogens\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"gutp\",\"sideBox\":\"Learn more about [Gut Pathogens](http://gutpathogens.biomedcentral.com/)\",\"snPcode\":\"13099\",\"submissionUrl\":\"https://submission.nature.com/new-submission/13099/3\",\"title\":\"Gut Pathogens\",\"twitterHandle\":\"@GutPathogens\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"27b5ba17-7d15-4180-8ddc-6121d7b3f48f\",\"owner\":[],\"postedDate\":\"November 24th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-02-23T16:01:29+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-7945968\",\"link\":\"https://doi.org/10.1186/s13099-026-00808-6\",\"journal\":{\"identity\":\"gut-pathogens\",\"isVorOnly\":false,\"title\":\"Gut Pathogens\"},\"publishedOn\":\"2026-02-21 15:57:30\",\"publishedOnDateReadable\":\"February 21st, 2026\"},\"versionCreatedAt\":\"2025-11-24 14:56:10\",\"video\":\"\",\"vorDoi\":\"10.1186/s13099-026-00808-6\",\"vorDoiUrl\":\"https://doi.org/10.1186/s13099-026-00808-6\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7945968\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7945968\",\"identity\":\"rs-7945968\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}