Exosomal miR-146a-5p mediates macrophage polarization through TRAF6/NF-κB signaling in endometriosis

doi:10.21203/rs.3.rs-4988177/v1

Download PDF

Article

Exosomal miR-146a-5p mediates macrophage polarization through TRAF6/NF-κB signaling in endometriosis

https://doi.org/10.21203/rs.3.rs-4988177/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Exosomes play significant roles in immune responses, neurogenesis, and angiogenesis, directly impacting the progression and symptomatic manifestations of endometriosis. This study aimed to investigate the role of exosomal miR-146a-5p in the pathogenesis of endometriosis. Through high-throughput RNA sequencing and qRT‒PCR, we revealed significant upregulation of miR-146a-5p in ectopic endothelial tissues, and Gene Ontology (GO) analysis revealed that miR-146a-5p has only one target, TNF receptor associated factor 6 (TRAF6). KEGG pathway analysis indicated that the NF-κB signaling pathway is the key signaling pathway involved. The study revealed that the upregulation of miR-146a-5p in macrophages is associated with an increase in M2 macrophages. In the U937 macrophage line, miR-146a-5p was capable of suppressing TRAF6 expression, which in turn decreased the phosphorylation level of NF-κB, whereas the overexpression of TRAF6 increased the activity of this pathway. Furthermore, we incorporated the NF-κB inhibitor EVP4593 into a macrophage culture, which revealed that blocking this pathway significantly induced both M1 and M2 macrophage polarization, particularly enhancing M2 polarization. In conclusion, exosome-derived miR-146a-5p promotes M2 polarization of macrophages by regulating the TRAF6/NF-κB pathway in endometriosis.

Health sciences/Medical research/Biomarkers/Predictive markers

Biological sciences/Molecular biology/Non coding rnas/Mirnas

Health sciences/Diseases/Reproductive disorders

Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages

Exosome

Endometriosis

miR-146a-5p

Macrophage polarization

Endometrial stromal cells

Signaling pathway

Endometriosis is an estrogen-dependent disease characterized by the presence of endometrial stromal cells (ESCs) and glands outside the uterine cavity, which can cause pelvic pain and infertility^1,2. Although endometriosis is a benign disease, it is associated with malignant biological behaviors such as distant metastasis, adhesion, invasion, implantation, and recurrence³. This process is associated with genes related to pathways such as cell proliferation, angiogenesis, neurogenesis, and inflammation⁴. Recently, emerging evidence has suggested that genetic predispositions and associated immunological abnormalities are key to the pathogenesis of endometriosis⁵.

Under normal conditions, peritoneal macrophages are capable of eliminating endometrial tissue that enters the peritoneal cavity via retrograde blood flow⁶. However, in patients with endometriosis, dysfunctional immune responses lead to a decrease in the phagocytic capacity of macrophages or alterations in the cytokine environment, often resulting in the ineffective surveillance and clearance of endometrial tissue deposited within the peritoneal cavity^7,8. This defect in immunosurveillance allows the growth of endometriotic lesions and the persistence of disease symptoms⁹. The polarization state of macrophages is reversible, allowing them to change phenotypes according to the needs of the microenvironment^10,11. Macrophages initially present as the M1 phenotype, secreting proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 that trigger an inflammatory response, whereas M2-type macrophages secrete anti-inflammatory factors to suppress inflammation and maintain homeostasis^12–14. The ability of macrophages to dynamically switch their phenotype and function in response to microenvironment signals makes them potential therapeutic targets.

Notably, the role of microRNAs in regulating macrophage polarization has been well defined, with the overexpression of miRNA-155 shown to inhibit C/EBPβ protein expression and regulate the production of inflammatory cytokines in TAMs by targeting C/EBP¹⁵. Furthermore, numerous studies have investigated the function of microRNAs in endometriosis, with findings indicating that microRNAs are reliable biomarkers for this disease^16,17. Therefore, we suspect that the dynamic switching of macrophage phenotypes may be influenced by microRNAs.

Our preliminary experimental results suggested that exosomes derived from ectopic ESCs promote M2 macrophage polarization by delivering miR-146a-5p, which targets TRAF6, in the pathological process of endometriosis¹⁵. TRAF6 can activate multiple signaling pathways, such as the NF-κB pathway¹⁸. Additionally, research supports a close relationship between microRNAs and the NF-κB signaling pathway in certain diseases, including rheumatoid arthritis, cervical cancer, and colorectal cancer^19–23. Thus, we hypothesized that miR-146a-5p may play an important role in the polarization process of endometriosis-related macrophages.

Exosomes from ectopic ESCs enter macrophages. To investigate the impact of ESC-derived exosomes on macrophages, we isolated exosomes from eutopic and ectopic ESCs (vimentin positive, CK7 negative; Fig. 1A). Transwell assays and exosome tracking experiments verified that the exosomes derived from the mesenchymal stromal cells of the donor could reach the recipient cells and be internalized, along with their contained cellular contents. The internalization of exosomes by U937 macrophages was visualized through Cy3 red fluorescence labeling (Fig. 1B).

Exosomes induce macrophage polarization toward the M2 phenotype by secreting miR-146a-5p. In our previous study, miR-146a-5p was silenced in inhibitor-transfected ectopic ESCs and overexpressed in mimic-transfected ESCs. Consequently, in the exosomes of these transfected ESCs, the expression of miR-146a-5p was also correspondingly silenced and overexpressed²⁴, indicating that miR-146a-5p facilitates the polarization of M2 macrophages (data not shown). Flow cytometry analysis revealed that after treatment with the exosome inhibitor GW4869, ectopic ESCs significantly increased the expression of CD86 and iNOS (Fig. 2A, B), whereas the proportions of M2 macrophages expressing CD163 and CD206 were reduced (Fig. 2C, D). Flow cytometry plots of the control and GW4869 groups are shown.

miR-146a-5p inhibits TRAF6 expression by targeting its 3′UTR in macrophages. U937 macrophages treated with exosomes from miR-146a-5p mimic- or inhibitor-transfected ectopic ESCs were used to detect miR-146a-5p and TRAF6 levels. Exosomes transfected with the miR-146a-5p inhibitor significantly increased TRAF6 expression, whereas exosomes transfected with the miR-146a-5p mimic significantly suppressed TRAF6 expression (Fig. 3A, B). The exosomes transfected with the miR-146a-5p inhibitor significantly increased the protein expression of TRAF6 (Fig. 3C, D), which is consistent with the changes observed at the mRNA level. Bioinformatics analysis revealed that miR-146a-5p has several binding sites in the TRAF6 3′UTR (data not shown)²⁴. To assess the binding of miR-146a-5p to the TRAF6 promoter, we used luciferase reporter gene plasmid transfection. Transfection with the wild-type TRAF6 promoter led to significant inhibition of TRAF6 transcription by the miR-146a-5p mimic and significant promotion by the miR-146a-5p inhibitor (Fig. 3E). However, after transfection with the mutated TRAF6 promoter, the regulatory effects of the miR-146a-5p mimic and inhibitor on TRAF6 transcription vanished. To investigate the regulatory effects of miR-146a-5p on TRAF6 expression, we separately detected TRAF6 expression at the mRNA (Fig. 3F) and protein (Fig. 3G, H) levels. The data revealed that the siRNA promoted TRAF6 expression at both the mRNA and protein levels, whereas the mimics had the opposite effect.

Inhibition of the NF-κB pathway can induce macrophage polarization toward the M2 phenotype. To further investigate the role of TRAF6, we synthesized TRAF6 overexpression plasmids and interference fragments in vitro. After transfection, we assessed the efficiency of TRAF6 overexpression and interference by qRT‒PCR and Western blotting. The oeTRAF6 construct significantly increased TRAF6 expression at both the mRNA (Fig. 4A) and protein (Fig. 4B, C) levels, whereas siTRAF6 significantly decreased TRAF6 expression at the mRNA (Fig. 4D) and protein (Fig. 4E, F) levels. To examine the downstream regulatory pathways of TRAF6, following the transfection of the TRAF6 interference fragment and overexpression plasmids, we conducted Western blot analysis to assess and compare the NF-κB protein expression and phosphorylation levels among the overexpression group, the downregulation group, and their respective control groups, which revealed that TRAF6 increased NF-κB signaling pathway activation (Fig. 4G, H).

In the U937 macrophage line culture system, the NF-κB pathway inhibitor EVP4593 was introduced, and the results were compared with those of a blank control group. Flow cytometry at 0, 24, and 48 h posttreatment revealed increased expression levels of CD86, INOS, CD163, and CD206 (Fig. 5A-D). The M1/M2 ratio graph revealed a significant increase in M2 cells at 48 h, and the line graph indicates that the inhibitor induced both M1 and M2 polarization, with increased M2 induction (Fig. 5E). A flow cytometry plot at 48 h is shown (Fig. 5F). The data revealed significant differences between type M1 macrophages (CD86 and iNOS) and type M2 macrophages (CD163 and CD206) after 48 h (Fig. 5G).

In our present study, through high-throughput sequencing and bioinformatics analyses, we identified miR-146a-5p for further study, and we showed that both miR-146a-5p in ectopic endometrial stromal cells and exosomal miR-146a-5p were highly expressed in endometriosis. miR-146a-5p is involved in inflammation and regulates the differentiation and function of immune cells^25–28. Further GO analysis of miR-146a-5p was conducted to examine its gene functions in biological processes, cellular components, and molecular functions and to predict its target genes. The results showed that its target was exclusively TRAF6, a key signaling adaptor protein that plays roles primarily in immune and inflammatory responses. KEGG pathway analysis revealed key pathways, including the MAPK, JNK, and NF-κB signaling pathways. A previous study revealed a significant association between miR-146a and an increased incidence of endometriosis²⁹, and the role of miRNAs in regulating macrophage polarization has been confirmed³⁰. Thus, we speculate that miR-146a-5p plays a significant role in the pathogenesis of endometriosis by mediating TRAF6. This study utilizes the signaling function of exosomes to connect the pathogenicity of endometriosis with the functional transformation of macrophages, investigating the role and molecular mechanisms of macrophage functional transformation in endometriosis.

GW4869 is a noncompetitive inhibitor of N-Smase (neutral sphingomyelinase) that can reduce the amount of exosomes released into conditioned medium³¹. By adding GW4689 to inhibit the secretion of exosomes and the release of miR-146a-5p from ectopic ESCs and detecting the levels of macrophage markers, we found that the polarization of macrophages toward the M1 phenotype was increased. Second, inhibitors and mimics were used to alter miR-146a-5p levels in ESCs and their exosomes, and we observed that the miR-146a-5p inhibitor significantly increased TRAF6 expression in macrophages. TRAF6 is an E3 ubiquitin ligase that is involved in numerous inflammatory diseases, and its primary function is focused on the regulation of the immune system³². Third, we overexpressed TRAF6 and transfected interference plasmids in U937 cells, and the results showed that an inhibitor of TRAF6 led to the suppression of the NF-κB pathway. This effect was similar to that of the NF-κB pathway-specific inhibitor EVP4593, resulting in an increased proportion of M2-type macrophages. The inhibition of this signaling cascade has been proven to suppress M1 polarization activity³³. When the NF-κB pathway inhibitor EVP4593 was added to the U937 macrophage culture system, inhibition of the NF-κB pathway induced the polarization of macrophages toward the M2 phenotype. NF-κB is known to regulate immune responses and control the expression of inflammatory cytokines^34,35, and NF-κB activation stimulates inflammation and cell proliferation and inhibits apoptosis, thereby promoting the development and maintenance of endometriosis³⁶. The similarity between the invasive growth of ectopic endometrial tissue and the ease of cancer spread may be due to the active involvement of the stromal microenvironment in controlling invasion, whether by embryos or cancer cells^37,38.

Previous work has shown that the NF-κB signaling pathway is involved in the phenotypic conversion of M2-type macrophages to M1-type macrophages³⁹. The switch from the M1 phenotype to the M2 phenotype mediates the immunosuppressive process, which is a marker of disease progression⁴⁰. M1/M2 polarization imbalance plays a significant role in autoimmune diseases. Consequently, modulating the polarization of macrophages can ameliorate the pathogenesis of these conditions. Recent studies have shown that inhibiting the activation of NF-κB P65, thereby reducing M1 polarization, aids in the treatment of experimental autoimmune uveitis (EAU). Similar to these studies, our data showed that inhibition of the NF-κB pathway can induce macrophage polarization toward the M2 phenotype.

The application of miR-146a-5p in endometriosis needs to be further verified through animal models and in vivo experiments. Moreover, the downstream JNK and MAPK pathways affected by TRAF6 are worthy of further investigation.

In conclusion, our findings indicate that exosome-derived miR-146a-5p-mediated TRAF6 promotes the polarization of macrophages toward the M2 phenotype by regulating the activation of NF-κB. An increase in miR-146a-5p leads to a decrease in TRAF6 expression, which in turn reduces the phosphorylation of NF-κB. The present study may contribute to uncovering the pathogenesis of endometriosis and offer new insights for developing diagnostic and therapeutic strategies for this disease.

Ethical approval. The use of human samples was approved by the Women’s Hospital of Hangzhou Normal University. All the subjects signed a written informed consent. The experimental protocols and procedures related to humans were approved by the Ethics Committee of the Women’s Hospital of Hangzhou Normal University (No. 2022-A-03). All methods were performed in accordance with the relevant guidelines and regulations.

Clinical samples. A total of 5 patients with endometriosis were recruited to provide ectopic and eutopic endometrial tissue when they underwent laparoscopy/hysteroscopy treatment at the Women’s Hospital of Hangzhou Normal University. These samples were obtained during the proliferative phase of the menstrual cycle, which was determined based on preoperative medical history and histological examination. The inclusion and exclusion criteria were as follows: regular menstruation for a period of 28–32 days; and patients who had received hormone therapy and contraception.

Coculture of macrophages and ESCs. Primary ESCs of ectopic and eutopic endometria were cultured as described in previous studies²⁴. The human histiocytic lymphoma cell line U937 was obtained from the cell bank of the Shanghai Academy of Biological Sciences. The medium consisted of RPMI 1640 (Millipore, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and a 1% dual-antibiotic penicillin‒streptomycin mixture (PS) (Solarbio, China). The ectopic ESC culture conditions were 10% FBS + 1% PS + DMEM/F12 (HyClone, USA). All the cells were cultured in a humidified incubator containing 5% CO₂ at 37 °C.

Exosome extraction and characterization. Specimens of ovarian endometrial tissue were collected, and ectopic ESCs were isolated and cultured with exosome-free serum in primary culture for 48 h. Exosomes were isolated with an exosome isolation kit (Invitrogen, USA). The isolated exosomes were stored at −80 °C as a backup. The purity of the cells was determined by immunofluorescence using vimentin and C-cell keratin factor (CK7).

Transwell assays. Transwell inserts (Corning, 3413) were used to detect cell migration and invasion. The isolated ectopic endothelial mesenchymal stromal cells were cocultured with the U937 macrophage line. Cy3-labeled miR-146a-5p molecules were transfected into mesenchymal stromal cells, which were then cultured in the upper chamber. U937 macrophages that did not express Cy3 were placed in the lower chamber, and red fluorescent Cy3-miR-146a-5p molecules were detected in the lower chamber after 12 hours.

Plasmid construction and cell transfection. The miR-146a-5p mimics (5’-UGAGAACUGAAUUCCAUGGGUU-3′), the miR-146a-5p siRNA (5′- AACCCAUGGAAUUCAGUUCUCA-3′), the miRNA negative control (NC) (5′-GUACGCCAAAAGUUAAACC-3′), the TRAF6 siRNA sequence (5’-GGUGAAAUGUCCAAAUGAAGGUUCAUUUGGACAUUUCACCAU-3ʹ), and the siNC (5’- UUGUACUACACAAAAGUACUG-3’) were obtained from Beyotime (Beijing, China). Cell transfection was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Quantitative real-time PCR (qRT‒PCR). mRNA levels were detected by qRT‒PCR. Total RNA from cells and tissues was isolated using TRIzol Reagent (Invitrogen, USA) and then converted to cDNA using the Hifair® II 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai) following the manufacturer’s instructions. Quantitative real-time PCR was then performed using Hieff® qPCR SYBR Master Mix (Yeasen Biotechnology, Shanghai) on an ABI 7500 real-time PCR system (Applied Biosystems, Foster, USA) according to the manufacturer’s instructions. qRT‒PCR for miRNA was performed using a stem‒loop RT primer and the Hifair® miRNA 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai) following the manufacturer’s protocols. The 2-ΔΔCt method was used to calculate the relative expression levels of the targeted genes. The internal controls for mRNA and miRNA were GAPDH and U6, respectively. The primers used are shown in Tables S1 and S2.

Western blot analysis. Protein levels were detected by Western blot analysis. Protein samples to be measured were isolated from whole-cell lysates or exosomes using RIPA lysis buffer (Millipore, USA). The total protein content was quantified using a Pierce™ BCA protein quantification kit (Thermo Fisher Scientific, USA). Briefly, the lysates were separated by gel electrophoresis. Proteins were then transferred onto polyvinylidene fluoride membranes and blocked with 5% nonfat milk. The membranes were then sequentially incubated with optimally diluted primary and secondary antibodies. Immunoreactivity signals of the targeted proteins were visualized in a chemiluminescent assay with an enhanced chemiluminescence (ECL) chromogenic substrate (Bio-Rad Laboratories, USA). The antibodies used for Western blotting are listed in Table S3.

Exosome blockade. The ectopic ESCs cultured in vitro were supplemented with the exosome inhibitor GW4689 for culture as the exosome inhibition group, and a blank control group was set up to obtain 40 ml of supernatant and isolate the exosomes. The obtained exosomes were added to each group of macrophages for 24 h, and the macrophages were subjected to RNA extraction and subsequent experiments.

Exosome regulation. Upregulated or downregulated miR-146a-5p molecules were transfected into ectopic ESCs cultured in vitro using mimics/siRNAs to obtain 40 ml of supernatant, which was subsequently isolated to obtain exosomes. In the U937 macrophage culture system, 50 μg of exosomes from each of the above groups were added separately and cultured for 24 h. Macrophage RNA and proteins were extracted for subsequent experiments.

FCM analysis. After treatment with the exosome inhibitor GW4869 or the NF-κB pathway inhibitor EVP4593 at different time points, U937 cells were collected at 48, 24, and 0 h for FCM analysis of macrophage polarization. The markers CD86 and iNOS were used to detect M1 macrophages, and CD163 and CD206 were used to detect M2 macrophages⁴¹. The cells were washed twice with PBS, and then, the cell pellet was collected by centrifugation and incubated with iNOS monoclonal antibody (CXNFT), Alexa Fluor 488 (53-5920-80, eBioscience, USA), CD86 monoclonal antibody (PO3), PE (MA516921, eBioscience, USA), CD163 monoclonal antibody (MAC 2-158), Alexa Fluor 488(53-1637-42, eBioscience, USA), CD206 monoclonal antibody (685641), and PE (MA5-23594, eBioscience, USA) at 4 °C for 15–30 min in the dark. After being washed with precooled PBS, the cells were resuspended in PBS for FCM analysis. After incubation, the cells were washed and placed in a Beckman flow cytometer. The data were analyzed with FlowJo software (FlowJo LLC, USA).

Luciferase assay. Luciferase gene reporter gene analysis was used to detect the posttranscriptional regulation of TRAF6 by miR-146a-5p. The luciferase reporter vector pcDNA3.1(+) (Addgene, USA) was used to construct TRAF6 wild-type (WT) or mutant 3'-UTRs. Recombinant reporter vectors with miR-146a-5p mimics or inhibitors were combined with Lipofectamine 2000 Reagent to transduce U937 cells. Luciferase activity was assayed using a Luciferase Assay Kit (Promega, USA).

Statistical analysis. The data were statistically analyzed using GraphPad Prism software (version 9.0.0, USA). At least three independent experiments were performed for each result. The data are presented as the means ± SDs. One-way ANOVA and Student’s t test were applied for the comparisons of mean values. P < 0.05 was regarded as statistically significant.

Acknowledgments

This study was supported by grants from the National Nature Science Foundation of China (82171641, 81873825).

Author contributions

Y.M. performed the experiments, analyzed the data and wrote and edited the manuscript.

L.X., T.Y. and L.J. oversaw collection of and provided human samples and performed the experiments. L.K. conceived the ideas and designed the experiments.

Competing interests

The authors declare that they have no competing interests.

Data availability

Data is provided within the manuscript or supplementary information files.

Zondervan, K. T., Becker, C. M. & Missmer, S. A. Endometriosis. N Engl J Med 382, 1244–1256 (2020).
Chen, S. et al. Peritoneal immune microenvironment of endometriosis: Role and therapeutic perspectives. Front. Immunol. 14, 1134663 (2023).
Chen, S., Luo, Y., Cui, L. & Yang, Q. miR-96-5p regulated TGF-β/SMAD signaling pathway and suppressed endometrial cell viability and migration via targeting TGFBR1. Cell Cycle 19, 1740–1753 (2020).
Saunders, P. T. K. & Horne, A. W. Endometriosis: Etiology, pathobiology, and therapeutic prospects. Cell 184, 2807–2824 (2021).
Vallvé-Juanico, J., Houshdaran, S. & Giudice, L. C. The endometrial immune environment of women with endometriosis. Human Reproduction Update 25, 565–592 (2019).
Ramírez-Pavez, T. N. et al. The Role of Peritoneal Macrophages in Endometriosis. IJMS 22, 10792 (2021).
Symons, L. K. et al. The Immunopathophysiology of Endometriosis. Trends in Molecular Medicine 24, 748–762 (2018).
Zhang, T., De Carolis, C., Man, G. C. W. & Wang, C. C. The link between immunity, autoimmunity and endometriosis: a literature update. Autoimmunity Reviews 17, 945–955 (2018).
Riccio, L. D. G. C. et al. Immunology of endometriosis. Best Practice & Research Clinical Obstetrics & Gynaecology 50, 39–49 (2018).
Artemova, D. et al. Endometriosis and Cancer: Exploring the Role of Macrophages. IJMS 22, 5196 (2021).
Hogg, C. et al. Macrophages inhibit and enhance endometriosis depending on their origin. Proc. Natl. Acad. Sci. U.S.A. 118, e2013776118 (2021).
Toledo, B. et al. Deciphering the performance of macrophages in tumour microenvironment: a call for precision immunotherapy. J Hematol Oncol 17, 44 (2024).
Shapouri-Moghaddam, A. et al. Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol 233, 6425–6440 (2018).
Bazzi, S. et al. Cytokine/Chemokine Release Patterns and Transcriptomic Profiles of LPS/IFNγ-Activated Human Macrophages Differentiated with Heat-Killed Mycobacterium obuense, M-CSF, or GM-CSF. IJMS 22, 7214 (2021).
He, M., Xu, Z., Ding, T., Kuang, D.-M. & Zheng, L. MicroRNA-155 Regulates Inflammatory Cytokine Production in Tumor-associated Macrophages via Targeting C/EBPβ. Cell Mol Immunol 6, 343–352 (2009).
Yu, M. Y. et al. Exosomal miRNAs-mediated macrophage polarization and its potential clinical application. International Immunopharmacology 117, 109905 (2023).
Jiao, Y. et al. Exosomal miR-30d-5p of neutrophils induces M1 macrophage polarization and primes macrophage pyroptosis in sepsis-related acute lung injury. Crit Care 25, 356 (2021).
Deng, L. et al. Activation of the IκB Kinase Complex by TRAF6 Requires a Dimeric Ubiquitin-Conjugating Enzyme Complex and a Unique Polyubiquitin Chain. Cell 103, 351–361 (2000).
Panir, K., Schjenken, J. E., Robertson, S. A. & Hull, M. L. Non-coding RNAs in endometriosis: a narrative review. Human Reproduction Update 24, 497–515 (2018).
Wu, J., Ding, J., Yang, J., Guo, X. & Zheng, Y. MicroRNA Roles in the Nuclear Factor Kappa B Signaling Pathway in Cancer. Front Immunol 9, 546 (2018).
Hoover, A. A. et al. Increased canonical NF-kappaB signaling specifically in macrophages is sufficient to limit tumor progression in syngeneic murine models of ovarian cancer. BMC Cancer 20, 970 (2020).
Wu, M. & Zhang, Y. MiR-182 inhibits proliferation, migration, invasion and inflammation of endometrial stromal cells through deactivation of NF-κB signaling pathway in endometriosis. Mol Cell Biochem 476, 1575–1588 (2021).
Chu, Q., Bi, D., Zheng, W. & Xu, T. MicroRNA negatively regulates NF-κB-mediated immune responses by targeting NOD1 in the teleost fish Miichthys miiuy. Sci. China Life Sci. 64, 803–815 (2021).
Ji, J. et al. Exosomes from ectopic endometrial stromal cells promote M2 macrophage polarization by delivering miR-146a-5p. International Immunopharmacology 128, 111573 (2024).
Shademan, B. et al. Investigation of the miRNA146a and miRNA155 gene expression levels in patients with multiple sclerosis. Journal of Clinical Neuroscience 78, 189–193 (2020).
Sanada, T. et al. Anti-inflammatory effects of miRNA-146a induced in adipose and periodontal tissues. Biochemistry and Biophysics Reports 22, 100757 (2020).
Li, J. et al. Cationic phosphorus dendron nanomicelles deliver microRNA mimics and microRNA inhibitors for enhanced anti-inflammatory therapy of acute lung injury. Biomater. Sci. 11, 1530–1539 (2023).
Hua, T. et al. Huc-MSCs-derived exosomes attenuate inflammatory pain by regulating microglia pyroptosis and autophagy via the miR-146a-5p/TRAF6 axis. J Nanobiotechnol 20, 324 (2022).
Farsimadan, M. et al. MicroRNA variants in endometriosis and its severity. British Journal of Biomedical Science 78, 206–210 (2021).
Liu, G. & Abraham, E. MicroRNAs in Immune Response and Macrophage Polarization. ATVB 33, 170–177 (2013).
Chen, Y. et al. MicroRNA-146a-5p Negatively Regulates Pro-Inflammatory Cytokine Secretion and Cell Activation in Lipopolysaccharide Stimulated Human Hepatic Stellate Cells through Inhibition of Toll-Like Receptor 4 Signaling Pathways. IJMS 17, 1076 (2016).
Lu, Y. et al. TRAF6 upregulation in spinal astrocytes maintains neuropathic pain by integrating TNF-α and IL-1β signaling. Pain 155, 2618–2629 (2014).
Lu, H. et al. Quercetin ameliorates kidney injury and fibrosis by modulating M1/M2 macrophage polarization. Biochemical Pharmacology 154, 203–212 (2018).
Lai, J. et al. Indirubin Inhibits LPS-Induced Inflammation via TLR4 Abrogation Mediated by the NF-kB and MAPK Signaling Pathways. Inflammation 40, 1–12 (2017).
El-Shitany, N. A. & Eid, B. G. Icariin modulates carrageenan-induced acute inflammation through HO-1/Nrf2 and NF-kB signaling pathways. Biomedicine & Pharmacotherapy 120, 109567 (2019).
González-Ramos, R., Defrère, S. & Devoto, L. Nuclear factor–kappaB: a main regulator of inflammation and cell survival in endometriosis pathophysiology. Fertility and Sterility 98, 520–528 (2012).
Pavličev, M. et al. A common allele increases endometrial Wnt4 expression, with antagonistic implications for pregnancy, reproductive cancers, and endometriosis. Nat Commun 15, 1152 (2024).
Suhail, Y., Afzal, J., & Kshitiz. Evolved Resistance to Placental Invasion Secondarily Confers Increased Survival in Melanoma Patients. JCM 10, 595 (2021).
Liu, Y., Wang, J. & Zhang, X. An Update on the Multifaceted Role of NF-kappaB in Endometriosis. Int. J. Biol. Sci. 18, 4400–4413 (2022).
Viola, A., Munari, F., Sánchez-Rodríguez, R., Scolaro, T. & Castegna, A. The Metabolic Signature of Macrophage Responses. Front. Immunol. 10, 1462 (2019).
Wu, D., Lu, P., Mi, X. & Miao, J. Exosomal miR-214 from endometrial stromal cells inhibits endometriosis fibrosis. MHR: Basic science of reproductive medicine (2018) doi:10.1093/molehr/gay019.

No competing interests reported.

Table.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Exosomal miR-146a-5p mediates macrophage polarization through TRAF6/NF-κB signaling in endometriosis

Status:

Version 1

Abstract

Figures

Introduction

Results

Discussion

Materials and Methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1