MiR-9-5p promotes M1 cell polarization in osteoarthritis progression by regulating NF-κB and AMPK signaling pathways by targeting SIRT1

doi:10.1016/j.intimp.2021.108207

International Immunopharmacology

Volume 101, Part A, December 2021, 108207

https://doi.org/10.1016/j.intimp.2021.108207 Get rights and content

Highlights:

•
miR-9 could regulate sirt1 mRNA degradation.
•
miR-9 regulates M1 and M2 macrophage polarization via AMPK and NF-κB pathway.
•
Inhibition of miR-9 expression could alleviate the impairment of OA in mouse model.

Abstract

Objective

To investigate the roles and regulatory mechanisms of miR-9-5p in the development of osteoarthritis (OA).

Methods

Synovial tissues from mouse OA model and control groups were collected and miR-9-5p expression levels and macrophage markers were measured with qPCR. The function of miR-9-5p in macrophage polarization was analyzed by flow cytometry and qPCR. Various databases were employed to screen the target genes one of which was validated with dual-luciferase analysis. Following the validation, rescue research was applied, and the signaling pathways were analyzed with Western blotting. Finally, the role of miR-9-5p in the progression of OA was validated in the mouse model.

Results

MiR-9-5p was highly expressed in the synovial tissues of the OA model and was positively associated with M1 markers. Function analysis demonstrated that miR-9-5p could promote the progression of OA by promoting M1 polarization and inhibiting M2 polarization in vivo and in vitro. The mechanism analysis demonstrated that miR-9-5p could regulate macrophage polarization via NF-κB and AMPK signaling pathways by inhibiting SIRT1 expression.

Conclusions

MiR-9-5p could promote M1 polarization and OA progression by regulating NF-κB and AMPK signaling pathways by inhibiting SIRT1 expression.

Introduction

Osteoarthritis (OA) is a kind of degenerative disease in older adults and has been a major cause of disability. The origin and progression of OA are associated with age, BMI, joint trauma history, and excessive use of joints [1]. Current treatment methods combine pain and inflammation management and joint replacement surgery for the late phase of the disease [2]. However, the molecular mechanism of OA remains unclear until now.

Inflammation is the main symptom of OA, and the inflammatory response could induce cartilage damage [3], [4]. Macrophage is the main synovial immune cells that participate in various chronic bone diseases such as gouty arthritis (GA), rheumatoid arthritis (RA) and OA [5], [6], [7]. Activated macrophages could polarize to types M1 and M2. M1 could enhance the inflammatory response by secreting IFN-γ, IL-1, and TNFα [8]. M2 cells could resolve inflammation and promote tissue repair and play a vital role in the resolution of the inflammatory phase [9].While improper activation of M1-mediated inflammation would lead to the destruction of normal tissue homeostasis and lead to cytokines storm [9]. Macrophages always remain quiescent with fibroblasts in normal synovial tissue. During the progression of OA, macrophages would polarize into the M1 type and play a central role in the inflammatory response by secreting pro-inflammatory factors and promote the progression of OA [10], [11].

MiRNA is a short RNA that comprises 18–25 nucleotides and participated in various tissue development and pathogenesis development. Plenty of research has shown that miRNA participated in OA progression [12]. MiR-204 could down-regulate chondrocyte sulfated glycosaminoglycan production by directly targeting genes that participated in cartilage proteoglycan biosynthesis [13]. miR-138, miR-93-5p, and miR-324-5p could participate in OA progression by regulating cartilage matrix degradation and hedgehog signaling pathway by targeting RhoC, TCF4, SMO, and GLI1 [14], [15], [16]. MiR-9-5p is highly expressed in OA tissue and might regulate genes that participated in the inflammatory process [17]. Further research demonstrated that miR-9-5p could promote the progression of OA by inhibiting chondrocyte apoptosis. As shown in peer research, miR-9-5p could induce macrophage M1 polarization and increase tumor radiosensitivity [18]. However, it is unknown whether and how miR-9-5p regulates macrophage polarization and promotes OA progression.

In this research, we confirm the role of miR-9-5p in OA progression and analyze the role of miR-9-5p in macrophage polarization and analyze the mechanism by which miR-9-5p regulates macrophage in vitro and in vivo.

Section snippets

Macrophage cell culture and differentiation into M1 and M2 Macrophages

Cells were cultured in RPMI 1640 medium with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. For the polarization of macrophage type M1, 20 ng/ml of 12-myristate 13-acetate phorbol (PMA, EMD) was added to the THP-1 culture medium and 6 h later, 100 ng/ml lipopolysaccharide (LPS, Sigma) and 20 ng/ml interferon-γ (IFN-γ, Sigma) were added to the culture medium, and cells were cultured for 48 h. To obtain the M2 macrophage type, 20 ng/ml PMA was added to the culture medium of THP-1 cells

The expression of miR-9-5p is increased in OA models and is associated with macrophage polarization of M1 cells

First, we examined the expression of miR-9-5p in synovial tissue between OA model mice and the negative control group. We found that miR-9-5p was highly expressed in the synovial tissue of the OA model (Fig. 1A). During the progression of OA, macrophage cells participated in the inflammation process and chondrocyte apoptosis. The markers of M1 and M2 cells were also measured with qPCR between the OA model group and the negative control group. As shown in Fig. 1B, the expression of M1 markers

Discussion

Here, we find that miR-9-5p is highly expressed in OA mouse models and is positively associated with OA progression by modulating macrophage polarization. Further cell and animal analysis demonstrated that miR-9-5p could regulate macrophage polarization by regulating AMPK and NF-κB signaling pathways by degrading SIRT1 expression.

MiR-9-5p is associated with various cancer progressions such as glioma, breast cancer, and osteosarcoma [22], [23], [24]. And in inflammatory diseases, miR-9 also

Funding

This study was supported by the National Natural Science Foundation of China (Grant No.: 81760407) and the Associated Project of the Yunnan Province Department of Science and Technology and Kunming Medical University Basic Research for Application [Grant No.: 2018FE001(-179)].

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References (38)

S. Glyn-Jones et al.
Osteoarthritis
Lancet (London, England)
(2015)
C.R. Scanzello et al.
The role of synovitis in osteoarthritis pathogenesis
Bone
(2012)
T.A. Wynn et al.
Macrophages in Tissue Repair, Regeneration, and Fibrosis
Immunity
(2016)
H. Xue et al.
miR-93-5p attenuates IL-1β-induced chondrocyte apoptosis and cartilage degradation in osteoarthritis partially by targeting TCF4
Bone
(2019)
S. Woods et al.
miR-324-5p is up regulated in end-stage osteoarthritis and regulates Indian Hedgehog signalling by differing mechanisms in human and mouse
Matrix Biol.: J. Int. Society Matrix Biol.
(2019)
M. Nugent
MicroRNAs: exploring new horizons in osteoarthritis
Osteoarthritis Cartilage
(2016)
F. Tong et al.
HPV + HNSCC-derived exosomal miR-9 induces macrophage M1 polarization and increases tumor radiosensitivity
Cancer Lett.
(2020)
J.N. Rogart et al.
Articular collagen degradation in the Hulth-Telhag model of osteoarthritis
Osteoarthritis Cartilage
(1999)
S.Y. Park et al.
SIRT1/Adenosine Monophosphate-Activated Protein Kinase α Signaling Enhances Macrophage Polarization to an Anti-inflammatory Phenotype in Rheumatoid Arthritis
Front. Immunol.
(2017)
X. Chen et al.
MiR-9 promotes tumorigenesis and angiogenesis and is activated by MYC and OCT4 in human glioma
J. Exp. Clin. Cancer Res.: CR
(2019)

X. Li et al.

MicroRNA-9 and breast cancer

Biomed. Pharmacotherapy = Biomedecine & pharmacotherapie

(2020)

S.W. Jones et al.

The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13

Osteoarthritis Cartilage

(2009)

W.F.T. Carson et al.

Enhancement of macrophage inflammatory responses by CCL2 is correlated with increased miR-9 expression and downregulation of the ERK1/2 phosphatase Dusp6

Cell. Immunol.

(2017)

J. Lv et al.

miRNA expression patterns in chemoresistant breast cancer tissues

Biomed. Pharmacotherapy = Biomedecine & pharmacotherapie

(2014)

A.S. Wellman et al.

Intestinal Epithelial Sirtuin 1 Regulates Intestinal Inflammation During Aging in Mice by Altering the Intestinal Microbiota

Gastroenterology

(2017)

Z. Yang et al.

Macrophage alpha1 AMP-activated protein kinase (alpha1AMPK) antagonizes fatty acid-induced inflammation through SIRT1

J. Biol. Chem.

(2010)

S.I. Macías-Hernández et al.

Glenohumeral osteoarthritis: overview, therapy, and rehabilitation

Disability Rehabilit.

(2017)

P.G. Conaghan et al.

Therapeutic options for targeting inflammatory osteoarthritis pain

Nat. Rev. Rheumatol.

(2019)

P.J. Murray et al.

Protective and pathogenic functions of macrophage subsets

Nat. Rev. Immunol.

(2011)

Cited by (13)

Linc01588 deletion inhibits the malignant biological characteristics of hydroquinone-induced leukemic cells via miR-9-5p/SIRT1
2024, Ecotoxicology and Environmental Safety
Leukemia caused by environmental chemical pollutants has attracted great attention, the malignant leukemic transformation model of TK6 cells induced by hydroquinone (HQ) has been previously found in our team. However, the type of leukemia corresponding to this malignant transformed cell line model needs further study and interpretation. Furthermore, the molecular mechanism of malignant proliferation of leukemic cells induced by HQ remains unclear. This study is the first to reveal the expression of aberrant genes in leukemic cells of HQ-induced malignant transformation, which may correspond to chronic lymphocytic leukemia (CLL). The expression of Linc01588, a long non-coding RNA (lncRNA), was significantly up-regulated in CLL patients and leukemic cell line model which previously described. After gain-of-function assays and loss-of-function assays, feeble cell viability, severe apoptotic phenotype and the increased secretion of TNF-α were easily observed in malignant leukemic TK6 cells with Linc01588 deletion after HQ intervention. The tumors derived from malignant TK6 cells with Linc01588 deletion inoculated subcutaneously in nude mice were smaller than controls. In CLL and its cell line model, the expression of Linc01588 and miR-9–5p, miR-9–5p and SIRT1 were negative correlation respectively in CLL and cell line model, while the expression of Linc01588 and SIRT1 were positive correlation. The dual-luciferase reporter assay showed that Linc01588 & miR-9–5p, miR-9–5p & SIRT1 could bind directly, respectively. Furthermore, knockdown of miR-9–5p successfully rescued the severe apoptotic phenotype and the increased secretion of TNF-α caused by the Linc01588 deletion, the deletion of Linc01588 in human CLL cell line MEC-2 could also inhibit malignant biological characteristics, and the phenotype caused by the deletion of Linc01588 could also be rescued after overexpression of SIRT1. Moreover, the regulation of SIRT1 expression in HQ19 cells by Linc01588 and miR-9–5 P may be related to the Akt/NF-κB pathway. In brief, Linc01588 deletion inhibits the malignant biological characteristics of HQ-induced leukemic cells via miR-9–5p/SIRT1, and it is a novel and hopeful clue for the clinical targeted therapy of CLL.
Sirtuins in macrophage immune metabolism: A novel target for cardiovascular disorders
2024, International Journal of Biological Macromolecules
Sirtuins (SIRT1-SIRT7), as a family of NAD⁺-dependent protein modifying enzymes, have various catalytic functions, such as deacetylases, dealkalylases, and deribonucleases. The Sirtuins family is directly or indirectly involved in pathophysiological processes such as glucolipid metabolism, oxidative stress, DNA repair and inflammatory response through various pathways and assumes an important role in several cardiovascular diseases such as atherosclerosis, myocardial infarction, hypertension and heart failure. A growing number of studies supports that metabolic and bioenergetic reprogramming directs the sequential process of inflammation. Failure of homeostatic restoration leads to many inflammatory diseases, and that macrophages are the central cells involving the inflammatory response and are the main source of inflammatory cytokines. Regulation of cellular metabolism has emerged as a fundamental process controlling macrophage function, but its exact signaling mechanisms remain to be revealed. Understanding the precise molecular basis of metabolic control of macrophage inflammatory processes may provide new approaches for targeting immune metabolism and inflammation. Here, we provide an update of studies in cardiovascular disease on the function and role of sirtuins in macrophage inflammation and metabolism, as well as drug candidates that may interfere with sirtuins, pointing to future prospects in this field.
The potential role of miRNA in regulating macrophage polarization
2023, Heliyon
Macrophage polarization is a dynamic process determining the outcome of various physiological and pathological situations through inducing pro-inflammatory responses or resolving inflammation via exerting anti-inflammatory effects. The miRNAs are epigenetic regulators of different biologic pathways that target transcription factors and signaling molecules to promote macrophage phenotype transition and regulate immune responses. Modulating the macrophage activation, differentiation, and polarization by miRNAs is crucial for immune responses in response to microenvironmental signals and under various physiological and pathological conditions. In term of clinical significance, regulating macrophage polarization via miRNAs could be utilized for inflammation control. Also, understanding the role of miRNAs in macrophage polarization can provide insights into diagnostic strategies associated with dysregulated miRNAs and for developing macrophage-centered therapeutic methods. In this case, targeting miRNAs to further regulate of macrophage polarization may become an efficient strategy for treating immune-associated disorders. The current review investigated and categorized various miRNAs directly or indirectly involved in macrophage polarization by targeting different transcription factors and signaling pathways. In addition, prospects for regulating macrophage polarization via miRNA as a therapeutic choice that could be implicated in various pathological conditions, including cancer or inflammation-mediated injuries, were discussed.
MicroRNA-15a/β1,4-GalT-I axis contributes to cartilage degeneration via NF-κB signaling in osteoarthritis
2023, Clinics
Osteoarthritis is a condition characterized by articular cartilage degradation. The increased expression of β1,4-Galactosyltransferase-I (β1,4-GalT-I) in the articular cartilage of osteoarthritis patients was related to an inflammatory response. The aim of this study was to elucidate the role of β1,4-GalT-I in osteoarthritis. This study aimed to determine the function of 1,4-GalT-I in osteoarthritis.
The osteoarthritis mouse model with the destabilization of the medial meniscus was established by microsurgical technique. Pathological changes in articular cartilage were observed by hematoxylin and eosin staining and safranin O-fast green staining. Quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assays were used to observe mRNA and protein expression, respectively. RNA interactions were verified by a luciferase reporter assay. SA-β-Gal staining was used to assess chondrocyte senescence. Immunofluorescence staining was conducted to observe the localization of Nuclear Factor-kappaB (NF-κB).
β1,4-GalT-I and microRNA-15a (miR-15a) show high and low expression in the articular cartilage of osteoarthritis, respectively. MiR-15a inhibits the mRNA translation of β1,4-GalT-I. β1,4-GalT-I promotes extracellular matrix degradation, senescence, and NF-κB activation in IL-1β-stimulated chondrocytes, which can be reversed by overexpression of miR-15a. Intra-articular injection of microRNA-15a ameliorates cartilage degeneration by inhibiting β1,4-GalT-I and phosphorylation of NF-κB in vivo.
The authors clarified that the miR-15a/β1,4-GalT-I axis inhibits the phosphorylation of NF-κB thereby inhibiting extracellular matrix degradation and senescence in chondrocytes to alleviate cartilage degeneration in osteoarthritis. MiR-15a and β1,4-GalT-I may serve as potentially effective targets for the future treatment of osteoarthritis.
Opsonized nanoparticles target and regulate macrophage polarization for osteoarthritis therapy: A trapping strategy
2022, Journal of Controlled Release
Citation Excerpt :
Even though the molecular mechanism of OA is still not clarified, inflammation is the main symptom. Macrophages, the primary synovial immune cells, participate in the chronic progression of OA [14]. Joint damage resulted from long-term wear and tear or accidental injury leads to the production of pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), and inflammatory vesicles in the joint cavity [15].
Osteoarthritis (OA) is a chronic disease caused by joint inflammation. Its occurrence and development depend on a continuous inflammation environment. The activated M1 macrophages play a critical role in the inflammatory response of OA. Regulating the pro-inflammatory M1 to anti-inflammatory M2 macrophages in the OA articular cavity could be a rational strategy for OA treatment. It has been acknowledged that activated macrophages could proactively capture opsonized nanoparticles in the bloodstream and then accumulate into the reticuloendothelial system (RES) organs. Based on this fact, a trapping strategy is proposed, which transforms a normal nanoparticle into an opsonized attractant to target and regulate macrophage polarization. In this study, the opsonized nanoparticle (IgG/Bb@BRPL) had several key features, including an immunoglobulin IgG (the opsonized layer), an anti-inflammatory agent berberine (Bb), and an oxidative stress-responsive bilirubin grafted polylysine biomaterial (BR-PLL) for drug loading (the inner nanocore). In vitro studies confirmed that IgG/Bb@BRPL prefer to be phagocytosed by M1 macrophage, not M0. And the internalized IgG/Bb@BRPL effectively promoted macrophage polarization toward the M2 phenotype and protected nearby chondrocytes. In vivo studies suggested that IgG/Bb@BRPL significantly enhanced therapeutic outcomes by suppressing inflammation and promoting cartilage repair while not prolonging the retention period compared to non-opsonized counterparts. This proof-of-concept study provided a novel opsonization trapping strategy for OA drug delivery and treatment.
MicroRNA-9-5p Is Involved in Lipopolysaccharide-Induced Acute Lung Injury Via the Regulation of Macrophage Polarization
2023, International Journal of Toxicology

View all citing articles on Scopus

View full text

MiR-9-5p promotes M1 cell polarization in osteoarthritis progression by regulating NF-κB and AMPK signaling pathways by targeting SIRT1

Highlights:

Abstract

Objective

Methods

Results

Conclusions

Introduction

Section snippets

Macrophage cell culture and differentiation into M1 and M2 Macrophages

The expression of miR-9-5p is increased in OA models and is associated with macrophage polarization of M1 cells

Discussion

Funding

Declaration of Competing Interest

Lancet (London, England)

Bone

Immunity

Bone

Matrix Biol.: J. Int. Society Matrix Biol.

Osteoarthritis Cartilage

Cancer Lett.

Osteoarthritis Cartilage

Front. Immunol.

J. Exp. Clin. Cancer Res.: CR

Biomed. Pharmacotherapy = Biomedecine & pharmacotherapie

Osteoarthritis Cartilage

Cell. Immunol.

Biomed. Pharmacotherapy = Biomedecine & pharmacotherapie

Gastroenterology

J. Biol. Chem.

Glenohumeral osteoarthritis: overview, therapy, and rehabilitation

Disability Rehabilit.

Therapeutic options for targeting inflammatory osteoarthritis pain

Nat. Rev. Rheumatol.

Protective and pathogenic functions of macrophage subsets

Nat. Rev. Immunol.