Unveiling the miR-26b-3p/XAF1 Axis: A New Frontier in Systemic Lupus Erythematosus Inflammation Running Head: miR-26b-3p–XAF1 regulation in lupus

Unveiling the miR-26b-3p/XAF1 Axis: A New Frontier in Systemic Lupus Erythematosus Inflammation Running Head: miR-26b-3p–XAF1 regulation in lupus | 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 Article Unveiling the miR-26b-3p/XAF1 Axis: A New Frontier in Systemic Lupus Erythematosus Inflammation Running Head: miR-26b-3p–XAF1 regulation in lupus Shunsheng Lin, Cuiyan Wang, Haike Lu, Zhi-Xin Huang, Tingjing Huang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7272649/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by dysregulated inflammatory responses, yet the molecular mechanisms underlying inflammation regulation remain incompletely understood. XAF1, traditionally recognized as a tumor suppressor, and miR-26b-3p, a microRNA with emerging immunoregulatory functions, have not been previously investigated in SLE pathogenesis. Aims To investigate the regulatory relationship between miR-26b-3p and XAF1 and determine their functional significance in modulating inflammatory responses associated with SLE. Methods THP-1 monocytes were differentiated into macrophages and stimulated with lipopolysaccharide (LPS) to establish an inflammatory model. Lentiviral vectors were employed to generate XAF1 overexpression and knockdown cell lines. Peripheral blood mononuclear cells (PBMCs) from SLE patients (n = 26) and healthy controls (n = 30) were analyzed. The miR-26b-3p-XAF1 interaction was validated using quantitative PCR, western blotting, dual-luciferase reporter assays, and gain/loss-of-function studies. Pro-inflammatory cytokine levels (IL-6, IL-15, TNF-α) were quantified by ELISA. Results LPS stimulation significantly upregulated XAF1 expression (2.7-fold mRNA, 2.4-fold protein, P < 0.05) alongside marked elevation of pro-inflammatory cytokines IL-6, IL-15, and TNF-α (2.9-4.2-fold increases, P < 0.01) in THP-1 cells. SLE patient PBMCs demonstrated significantly reduced miR-26b-3p expression (0.42-fold, P < 0.01) with reciprocal XAF1 upregulation, showing strong inverse correlation (r=-0.68, P < 0.001). Dual-luciferase reporter assays confirmed direct targeting of XAF1 3'UTR by miR-26b-3p (0.42-fold luciferase activity, P < 0.01). Functional studies revealed that XAF1 overexpression enhanced cytokine production (2.4-3.2-fold, P < 0.01), while miR-26b-3p mimics effectively suppressed both XAF1 expression and inflammatory cytokine secretion (40–55% reduction, P < 0.05). Conclusion Our findings establish the miR-26b-3p/XAF1 axis as a novel regulatory mechanism in SLE inflammation, with miR-26b-3p functioning as a negative regulator of XAF1-mediated inflammatory responses. This discovery highlights the therapeutic potential of targeting this axis for SLE treatment and advances our understanding of microRNA-mediated regulation in autoimmune inflammation. Health sciences/Diseases Biological sciences/Immunology Systemic Lupus Erythematosus XAF1 Inflammation Regulation THP-1 Cells LPS Stimulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Systemic lupus erythematosus (SLE) represents a complex autoimmune disorder characterized by multi-organ inflammation and tissue damage [ 1 ]. Despite advances in understanding its pathogenesis, the molecular mechanisms underlying SLE remain incompletely understood [ 2 , 3 ]. Recent evidence suggests that dysregulated inflammatory responses play a central role in disease progression and severity [ 3 ]. The immunopathogenesis of SLE involves intricate interactions between innate and adaptive immunity [ 4 – 6 ]. Notably, aberrant B cell activation leads to autoantibody production, while dysregulated T cell responses amplify inflammatory cascades through co-stimulatory pathways and cytokine production [ 7 , 8 ]. Within the innate immune compartment, macrophage polarization states critically influence disease progression, particularly through their role in cytokine regulation and immune complex processing [ 9 , 10 ]. Inflammatory cytokines serve as key mediators in SLE pathogenesis [ 11 ]. Elevated levels of IL-6 correlate with disease activity, while increased IL-15 promotes lymphocyte proliferation and survival [ 12 , 13 ]. Similarly, TNF-α contributes to vascular dysfunction and organ damage, highlighting the complex cytokine networks in SLE progression [ 14 ]. Recent evidence has implicated XAF1, a negative regulator of the anti-apoptotic protein XIAP, in various pathological conditions. While XAF1's role as a tumor suppressor is well-documented, its function in autoimmune inflammation remains unexplored [ 15 ]. Notably, XAF1 activation occurs under conditions of cellular stress and cytokine stimulation, suggesting potential involvement in inflammatory responses. The bacterial endotoxin lipopolysaccharide (LPS) represents a crucial environmental trigger in SLE, activating Toll-like receptor signaling pathways and potentially precipitating disease flares [ 16 – 18 ]. This mechanism provides a valuable experimental model for investigating inflammatory responses in SLE [ 19 , 20 ]. MicroRNAs have emerged as critical regulators of immune responses in SLE. While several microRNAs, including miR-21 and miR-155, demonstrate altered expression patterns in SLE patients, the role of miR-26b-3p remains undefined [ 21 ]. Bioinformatic analyses suggest potential interaction between miR-26b-3p and XAF1, raising the possibility of a novel regulatory axis in SLE inflammation [ 22 , 23 ]. Here, we investigate the functional relationship between miR-26b-3p and XAF1 in SLE-associated inflammation. Using cellular models and patient-derived samples, we characterize the regulatory mechanisms of this axis and its potential implications for therapeutic intervention in SLE. 2. Materials and Methods 2.1 Cell Culture and Model Generation THP-1 cells (Wuhan PriCells Biotechnology) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.05 mM β-mercaptoethanol, and 1% penicillin-streptomycin at 37°C and 5% CO2. To generate an SLE inflammation model, THP-1 cells were first differentiated into macrophages using 1 µg/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 48 h, followed by stimulation with 100 ng/mL lipopolysaccharide (LPS, Sigma) for 24 h. 2.2 Lentiviral-Mediated Gene Manipulation Custom lentiviral vectors for XAF1 overexpression (OE-XAF1) and knockdown (SI-XAF1) were engineered by Shanghai GeneChem. Stable cell lines were established through lentiviral transduction using HiTransB-1 infection enhancer, followed by puromycin selection. Transduction efficiency was verified by quantifying XAF1 expression levels. 2.3 Experimental Methods 2.3.1 Cell Culture and Passage THP-1 cells were rapidly thawed at 37°C in a water bath and cultured in complete medium. Once the cells reached the logarithmic growth phase, achieving approximately 80% confluency, they were gently detached using a Pasteur pipette and passaged into new culture flasks at a 1:2 or 1:3 ratio. 2.3.2 RNA Extraction and RT-qPCR Total RNA isolation was performed using TRIzol reagent, followed by cDNA synthesis with a reverse transcription kit. The expression levels of XAF1, IL-6, IL-15, TNF-α, and β-actin were quantified using SYBR Green PCR Master Mix. The specific primers used for amplification are as follows: XAF1: Forward 5’-GTGTCCTGCTTGGTGCCTGAATC-3’, Reverse 5’-GTCCTTCCGTCCCTTTCTACAGTTC-3’. IL-6: Forward 5’-GACAGCCACTCACCTCTTCAGAAC-3’, Reverse 5’-GCCTCTTTGCTGCTTTCACACATG-3’. IL-15: Forward 5’-GCTGTTTCAGTGCAGGGCTTCC-3’, Reverse 5’- AACTGGGGTGAACATCACTTTCCG-3’. TNF-α: Forward 5’-AGCCCTGGTATGAGCCCATCTATC-3’, Reverse 5’-TCCCAAAGTAGACCTGCCCAGAC-3’ β-actin: Forward 5’-CAGGCACCAGGGCGTGAT-3’, Reverse 5’-TAGCAACGTACATGGCTGGG-3’. 2.3.3 Protein Extraction and Western Blot Analysis Cellular proteins were extracted using RIPA lysis buffer and quantified by BCA assay (Beyotime Biotechnology). Proteins were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and immunoblotted with anti-XAF1 antibody (Cell Signaling Technology). Protein bands were quantified using ImageJ software. 2.3.4 Dual-Luciferase Reporter Assay To validate direct interaction between miR-26b-3p and XAF1, wild-type and mutant XAF1 3'UTR sequences were cloned into pmiRGLO vectors. 293T cells [HEK- 293T] (Pricella, CL-0005) were transfected with these constructs, and luciferase activity was measured after 48 h using the Dual-Lumi™ kit (Beyotime Biotechnology). 2.3.5 Cytokine Quantification IL-6, IL-15, and TNF-α levels in culture supernatants were measured using specific ELISA kits (RayBiotech) following manufacturer's protocols. 2.3.6 Statistical Analysis Densitometric analysis of Western blot bands was conducted using ImageJ software. Relative gene expression levels from RT-qPCR data were calculated using the 2^-△△CT method. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) with SPSS 23.0 software. A P-value ≤ 0.05 was considered statistically significant. 3. Results 3.1 Establishment and Validation of XAF1-Modified THP-1 Cell Lines We engineered THP-1 cells to modulate XAF1 expression through lentiviral-mediated gene transfer, establishing stable cell lines with XAF1 overexpression (OE) and knockdown (SI). Fluorescence microscopy revealed robust viral transduction efficiency exceeding 80%, as evidenced by GFP expression (Fig. 1 ). Quantitative analysis demonstrated that XAF1 mRNA levels in the OE group increased by 3.2-fold (P < 0.05), while protein levels showed a 2.8-fold elevation (P < 0.01) compared to controls. Conversely, the SI group exhibited 75% reduction in XAF1 mRNA (P < 0.05) and 70% decrease in protein expression (P < 0.01) (Fig. 2 ). These results confirm successful bidirectional manipulation of XAF1 expression in THP-1 cells. 3.2 Establishment and Validation of LPS-Induced Inflammatory THP-1 Model We established a macrophage-based inflammatory model by initially differentiating THP-1 monocytes into adherent macrophages using PMA, followed by LPS stimulation (100 ng/mL). Cell viability assays revealed that THP-1 cells maintained > 95% viability during the first 24 hours of LPS exposure, with a significant decline observed only after 30 hours (27% reduction, P < 0.01) (Fig. S1 ). Immunofluorescence analysis of iNOS expression, a canonical M1 macrophage marker, showed a 4.2-fold increase in fluorescence intensity following LPS treatment, confirming successful M1 polarization (Fig. S2 ). This optimized protocol ensures robust inflammatory activation while maintaining cell viability within the experimental window. 3.3 LPS-Induced Upregulation of XAF1 and Pro-inflammatory Cytokines LPS stimulation significantly altered the expression profile of XAF1 and associated inflammatory mediators in both THP-1 cells and primary PBMCs. In THP-1 cells, XAF1 expression increased by 2.7-fold at the mRNA level and 2.4-fold at the protein level (P < 0.05) (Fig. 3 a-c). This upregulation was accompanied by marked elevation of SLE-associated cytokines, including IL-6 (3.8-fold), IL-15 (2.9-fold), and TNF-α (4.2-fold) (P < 0.01) (Fig. 3 d-i). Notably, similar XAF1 induction patterns were observed in PBMCs (2.5-fold increase, P < 0.01) (Fig. 4 ), suggesting a conserved regulatory mechanism in primary immune cells. These findings establish XAF1 as a potential mediator in LPS-induced inflammatory responses relevant to SLE pathogenesis. 3.4 Inverse Correlation between miR-26b-3p and XAF1 Expression in SLE Patients Comparative analysis of PBMC samples from SLE patients (n = 26) and healthy controls (n = 30) revealed a striking dysregulation of the miR-26b-3p/XAF1 axis. miR-26b-3p expression was significantly diminished in SLE patients (0.42-fold compared to controls, P < 0.01), while XAF1 levels showed reciprocal upregulation (Fig. S3a). In silico analysis identified evolutionarily conserved miR-26b-3p binding sites within the XAF1 3'UTR region (Fig. S3b). The inverse correlation between miR-26b-3p and XAF1 expression (r = -0.68, P < 0.001) suggests a potential regulatory mechanism in SLE pathogenesis. This clinical association provides the first evidence linking miR-26b-3p dysregulation to aberrant XAF1 expression in SLE patients. 3.5 Mechanistic Validation of miR-26b-3p-Mediated XAF1 Regulation To delineate the regulatory relationship between miR-26b-3p and XAF1, we first examined their expression dynamics in the LPS-induced inflammatory model. LPS stimulation resulted in a significant reduction in miR-26b-3p levels (0.45-fold, P < 0.05) (Fig. S4a), mirroring the pattern observed in SLE patients. Gain- and loss-of-function studies using miR-26b-3p mimics and inhibitors demonstrated robust modulation of miR-26b-3p expression (3.8-fold increase with mimics, P < 0.05; 0.3-fold decrease with inhibitors, P < 0.01) (Fig. S4b-c). Western blot analysis revealed that miR-26b-3p overexpression significantly suppressed XAF1 protein levels (0.4-fold, P < 0.01) (Fig. S4f-g), while miR-26b-3p inhibition enhanced XAF1 expression (2.6-fold, P < 0.01) (Fig. S4d-e). These reciprocal changes in XAF1 expression following miR-26b-3p manipulation establish a functional regulatory axis operating at both transcriptional and translational levels. 3.6 Direct Targeting of XAF1 by miR-26b-3p Confirmed by Luciferase Reporter Assays To establish direct molecular interaction between miR-26b-3p and XAF1, we employed dual-luciferase reporter assays using wild-type and mutant XAF1 3'UTR constructs. Co-transfection of miR-26b-3p mimics with wild-type XAF1 3'UTR resulted in significant suppression of luciferase activity (0.42-fold compared to control, P < 0.01). In contrast, mutation of the predicted miR-26b-3p binding site abolished this repressive effect (Fig. S5), maintaining luciferase activity at baseline levels. These results provide definitive evidence for direct targeting of XAF1 by miR-26b-3p through specific 3'UTR interaction, establishing the molecular basis for their functional relationship. 3.7 Functional Impact of the miR-26b-3p/XAF1 Axis on Inflammatory Cytokine Production To evaluate the functional significance of the miR-26b-3p/XAF1 regulatory axis in inflammation, we assessed its impact on pro-inflammatory cytokine production in LPS-stimulated THP-1 cells. XAF1 overexpression significantly enhanced the secretion of IL-6 (2.8-fold), IL-15 (2.4-fold), and TNF-α (3.2-fold) (P < 0.01) (Fig. 5 a-c), while XAF1 knockdown markedly attenuated cytokine production (reduction by 65–75%, P < 0.001) (Fig. 5 d-f). Consistent with its suppressive effect on XAF1, miR-26b-3p mimics significantly reduced inflammatory cytokine levels (40–55% reduction, P < 0.05) (Fig. 5 g-i), whereas miR-26b-3p inhibition enhanced cytokine production (1.8-2.3-fold increase, P < 0.05) (Fig. 5 j-l). Notably, miR-26b-3p mimics effectively reversed the enhanced cytokine production in XAF1-overexpressing cells (reduction by 60–70%, P < 0.001) (Fig. 5 m-o), demonstrating the dominant regulatory role of this microRNA. These findings establish the miR-26b-3p/XAF1 axis as a critical modulator of inflammatory responses in this cellular model of SLE. 4. Discussion Our findings reveal a previously unrecognized regulatory mechanism in SLE pathogenesis, demonstrating that the miR-26b-3p/XAF1 axis serves as a critical modulator of inflammatory responses. This work provides several key insights into the molecular basis of SLE-associated inflammation and suggests potential therapeutic strategies. Novel Role of XAF1 in SLE Inflammation We demonstrate that XAF1 expression is significantly elevated in both LPS-stimulated THP-1 cells and patient-derived PBMCs [ 24 ], correlating with increased production of pro-inflammatory cytokines [ 25 ]. This observation extends beyond XAF1's known function as a tumor suppressor, revealing its novel role in autoimmune inflammation. The consistent upregulation of XAF1 across different experimental models and patient samples suggests its potential utility as a biomarker for disease activity. miR-26b-3p as a Novel Regulator of XAF1 Through multiple complementary approaches, we establish miR-26b-3p as a direct regulator of XAF1 [ 23 ]. The inverse correlation between miR-26b-3p and XAF1 expression in SLE patient samples, combined with our mechanistic studies using mimics and inhibitors, provides compelling evidence for this regulatory relationship. The dual-luciferase reporter assays definitively confirm direct binding between miR-26b-3p and XAF1's 3'UTR, establishing a molecular basis for this regulation. Functional Impact on Inflammatory Responses Our gain- and loss-of-function experiments reveal that manipulating the miR-26b-3p/XAF1 axis directly affects the production of key inflammatory mediators (IL-6, IL-15, and TNF-α) [ 26 , 27 ]. Particularly noteworthy is the rescue effect observed when combining XAF1 overexpression with miR-26b-3p mimics, supporting the specificity of this regulatory pathway in controlling inflammatory responses. Experimental Model Validation The LPS-stimulated THP-1 cell model effectively recapitulates key aspects of SLE inflammation [ 28 , 29 ], as evidenced by the parallel changes in inflammatory markers observed in patient samples. While this model system has limitations, its reproducibility and controllability make it valuable for mechanistic studies of SLE pathogenesis. Study Limitations and Future Directions Several limitations of our study warrant consideration. First, while our in vitro models provide valuable mechanistic insights, validation in additional patient cohorts and alternative cell systems would strengthen our findings. Second, the broader network of molecules and signaling pathways that interact with the miR-26b-3p/XAF1 axis remains to be fully elucidated. Future studies should explore: 1) The upstream regulators of miR-26b-3p in SLE; 2) The complete spectrum of XAF1 targets in immune cells; 3) The potential therapeutic applications of targeting this pathway; 4) The role of this axis in specific SLE manifestations. 5. Conclusions The identification of the miR-26b-3p/XAF1 regulatory axis opens new avenues for therapeutic intervention in SLE. Strategies aimed at modulating this pathway, either through miRNA-based approaches or direct targeting of XAF1, merit further investigation as potential treatments for SLE and possibly other autoimmune disorders. Declarations Ethics declaration : Peripheral blood mononuclear cells (PBMCs) were obtained from SLE patients (n = 26) and healthy controls (n = 30) with written informed consent. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Ethics approval number: 2021-KY-95) and conducted in accordance with the Declaration of Helsinki. Conflict of Interest: The authors declare no conflicts of interest related to this manuscript. Consent to Publish declaration : Not applicable. Funding: This work was supported by the Science and Technology Program of Guangzhou, China (grant numbers 2024B03J0436); Research Funds of Centre for Leading Medicine and Advanced Technologies of IHM (grant number 2023IHM01052). Author Contribution SL and ZXH conceived the study design. SL, CW, HL, and TH wrote the initial draft of the manuscript, which was subsequently revised by SL, CW, HL, ZXH, and TH conducted the data analysis and jointly carried out the experiments. All authors have reviewed and approved the final version of the manuscript. Acknowledgment: None. Data Availability All data in this study are genuine and reliable. The data supporting the findings of this study can be obtained from the corresponding author upon reasonable request. References Accapezzato, D. et al. Advances in the pathogenesis and treatment of systemic lupus erythematosus. Int. J. Mol. Sci. 24 (7), 6578 (2023). Goulielmos, G. N. et al. The genetics and molecular pathogenesis of systemic lupus erythematosus (SLE) in populations of different ancestry. Gene 668 , 59–72 (2018). Hoi, A., Igel, T., Mok, C. C. & Arnaud, L. Systemic lupus erythematosus. Lancet 403 (10441), 2326–2338 (2024). Sutanto, H. & Yuliasih, Y. 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15:48:50","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80573,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7272649/v1/09058e4e04bc8d1a9a6db6b1.html"},{"id":94687560,"identity":"c2a01c1b-f710-4dfc-80c3-0deabb80f2f6","added_by":"auto","created_at":"2025-10-29 15:48:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":185155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroscopic presentation of THP-1 cells after virus transfection. \u003c/strong\u003e(a1) Bright field performance of OENC group. (a2) Fluorescence performance of OENC group in dark field. (b1) Bright field performance of OE group. (b2) Fluorescence performance of OE group in dark field. (c1) Bright field performance of SINC group. (c2) Fluorescence performance of SINC group in dark field. (d1) Bright field performance of SI group. (d2) Fluorescence performance of SI group in dark field. Note: OENC represents the Overexpression Empty Negative Control group; OE represents Overexpression group; SINC represents Scrambled Interfering Negative Control group; SI represents Scrambled Interfering group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7272649/v1/8aec30783f1bae1eb225dd57.png"},{"id":94687557,"identity":"8204fbd6-ee55-4961-a2d0-b6e4fd68ba72","added_by":"auto","created_at":"2025-10-29 15:48:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":379186,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of transfection efficiency of THP-1 cells transfected with inhibition and overexpression lentivirus.\u003c/strong\u003e (a) Compared with the OENC group, the expression level of XAF1 mRNA was significantly upregulated (P\u0026lt;0.05) in the OE group after transfection with XAF1 overexpression lentivirus. (b) Compared with the SINC group, the expression level of XAF1 mRNA was significantly downregulated (P\u0026lt;0.05) in the SI group after transfection with XAF1 knockdown virus. (c, d) Compared with the OENC group, the expression level of XAF1 protein was significantly upregulated (P\u0026lt;0.01) in the OE group. (E, F) Compared with the SINC group, the expression level of XAF1 protein was significantly downregulated (P\u0026lt;0.01) in the SI group. *P\u0026lt;0.05, **P\u0026lt;0.01. Note: OENC represents the Overexpression Empty Negative Control group; OE represents Overexpression group; SINC represents Scrambled Interfering Negative Control group; SI represents Scrambled Interfering group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7272649/v1/c347a4defdfb32f775ab1538.png"},{"id":94728547,"identity":"ef459beb-7187-4784-b987-014e139ff74f","added_by":"auto","created_at":"2025-10-30 07:04:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":286572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in XAF1 and cytokine expression after LPS stimulation of THP-1.\u003c/strong\u003e(a) Comparison of XAF1 mRNA expression between NC group and LPS group. (b c) Comparison of XAF1 protein expression between NC group and LPS group. (d) Comparison of the expression of IL6 mRNA between NC group and LPS group. (e) Comparison of the expression of IL6 mRNA between N group and LPS group. (f) Comparison of the expression of TNF-α mRNA between N group and LPS group. (g) Comparison of IL6 protein expression between NC group and LPS group. (h) Comparison of IL15 protein expression between NC group and LPS group. (i) Comparison of TNF-ɑ protein expression between NC group and LPS group. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001. Note: NC-24h represents Normal culture for 24 hours group; LPS-24h represents LPS stimulation for 24 hours group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7272649/v1/338f54d60ab664029472ada7.png"},{"id":94728686,"identity":"a6c7c45c-fee7-4c94-8676-d2d8bfdd4212","added_by":"auto","created_at":"2025-10-30 07:04:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":121307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in XAF1 expression after LPS stimulation of PBMC. \u003c/strong\u003e(a) Comparison of XAF1 mRNA expression between NC group and LPS group. (bc) Comparison of XAF-1 protein expression between NC group and LPS group. ** P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7272649/v1/d82e4cc50d321f07d3b32b49.png"},{"id":94687562,"identity":"c0cac1b1-cbde-40a7-9af6-d3c6d3a146b1","added_by":"auto","created_at":"2025-10-29 15:48:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":80188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of miR-26-3p and XAF1 on LPS-induced THP-1 production of inflammatory cytokines. \u003c/strong\u003e(a) Comparison of the expression of IL6 between OENC group and ON group. (b) Comparison of the expression of IL15 between OENC group and ON group. (c) Comparison of the expression of TNF-α between OENC group and ON group. (d) Comparison of the expression of IL6 between SINC group and SI group. (e) Comparison of the expression of IL15 between SINC group and SI group. (f) Comparison of the expression of TNF-α between SINC group and SI group. (g) Comparison of the expression of IL6 between mimicsNC group and mimics group. (h) Comparison of the expression of IL15 between mimicsNC group and mimics group. (i) Comparison of the expression of TNF-α between mimicsNC group and mimics group. (j) Comparison of the expression of IL6 between inhibitorNC group and inhibitor group. (k) Comparison of the expression of IL15 between inhibitorNC group and inhibitor group. (l) Comparison of the expression of TNF-α between inhibitorNC group and inhibitor group. (m) Comparison of the expression of IL6 between OE group and OE+mimics group. (n) Comparison of the expression of IL15 between OE group and OE+mimics group. (o) Comparison of the expression of TNF-α between OE group and OE+mimics group. *P＜0.05, **P＜0.01,***P＜0.001.Note: OE+mimics represents XAF-1 overexpressing cells transfected with miRNA mimics group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7272649/v1/7925df6972eaa49eaccfc5e4.png"},{"id":104401165,"identity":"c61ed6e1-408f-4d52-a044-7777e320d420","added_by":"auto","created_at":"2026-03-11 12:12:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2074244,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7272649/v1/6ea73379-a4ff-45f7-8174-5912f4cb29b0.pdf"},{"id":94728865,"identity":"73cc7278-9e09-4d84-ba2a-191b64825a7c","added_by":"auto","created_at":"2025-10-30 07:04:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1751168,"visible":true,"origin":"","legend":"","description":"","filename":"supplement.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7272649/v1/b6e646af156ddf462d657768.pdf"},{"id":94687574,"identity":"76714a63-e53b-4a09-8530-be3c4265c776","added_by":"auto","created_at":"2025-10-29 15:48:50","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2735874,"visible":true,"origin":"","legend":"","description":"","filename":"supplementalmaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7272649/v1/e5c65f9c47f550d640033f68.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unveiling the miR-26b-3p/XAF1 Axis: A New Frontier in Systemic Lupus Erythematosus Inflammation Running Head: miR-26b-3p–XAF1 regulation in lupus","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSystemic lupus erythematosus (SLE) represents a complex autoimmune disorder characterized by multi-organ inflammation and tissue damage [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite advances in understanding its pathogenesis, the molecular mechanisms underlying SLE remain incompletely understood [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Recent evidence suggests that dysregulated inflammatory responses play a central role in disease progression and severity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe immunopathogenesis of SLE involves intricate interactions between innate and adaptive immunity [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Notably, aberrant B cell activation leads to autoantibody production, while dysregulated T cell responses amplify inflammatory cascades through co-stimulatory pathways and cytokine production [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Within the innate immune compartment, macrophage polarization states critically influence disease progression, particularly through their role in cytokine regulation and immune complex processing [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eInflammatory cytokines serve as key mediators in SLE pathogenesis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Elevated levels of IL-6 correlate with disease activity, while increased IL-15 promotes lymphocyte proliferation and survival [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Similarly, TNF-α contributes to vascular dysfunction and organ damage, highlighting the complex cytokine networks in SLE progression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent evidence has implicated XAF1, a negative regulator of the anti-apoptotic protein XIAP, in various pathological conditions. While XAF1's role as a tumor suppressor is well-documented, its function in autoimmune inflammation remains unexplored [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Notably, XAF1 activation occurs under conditions of cellular stress and cytokine stimulation, suggesting potential involvement in inflammatory responses.\u003c/p\u003e\u003cp\u003eThe bacterial endotoxin lipopolysaccharide (LPS) represents a crucial environmental trigger in SLE, activating Toll-like receptor signaling pathways and potentially precipitating disease flares [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This mechanism provides a valuable experimental model for investigating inflammatory responses in SLE [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMicroRNAs have emerged as critical regulators of immune responses in SLE. While several microRNAs, including miR-21 and miR-155, demonstrate altered expression patterns in SLE patients, the role of miR-26b-3p remains undefined [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Bioinformatic analyses suggest potential interaction between miR-26b-3p and XAF1, raising the possibility of a novel regulatory axis in SLE inflammation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHere, we investigate the functional relationship between miR-26b-3p and XAF1 in SLE-associated inflammation. Using cellular models and patient-derived samples, we characterize the regulatory mechanisms of this axis and its potential implications for therapeutic intervention in SLE.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e2.1 Cell Culture and Model Generation\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTHP-1 cells (Wuhan PriCells Biotechnology) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.05 mM β-mercaptoethanol, and 1% penicillin-streptomycin at 37\u0026deg;C and 5% CO2. To generate an SLE inflammation model, THP-1 cells were first differentiated into macrophages using 1 \u0026micro;g/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 48 h, followed by stimulation with 100 ng/mL lipopolysaccharide (LPS, Sigma) for 24 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Lentiviral-Mediated Gene Manipulation\u003c/h2\u003e\u003cp\u003eCustom lentiviral vectors for XAF1 overexpression (OE-XAF1) and knockdown (SI-XAF1) were engineered by Shanghai GeneChem. Stable cell lines were established through lentiviral transduction using HiTransB-1 infection enhancer, followed by puromycin selection. Transduction efficiency was verified by quantifying XAF1 expression levels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Experimental Methods\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 Cell Culture and Passage\u003c/h2\u003e\u003cp\u003eTHP-1 cells were rapidly thawed at 37\u0026deg;C in a water bath and cultured in complete medium. Once the cells reached the logarithmic growth phase, achieving approximately 80% confluency, they were gently detached using a Pasteur pipette and passaged into new culture flasks at a 1:2 or 1:3 ratio.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 \u003cem\u003eRNA Extraction and RT-qPCR\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTotal RNA isolation was performed using TRIzol reagent, followed by cDNA synthesis with a reverse transcription kit. The expression levels of XAF1, IL-6, IL-15, TNF-α, and β-actin were quantified using SYBR Green PCR Master Mix. The specific primers used for amplification are as follows:\u003c/p\u003e\u003cp\u003eXAF1: Forward 5\u0026rsquo;-GTGTCCTGCTTGGTGCCTGAATC-3\u0026rsquo;,\u003c/p\u003e\u003cp\u003eReverse 5\u0026rsquo;-GTCCTTCCGTCCCTTTCTACAGTTC-3\u0026rsquo;.\u003c/p\u003e\u003cp\u003eIL-6: Forward 5\u0026rsquo;-GACAGCCACTCACCTCTTCAGAAC-3\u0026rsquo;,\u003c/p\u003e\u003cp\u003eReverse 5\u0026rsquo;-GCCTCTTTGCTGCTTTCACACATG-3\u0026rsquo;.\u003c/p\u003e\u003cp\u003eIL-15: Forward 5\u0026rsquo;-GCTGTTTCAGTGCAGGGCTTCC-3\u0026rsquo;,\u003c/p\u003e\u003cp\u003eReverse 5\u0026rsquo;- AACTGGGGTGAACATCACTTTCCG-3\u0026rsquo;.\u003c/p\u003e\u003cp\u003eTNF-α: Forward 5\u0026rsquo;-AGCCCTGGTATGAGCCCATCTATC-3\u0026rsquo;,\u003c/p\u003e\u003cp\u003eReverse 5\u0026rsquo;-TCCCAAAGTAGACCTGCCCAGAC-3\u0026rsquo;\u003c/p\u003e\u003cp\u003eβ-actin: Forward 5\u0026rsquo;-CAGGCACCAGGGCGTGAT-3\u0026rsquo;,\u003c/p\u003e\u003cp\u003eReverse 5\u0026rsquo;-TAGCAACGTACATGGCTGGG-3\u0026rsquo;.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3 Protein Extraction and Western Blot Analysis\u003c/h2\u003e\u003cp\u003eCellular proteins were extracted using RIPA lysis buffer and quantified by BCA assay (Beyotime Biotechnology). Proteins were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and immunoblotted with anti-XAF1 antibody (Cell Signaling Technology). Protein bands were quantified using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.3.4 Dual-Luciferase Reporter Assay\u003c/h2\u003e\u003cp\u003eTo validate direct interaction between miR-26b-3p and XAF1, wild-type and mutant XAF1 3'UTR sequences were cloned into pmiRGLO vectors. 293T cells [HEK- 293T] (Pricella, CL-0005) were transfected with these constructs, and luciferase activity was measured after 48 h using the Dual-Lumi\u0026trade; kit (Beyotime Biotechnology).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.3.5 Cytokine Quantification\u003c/h2\u003e\u003cp\u003eIL-6, IL-15, and TNF-α levels in culture supernatants were measured using specific ELISA kits (RayBiotech) following manufacturer's protocols.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.3.6 Statistical Analysis\u003c/h2\u003e\u003cp\u003eDensitometric analysis of Western blot bands was conducted using ImageJ software. Relative gene expression levels from RT-qPCR data were calculated using the 2^-△△CT method. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) with SPSS 23.0 software. A P-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Establishment and Validation of XAF1-Modified THP-1 Cell Lines\u003c/h2\u003e\u003cp\u003eWe engineered THP-1 cells to modulate XAF1 expression through lentiviral-mediated gene transfer, establishing stable cell lines with XAF1 overexpression (OE) and knockdown (SI). Fluorescence microscopy revealed robust viral transduction efficiency exceeding 80%, as evidenced by GFP expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Quantitative analysis demonstrated that XAF1 mRNA levels in the OE group increased by 3.2-fold (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while protein levels showed a 2.8-fold elevation (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to controls. Conversely, the SI group exhibited 75% reduction in XAF1 mRNA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 70% decrease in protein expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results confirm successful bidirectional manipulation of XAF1 expression in THP-1 cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Establishment and Validation of LPS-Induced Inflammatory THP-1 Model\u003c/h2\u003e\u003cp\u003eWe established a macrophage-based inflammatory model by initially differentiating THP-1 monocytes into adherent macrophages using PMA, followed by LPS stimulation (100 ng/mL). Cell viability assays revealed that THP-1 cells maintained\u0026thinsp;\u0026gt;\u0026thinsp;95% viability during the first 24 hours of LPS exposure, with a significant decline observed only after 30 hours (27% reduction, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Immunofluorescence analysis of iNOS expression, a canonical M1 macrophage marker, showed a 4.2-fold increase in fluorescence intensity following LPS treatment, confirming successful M1 polarization (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). This optimized protocol ensures robust inflammatory activation while maintaining cell viability within the experimental window.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3 LPS-Induced Upregulation of XAF1 and Pro-inflammatory Cytokines\u003c/h2\u003e\u003cp\u003eLPS stimulation significantly altered the expression profile of XAF1 and associated inflammatory mediators in both THP-1 cells and primary PBMCs. In THP-1 cells, XAF1 expression increased by 2.7-fold at the mRNA level and 2.4-fold at the protein level (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). This upregulation was accompanied by marked elevation of SLE-associated cytokines, including IL-6 (3.8-fold), IL-15 (2.9-fold), and TNF-α (4.2-fold) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-i). Notably, similar XAF1 induction patterns were observed in PBMCs (2.5-fold increase, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting a conserved regulatory mechanism in primary immune cells. These findings establish XAF1 as a potential mediator in LPS-induced inflammatory responses relevant to SLE pathogenesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Inverse Correlation between miR-26b-3p and XAF1 Expression in SLE Patients\u003c/h2\u003e\u003cp\u003eComparative analysis of PBMC samples from SLE patients (n\u0026thinsp;=\u0026thinsp;26) and healthy controls (n\u0026thinsp;=\u0026thinsp;30) revealed a striking dysregulation of the miR-26b-3p/XAF1 axis. miR-26b-3p expression was significantly diminished in SLE patients (0.42-fold compared to controls, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while XAF1 levels showed reciprocal upregulation (Fig. S3a). In silico analysis identified evolutionarily conserved miR-26b-3p binding sites within the XAF1 3'UTR region (Fig. S3b). The inverse correlation between miR-26b-3p and XAF1 expression (r = -0.68, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) suggests a potential regulatory mechanism in SLE pathogenesis. This clinical association provides the first evidence linking miR-26b-3p dysregulation to aberrant XAF1 expression in SLE patients.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Mechanistic Validation of miR-26b-3p-Mediated XAF1 Regulation\u003c/h2\u003e\u003cp\u003eTo delineate the regulatory relationship between miR-26b-3p and XAF1, we first examined their expression dynamics in the LPS-induced inflammatory model. LPS stimulation resulted in a significant reduction in miR-26b-3p levels (0.45-fold, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. S4a), mirroring the pattern observed in SLE patients. Gain- and loss-of-function studies using miR-26b-3p mimics and inhibitors demonstrated robust modulation of miR-26b-3p expression (3.8-fold increase with mimics, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; 0.3-fold decrease with inhibitors, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig. S4b-c).\u003c/p\u003e\u003cp\u003eWestern blot analysis revealed that miR-26b-3p overexpression significantly suppressed XAF1 protein levels (0.4-fold, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig. S4f-g), while miR-26b-3p inhibition enhanced XAF1 expression (2.6-fold, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig. S4d-e). These reciprocal changes in XAF1 expression following miR-26b-3p manipulation establish a functional regulatory axis operating at both transcriptional and translational levels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Direct Targeting of XAF1 by miR-26b-3p Confirmed by Luciferase Reporter Assays\u003c/h2\u003e\u003cp\u003eTo establish direct molecular interaction between miR-26b-3p and XAF1, we employed dual-luciferase reporter assays using wild-type and mutant XAF1 3'UTR constructs. Co-transfection of miR-26b-3p mimics with wild-type XAF1 3'UTR resulted in significant suppression of luciferase activity (0.42-fold compared to control, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, mutation of the predicted miR-26b-3p binding site abolished this repressive effect (Fig. S5), maintaining luciferase activity at baseline levels. These results provide definitive evidence for direct targeting of XAF1 by miR-26b-3p through specific 3'UTR interaction, establishing the molecular basis for their functional relationship.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Functional Impact of the miR-26b-3p/XAF1 Axis on Inflammatory Cytokine Production\u003c/h2\u003e\u003cp\u003eTo evaluate the functional significance of the miR-26b-3p/XAF1 regulatory axis in inflammation, we assessed its impact on pro-inflammatory cytokine production in LPS-stimulated THP-1 cells. XAF1 overexpression significantly enhanced the secretion of IL-6 (2.8-fold), IL-15 (2.4-fold), and TNF-α (3.2-fold) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c), while XAF1 knockdown markedly attenuated cytokine production (reduction by 65\u0026ndash;75%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-f).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConsistent with its suppressive effect on XAF1, miR-26b-3p mimics significantly reduced inflammatory cytokine levels (40\u0026ndash;55% reduction, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg-i), whereas miR-26b-3p inhibition enhanced cytokine production (1.8-2.3-fold increase, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej-l). Notably, miR-26b-3p mimics effectively reversed the enhanced cytokine production in XAF1-overexpressing cells (reduction by 60\u0026ndash;70%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003em-o), demonstrating the dominant regulatory role of this microRNA. These findings establish the miR-26b-3p/XAF1 axis as a critical modulator of inflammatory responses in this cellular model of SLE.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur findings reveal a previously unrecognized regulatory mechanism in SLE pathogenesis, demonstrating that the miR-26b-3p/XAF1 axis serves as a critical modulator of inflammatory responses. This work provides several key insights into the molecular basis of SLE-associated inflammation and suggests potential therapeutic strategies.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNovel Role of XAF1 in SLE Inflammation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe demonstrate that XAF1 expression is significantly elevated in both LPS-stimulated THP-1 cells and patient-derived PBMCs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], correlating with increased production of pro-inflammatory cytokines [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This observation extends beyond XAF1's known function as a tumor suppressor, revealing its novel role in autoimmune inflammation. The consistent upregulation of XAF1 across different experimental models and patient samples suggests its potential utility as a biomarker for disease activity.\u003c/p\u003e\u003cp\u003e\u003cb\u003emiR-26b-3p as a Novel Regulator of XAF1\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThrough multiple complementary approaches, we establish miR-26b-3p as a direct regulator of XAF1 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The inverse correlation between miR-26b-3p and XAF1 expression in SLE patient samples, combined with our mechanistic studies using mimics and inhibitors, provides compelling evidence for this regulatory relationship. The dual-luciferase reporter assays definitively confirm direct binding between miR-26b-3p and XAF1's 3'UTR, establishing a molecular basis for this regulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFunctional Impact on Inflammatory Responses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur gain- and loss-of-function experiments reveal that manipulating the miR-26b-3p/XAF1 axis directly affects the production of key inflammatory mediators (IL-6, IL-15, and TNF-α) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Particularly noteworthy is the rescue effect observed when combining XAF1 overexpression with miR-26b-3p mimics, supporting the specificity of this regulatory pathway in controlling inflammatory responses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental Model Validation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe LPS-stimulated THP-1 cell model effectively recapitulates key aspects of SLE inflammation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], as evidenced by the parallel changes in inflammatory markers observed in patient samples. While this model system has limitations, its reproducibility and controllability make it valuable for mechanistic studies of SLE pathogenesis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStudy Limitations and Future Directions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeveral limitations of our study warrant consideration. First, while our in vitro models provide valuable mechanistic insights, validation in additional patient cohorts and alternative cell systems would strengthen our findings. Second, the broader network of molecules and signaling pathways that interact with the miR-26b-3p/XAF1 axis remains to be fully elucidated. Future studies should explore: 1) The upstream regulators of miR-26b-3p in SLE; 2) The complete spectrum of XAF1 targets in immune cells; 3) The potential therapeutic applications of targeting this pathway; 4) The role of this axis in specific SLE manifestations.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThe identification of the miR-26b-3p/XAF1 regulatory axis opens new avenues for therapeutic intervention in SLE. Strategies aimed at modulating this pathway, either through miRNA-based approaches or direct targeting of XAF1, merit further investigation as potential treatments for SLE and possibly other autoimmune disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e: Peripheral blood mononuclear cells (PBMCs) were obtained from SLE patients (n\u0026thinsp;=\u0026thinsp;26) and healthy controls (n\u0026thinsp;=\u0026thinsp;30) with written informed consent. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Ethics approval number: 2021-KY-95) and conducted in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003ch2\u003eConflict of Interest:\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflicts of interest related to this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003edeclaration\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Science and Technology Program of Guangzhou, China (grant numbers 2024B03J0436); Research Funds of Centre for Leading Medicine and Advanced Technologies of IHM (grant number 2023IHM01052).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eSL and ZXH conceived the study design. SL, CW, HL, and TH wrote the initial draft of the manuscript, which was subsequently revised by SL, CW, HL, ZXH, and TH conducted the data analysis and jointly carried out the experiments. All authors have reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgment:\u003c/h2\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data in this study are genuine and reliable. The data supporting the findings of this study can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAccapezzato, D. et al. Advances in the pathogenesis and treatment of systemic lupus erythematosus. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e (7), 6578 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGoulielmos, G. N. et al. 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Rheumatol.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e (9), 515\u0026ndash;532 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Systemic Lupus Erythematosus, XAF1, Inflammation Regulation, THP-1 Cells, LPS Stimulation","lastPublishedDoi":"10.21203/rs.3.rs-7272649/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7272649/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSystemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by dysregulated inflammatory responses, yet the molecular mechanisms underlying inflammation regulation remain incompletely understood. XAF1, traditionally recognized as a tumor suppressor, and miR-26b-3p, a microRNA with emerging immunoregulatory functions, have not been previously investigated in SLE pathogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAims\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the regulatory relationship between miR-26b-3p and XAF1 and determine their functional significance in modulating inflammatory responses associated with SLE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTHP-1 monocytes were differentiated into macrophages and stimulated with lipopolysaccharide (LPS) to establish an inflammatory model. Lentiviral vectors were employed to generate XAF1 overexpression and knockdown cell lines. Peripheral blood mononuclear cells (PBMCs) from SLE patients (n = 26) and healthy controls (n = 30) were analyzed. The miR-26b-3p-XAF1 interaction was validated using quantitative PCR, western blotting, dual-luciferase reporter assays, and gain/loss-of-function studies. Pro-inflammatory cytokine levels (IL-6, IL-15, TNF-α) were quantified by ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLPS stimulation significantly upregulated XAF1 expression (2.7-fold mRNA, 2.4-fold protein, P \u0026lt; 0.05) alongside marked elevation of pro-inflammatory cytokines IL-6, IL-15, and TNF-α (2.9-4.2-fold increases, P \u0026lt; 0.01) in THP-1 cells. SLE patient PBMCs demonstrated significantly reduced miR-26b-3p expression (0.42-fold, P \u0026lt; 0.01) with reciprocal XAF1 upregulation, showing strong inverse correlation (r=-0.68, P \u0026lt; 0.001). Dual-luciferase reporter assays confirmed direct targeting of XAF1 3'UTR by miR-26b-3p (0.42-fold luciferase activity, P \u0026lt; 0.01). Functional studies revealed that XAF1 overexpression enhanced cytokine production (2.4-3.2-fold, P \u0026lt; 0.01), while miR-26b-3p mimics effectively suppressed both XAF1 expression and inflammatory cytokine secretion (40–55% reduction, P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur findings establish the miR-26b-3p/XAF1 axis as a novel regulatory mechanism in SLE inflammation, with miR-26b-3p functioning as a negative regulator of XAF1-mediated inflammatory responses. This discovery highlights the therapeutic potential of targeting this axis for SLE treatment and advances our understanding of microRNA-mediated regulation in autoimmune inflammation.\u003c/p\u003e","manuscriptTitle":"Unveiling the miR-26b-3p/XAF1 Axis: A New Frontier in Systemic Lupus Erythematosus Inflammation Running Head: miR-26b-3p–XAF1 regulation in lupus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 15:48:45","doi":"10.21203/rs.3.rs-7272649/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"939be831-ed42-4a99-803d-5fbe2b565a6f","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56901860,"name":"Health sciences/Diseases"},{"id":56901861,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2026-03-04T04:40:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-29 15:48:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7272649","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7272649","identity":"rs-7272649","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}