NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

Abstract

MicroRNAs (miRNAs) contribute to osteoarthritis (OA) development. Nevertheless, the function and mechanism of miR-30b-5p in OA are unclear. In the present article, we gauged the miR-30b-5p level in OA patients and analyzed its correlation with OA stages. Then, we conducted in-vivo and in-vitro gain-of-function assays to determine the function of miR-30b-5p, silent information regulator 2 homolog 1 (SIRT1) and Fox. Cell counting Kit-8 (CCK-8) assay, BrdU assay and flow cytometry were utilized to gauge cell viability and apoptosis of human chondrocyte (HC-A). The targeting association between miR-30b-5p and SIRT1 was validated through the dual-luciferase reporter assay and RNA immunoprecipitation (RIP) experiment. The results signified that miR-30b-5p was up-regulated in OA patients, OA rats and interleukin-1β (IL-1β)-induced chondrocytes. The higher miR-30b-5p expression brought about progressive stages of OA patients and enhanced levels of pro-inflammatory mediators in the synovial fluid. Functionally, overexpressing miR-30b-5p hampered cell viability, aggravated chondrocyte apoptosis and NLRP3 inflammasome activation induced by IL-1β, while down-regulating miR-30b-5p exerted the reverse effects. The in-vivo experiment exhibited that down-regulating miR-30b-5p improved joint pain and loss of articular cartilage in the rats with restrained inflammation and NLRP3 inflammasome activation. Mechanistically, miR-30b-5p targeted the 3’-non-translated region (3’UTR) of SIRT1, and miR-30b-5p was inducible with NF-κB phosphorylation enhancement. Overexpressing SIRT1 or inhibiting NF-κB relieved miR-30b-5p-induced apoptosis and NLRP3 inflammasome activation by promoting FoxO3a, while down-regulating SIRT1 or FoxO3a reversed miR-30b-5p-in-induced anti-inflammatory and apoptosis-suppressive effects. Collectively, NF-κB-induced miR-30b-5p modulates chondrocyte apoptosis and OA progression by regulating the SIRT1-FoxO3a-mediated NLRP3 inflammasome.

Downregulation of Neurofilament Light Chain Expression in Human Neuronal-Glial Cell Co-Cultures by a Microbiome-Derived Lipopolysaccharide-Induced miRNA-30b-5p

“…targets that maintain neuronal structure and synaptic signaling, such as the NF-L transcript, is significantly downregulated (42,(45)(46)(47)(48). We provide molecular-genetic evidence that LPS and miRNA-30b in HNG cells in primary culture both target the NF-L 3'-UTR, a process known to ultimately result in NF-L downregulation.…”

“…The expression of miRNA-30b is implicated in playing a crucial homeostatic regulatory role in tissue and organ development and the pathogenesis of an array of diseases from cancer to progressive inflammatory neurodegenerative disorders, such as AD (41)(42)(43)(44)(45)(46). Multiple independent reports indicate that the NF-kB-inducible miRNA-30b: (i) is upregulated in AD and animal models of AD (43)(44)(45)(46); (ii) that the overexpression of miRNA-30b in the hippocampus impairs basal synaptic transmission, long term potentiation (LTP), learning, and memory and is associated with a significant reduction in dendritic spine density (42,43); (iii) causes synaptic and cognitive dysfunction in AD and in AD animal models (https://www.ncbi.nlm.nih.gov/gene/407030; 2022; last accessed 24 April 2022; 42,44); (iv) is significantly upregulated by lipopolysaccharide (LPS) or protozoan-mediated infection of human epithelial cells (46); and (v) targets the 3'-UTR of the mRNA encoding sirtuin 1 (SIRT1), a ubiquitous deacetylase that regulates numerous cellular functions at the level of gene expression, including aging, lipid homeostasis, and inflammatory signaling (48). Because of the abundance of this NF-kB-upregulated miRNA-30b in the human brain and CNS neurons, with its significant over-expression in AD and this miRNA's known impact on human neurophysiological effects and pathways relevant to neurodegenerative disease, we miRNA-30b, a scrambled control miRNA-30b (miRNA-30b-sc) or control miRNA-183; see (14,18,39,49) and text for further details on all reagents and methods used in these experiments; (E) compared to control, HNG cells transfected with a scrambled (sc) control pLightSwitch-3'-UTR vector, the NF-L-mRNA-3 ′ -UTR vector exhibited decreased luciferase signal to a mean of 0.18-fold of controls in the presence of exogenous LPS and 0.11 in the presence of miRNA-30b; this same vector exhibited no change in relative luciferase yield in the presence of a control miRNA-30b-sc or miRNA-183; for each experiment (using different batches of HNG cells) a control luciferase signal was generated that included separate controls with each analysis; in addition a control vector β-actin-3 ′ -UTR showed no significant effects on the relative luciferase signal yield after treatment with either miRNA-183 or miRNA-30b (data not shown); a dashed horizontal line set to 1 is included for ease of comparison; N = 5; *p < 0.01 (ANOVA); values represent mean +/-1 standard deviation (S.D.…”

“…1 and Supplementary Table 1). Because of multiple previous studies verifying its brain involvement on CNS pathology, the brain-enriched NF-kB-sensitive miRNA-30b and NF-L 3'-UTR interaction was studied further (44)(45)(46)(47)(48). To validate a functional miRNA-30b-NF-L 3'-UTR interaction, we used HNG cells (at 2 weeks in culture) transfected with a miRNA-30b-NF-L 3'-UTR expression vector luciferase reporter assay (pLightSwitch-3…”

Zhilong Huoxue Tongyu Capsule Alleviated the Pyroptosis of Vascular Endothelial Cells Induced by ox-LDL through miR-30b-5p/NLRP3

“…The activation of inflammatory response is an important process in the apoptosis and injury of endothelial cells caused by ox-LDL, and ox-LDL-induced activation of inflammatory response can directly cause endothelial sclerosis and thus participate in the formation of atherosclerotic plaques [ 46 ]. Meanwhile miR-30b-5p has been implicated in activation of NLRP3 inflammasome in other studies [ 47 ]. Importantly, many research studies suggest that NLRP3 is involved in pyrophosis of AS [ 12 , 48 ].…”

DNA Methylation and Non-Coding RNAs during Tissue-Injury Associated Pain

“…They found an up-regulation of this miRNA, which seems to inhibit the expression of TGF-β, a known mediator of nociception, at the spinal cord, DRG, cerebrospinal fluid (CSF) and plasma of rodents subjected to SNI, and in CSF and plasma from patients. Similarly, two recent publications have studied the mechanism by which down-regulation of miR-183 and up-regulation of miR-30b-5p influence osteoarthritis pain by systematically looking at both a preclinical model and a clinical setting [ 75 , 81 ]. In both cases, reconstituting their expression to pre-injury levels ameliorated the painful condition in the murine model.…”