miR-526b targets 3′ UTR of MMP1 mRNA

Regulation of matrix metalloproteinases (MMPs) is important for many physiological processes involving cancers, inflammation, tissue remodeling and skin aging. Here, we report the novel finding that the expression of MMP1 mRNA is downregulated by the overexpression of miR-526b which is a member of chromosome 19 microRNA cluster (C19MC). Our analysis using reporter constructs containing the 3′ untranslated region (3′ UTR) of MMP1 and its mutant form showed that the region from 377–383 in the 3′ UTR of MMP1 is critical for targeting by miR-526b. In addition, the expression pattern of miR-526b and MMP1 mRNA showed reverse relation between adult dermal and neonatal fibroblasts. We show for the first time that miR-526b, an miRNA belonging to C19MC, can target the 377–383 region of the MMP1 3′ UTR.     

Crosstalk between FLS and chondrocytes is regulated by HIF-2α-mediated cytokines in arthritis

Rheumatoid arthritis (RA) and osteoarthritis (OA), two common types of arthritis, affect the joints mainly by targeting the synovium and cartilage. Increasing evidence indicates that a significant network connects synovitis and cartilage destruction during the progression of arthritis. We recently demonstrated that hypoxia-inducible factor (HIF)-2α causes RA and OA by regulating the expression of catabolic factors in fibroblast-like synoviocytes (FLS) or chondrocytes. To address the reciprocal influences of HIF-2α on FLS and chondrocytes, we applied an in vitro co-culture system using a transwell apparatus. When co-cultured with HIF-2α-overexpressing chondrocytes, FLS exhibited increased expression of matrix metalloproteinases and inflammatory mediators, similar to the effects induced by tumor-necrosis factor (TNF)-α treatment of FLS. Moreover, chondrocytes co-cultured with HIF-2α-overexpressing FLS exhibited upregulation of Mmp3 and Mmp13, which is similar to the effects induced by interleukin (IL)-6 treatment of chondrocytes. We confirmed these differential HIF-2α-induced effects via distinct secretory mediators using Il6-knockout cells and a TNF-α-blocking antibody. The FLS-co-culture-induced gene expression changes in chondrocytes were significantly abrogated by IL-6 deficiency, whereas TNF-α neutralization blocked the alterations in gene expression associated with co-culture of FLS with chondrocytes. Our results further suggested that the observed changes might reflect the HIF-2α-induced upregulation of specific receptors for TNF-α (in FLS) and IL-6 (in chondrocytes). This study broadens our understanding of the possible regulatory mechanisms underlying the crosstalk between the synovium and cartilage in the presence of HIF-2α, and may suggest potential new anti-arthritis therapies.     

Molecular and Cellular Effects of Chemical Chaperone—TUDCA on ER-Stressed NHAC-kn Human Articular Chondrocytes Cultured in Normoxic and Hypoxic Conditions

Osteoarthritis (OA) is considered one of the most common arthritic diseases characterized by progressive degradation and abnormal remodeling of articular cartilage. Potential therapeutics for OA aim at restoring proper chondrocyte functioning and inhibiting apoptosis. Previous studies have demonstrated that tauroursodeoxycholic acid (TUDCA) showed anti-inflammatory and anti-apoptotic activity in many models of various diseases, acting mainly via alleviation of endoplasmic reticulum (ER) stress. However, little is known about cytoprotective effects of TUDCA on chondrocyte cells. The present study was designed to evaluate potential effects of TUDCA on interleukin-1β (IL-1β) and tunicamycin (TNC)-stimulated NHAC-kn chondrocytes cultured in normoxic and hypoxic conditions. Our results showed that TUDCA alleviated ER stress in TNC-treated chondrocytes, as demonstrated by reduced CHOP expression; however, it was not effective enough to prevent apoptosis of NHAC-kn cells in either normoxia nor hypoxia. However, co-treatment with TUDCA alleviated inflammatory response induced by IL-1β, as shown by down regulation of Il-1β, Il-6, Il-8 and Cox2, and increased the expression of antioxidant enzyme Sod2. Additionally, TUDCA enhanced Col IIα expression in IL-1β- and TNC-stimulated cells, but only in normoxic conditions. Altogether, these results suggest that although TUDCA may display chondoprotective potential in ER-stressed cells, further analyses are still necessary to fully confirm its possible recommendation as potential candidate in OA therapy.     

The enhanced expression of IL-17-secreting T cells during the early progression of atherosclerosis in ApoE-deficient mice fed on a western-type diet

Atherosclerosis is a chronic progressive inflammatory disorder and the leading cause of cardiovascular mortality. Here we assessed the dynamic changes of T-cell-derived cytokines, such as inteferon (IFN)-γ, interleukin (IL)-17 and IL-4, during the progression of atherosclerosis in apolipoprotein E-null (ApoE^−/−) mice, to understand the role of immune responses in different stages of atherosclerosis. Male ApoE^−/− mice were fed a high-fat, western-type diet (WD: 21% lipid, 1.5% cholesterol) after 5 weeks of age and were compared with C57BL/6 wild-type control mice fed a standard chow diet. Atherosclerotic lesions appeared in the aortic sinus of ApoE^−/− mice 4 weeks after WD and the lesions progressed and occupied >50% of the total sinus area 16 weeks after WD. Aortic IL-17 mRNA and protein expression started to increase in ApoE^−/− mice after 4 weeks on the WD and peaked at around 8–12 weeks on the WD. In terms of systemic expression of T-cell-derived cytokines, IL-17 production from splenocytes after anti-CD3/CD28 stimuli increased from 4 weeks on the WD, peaked at 12 weeks and returned to control levels at 16 weeks. The production of IFN-γ and IL-4 (Th1 and Th2 cytokines, respectively) from splenocytes was delayed compared with IL-17. Taken together, the present data indicate that Th17 cell response may be involved at an early stage in the development of atherosclerosis.     

miR-98 suppresses melanoma metastasis through a negative feedback loop with its target gene IL-6

Dysregulated microRNA (miRNA) expression has a critical role in tumor development and metastasis. However, the mechanism by which miRNAs control melanoma metastasis is unknown. Here, we report reduced miR-98 expression in melanoma tissues with increasing tumor stage as well as metastasis; its expression is also negatively associated with melanoma patient survival. Furthermore, we demonstrate that miR-98 inhibits melanoma cell migration in vitro as well as metastatic tumor size in vivo. We also found that IL-6 is a target gene of miR-98, and IL-6 represses miR-98 levels via the Stat3-NF-κB-lin28B pathway. In an in vivo melanoma model, we demonstrate that miR-98 reduces melanoma metastasis and increases survival in part by reducing IL-6 levels; it also decreases Stat3 and p65 phosphorylation as well as lin28B mRNA levels. These results suggest that miR-98 inhibits melanoma metastasis in part through a novel miR-98-IL-6-negative feedback loop.     

Extracts Prepared from Feed Supplements Containing Wood Lignans Improve Intestinal Health by Strengthening Barrier Integrity and Reducing Inflammation

Lignans are known to exhibit a broad spectrum of biological activities, indicating their potential as constituents of feed supplements. This study investigated two extracts derived from the feed supplements ‘ROI’ and ‘Protect’—which contain the wood lignans magnolol and honokiol (‘ROI’), or soluble tannins additional to the aforementioned lignans (‘Protect’)—and their impact on selected parameters of intestinal functionality. The antioxidant and anti-inflammatory properties of the extracts were determined by measuring their effects on reactive oxygen species (ROS) and pro-inflammatory cytokine production in vitro. The impact on intestinal barrier integrity was evaluated in Caco-2 cells and Drosophila melanogaster by examining leaky gut formation. Furthermore, a feeding trial using infected piglets was conducted to study the impact on the levels of superoxide dismutase, glutathione and lipid peroxidation. The Protect extract lowered ROS production in Caco-2 cells and reversed the stress-induced weakening of barrier integrity. The ROI extract inhibited the expression or secretion of interleukin-8 (IL-8), interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα). Moreover, the ROI extract decreased leaky gut formation and mortality rates in Drosophila melanogaster. Dietary supplementation with Protect improved the antioxidant status and barrier integrity of the intestines of infected piglets. In conclusion, wood lignan-enriched feed supplements are valuable tools that support intestinal health by exerting antioxidant, anti-inflammatory and barrier-strengthening effects.     

The natural product salicin alleviates osteoarthritis progression by binding to IRE1α and inhibiting endoplasmic reticulum stress through the IRE1α-IκBα-p65 signaling pathway

Despite the high prevalence of osteoarthritis (OA) in older populations, disease-modifying OA drugs (DMOADs) are still lacking. This study was performed to investigate the effects and mechanisms of the small molecular drug salicin (SA) on OA progression. Primary rat chondrocytes were stimulated with TNF-α and treated with or without SA. Inflammatory factors, cartilage matrix degeneration markers, and cell proliferation and apoptosis markers were detected at the mRNA and protein levels. Cell proliferation and apoptosis were evaluated by EdU assays or flow cytometric analysis. RNA sequencing, molecular docking and drug affinity-responsive target stability analyses were used to clarify the mechanisms. The rat OA model was used to evaluate the effect of intra-articular injection of SA on OA progression. We found that SA rescued TNF-α-induced degeneration of the cartilage matrix, inhibition of chondrocyte proliferation, and promotion of chondrocyte apoptosis. Mechanistically, SA directly binds to IRE1α and occupies the IRE1α phosphorylation site, preventing IRE1α phosphorylation and regulating IRE1α-mediated endoplasmic reticulum (ER) stress by IRE1α-IκBα-p65 signaling. Finally, intra-articular injection of SA-loaded lactic-co-glycolic acid (PLGA) ameliorated OA progression by inhibiting IRE1α-mediated ER stress in the OA model. In conclusion, SA alleviates OA by directly binding to the ER stress regulator IRE1α and inhibits IRE1α-mediated ER stress via IRE1α-IκBα-p65 signaling. Topical use of the small molecular drug SA shows potential to modify OA progression.Subject terms: Osteoarthritis, Molecularly targeted therapy, Single-molecule biophysics, Drug development     

MiR-144-3p is associated with pathological inflammation in patients infected with Mycobacteroides abscessus

Infection with rapidly growing nontuberculous mycobacteria is emerging as a global health issue; however, key host factors remain elusive. Here, we investigated the characteristic immune profiles of peripheral blood mononuclear cells (PBMCs) from patients infected with Mycobacteroides abscessus subsp. abscessus (Mabc) and M. abscessus subsp. massiliense (Mmass). Using an integrated analysis of global mRNA and microRNA expression profiles, we found that several inflammatory cytokines/chemokines [interleukin (IL)-1β, IL-6, C-X-C motif chemokine ligand 2, and C-C motif chemokine ligand 2] and miR-144-3p were significantly upregulated in PBMCs from patients compared with those from healthy controls (HCs). Notably, there was a strong correlation between the expression levels of miR-144-3p and proinflammatory cytokines/chemokines. Similarly, upregulated expression of miR-144-3p and proinflammatory cytokines/chemokines was found in macrophages and lungs from mice after infection with Mabc and Mmass. We showed that the expression of negative regulators of inflammation (SARM1 and TNIP3) was significantly downregulated in PBMCs from the patients, although they were not putative targets of miR-144-3p. Furthermore, overexpression of miR-144-3p led to a marked increase in proinflammatory cytokines/chemokines and promoted bacterial growth in macrophages. Together, our results highlight the importance of miR-144-3p linking to pathological inflammation during M. abscessus infection.Subject terms: Bacterial infection, Bacterial infection, Chemokines, Mechanisms of disease     

LncRNA HSPA7 in human atherosclerotic plaques sponges miR-223 and promotes the proinflammatory vascular smooth muscle cell transition

Although there are many genetic loci in noncoding regions associated with vascular disease, studies on long noncoding RNAs (lncRNAs) discovered from human plaques that affect atherosclerosis have been highly limited. We aimed to identify and functionally validate a lncRNA using human atherosclerotic plaques. Human aortic samples were obtained from patients who underwent aortic surgery, and tissues were classified according to atherosclerotic plaques. RNA was extracted and analyzed for differentially expressed lncRNAs in plaques. Human aortic smooth muscle cells (HASMCs) were stimulated with oxidized low-density lipoprotein (oxLDL) to evaluate the effect of the identified lncRNA on the inflammatory transition of the cells. Among 380 RNAs differentially expressed between the plaque and control tissues, lncRNA HSPA7 was selected and confirmed to show upregulated expression upon oxLDL treatment. HSPA7 knockdown inhibited the migration of HASMCs and the secretion and expression of IL-1β and IL-6; however, HSPA7 knockdown recovered the oxLDL-induced reduction in the expression of contractile markers. Although miR-223 inhibition promoted the activity of Nf-κB and the secretion of inflammatory proteins such as IL-1β and IL-6, HSPA7 knockdown diminished these effects. The effects of miR-223 inhibition and HSPA7 knockdown were also found in THP-1 cell-derived macrophages. The impact of HSPA7 on miR-223 was mediated in an AGO2-dependent manner. HSPA7 is differentially increased in human atheroma and promotes the inflammatory transition of vascular smooth muscle cells by sponging miR-223. For the first time, this study elucidated the molecular mechanism of action of HSPA7, a lncRNA of previously unknown function, in humans.Subject terms: Mechanisms of disease, Atherosclerosis, Long non-coding RNAs     

Rosmarinic Acid Prevents Cisplatin-Induced Liver and Kidney Injury by Inhibiting Inflammatory Responses and Enhancing Total Antioxidant Capacity,Thereby Activating the Nrf2 Signaling Pathway

Drug-induced liver and kidney damage is an emergent clinical issue that should be addressed. Rosmarinic acid (RA) has obvious anti-inflammatory and antioxidant effects, so we evaluated the anti-inflammatory and antioxidant effects of RA pretreatment on serum and liver and kidney tissues of cisplatin (CP)-treated mice and explored the possible mechanisms. The results showed that RA pretreatment effectively downregulated the serum, liver, and kidney levels of ALT, AST, BUN, and CRE and the inflammatory factors IL-1β, IL-6, and TNF-α, and simultaneously enhanced the total antioxidant capacity of the liver and kidney. RA pretreatment significantly reduced the levels of MPO, MDA, and NO in liver and kidney tissue, inhibited the mRNA expression of IL-1β, IL-6, and TNF-α in liver and kidney tissue, activated the Nrf2 signaling pathway, and upregulated the mRNA expression of downstream target genes. Our findings show that RA could effectively prevent and alleviate acute liver and kidney injury caused by CP.     

METTL3 promotes adriamycin resistance in MCF-7 breast cancer cells by accelerating pri-microRNA-221-3p maturation in a m6A-dependent manner

Breast cancer (BC) is the most prevalent malignant neoplasm among women and is the fifth most common cause of cancer-associated death worldwide. Acquired chemoresistance driven by genetic and epigenetic alterations is a significant clinical challenge in treating BC. However, the mechanism of BC cell resistance to adriamycin (ADR) remains to be elucidated. In this study, we identified the methyltransferase-like 3/microRNA-221-3p/homeodomain-interacting protein kinase 2/Che-1 (METTL3/miR-221-3p/HIPK2/Che-1) axis as a novel signaling event that may be responsible for resistance of BC cells to ADR. A dual-luciferase reporter gene assay was employed to test the presence of miR-221-3p binding sites in the 3′UTR of HIPK2. Drug resistance was evaluated by immunoblotting multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP). Cultured ADR-resistant MCF-7 cells were assayed for their half maximal inhibitory concentration (IC50) values and apoptosis using an MTT assay and Annexin V-FITC/PI-labeled flow cytometry, and the cells were then xenografted into nude mice. METTL3 knockdown was shown to reduce the expression of miR-221-3p by reducing pri-miR-221-3p m6A mRNA methylation, thereby reducing the IC50 value of ADR-resistant MCF-7 cells, reducing the expression of MDR1 and BCRP, and inducing apoptosis. Mechanistically, miR-221-3p was demonstrated to negatively regulate HIPK2 and upregulate its direct target Che-1, thus leading to enhanced drug resistance in ADR-resistant MCF-7 cells. In vitro results were reproduced in nude mice xenografted with ADR-resistant MCF-7 cells. Our work elucidates an epigenetic mechanism of acquired chemoresistance in BC, in support of the METTL3/miR-221-3p/HIPK2/Che-1 axis as a therapeutic target for the improvement of chemotherapy.Subject terms: Breast cancer, Cell biology     

MicroRNA-103a-3p controls proliferation and osteogenic differentiation of human adipose tissue-derived stromal cells

The elucidation of the molecular mechanisms underlying the differentiation and proliferation of human adipose tissue-derived stromal cells (hADSCs) represents a critical step in the development of hADSCs-based cellular therapies. To examine the role of the microRNA-103a-3p (miR-103a-3p) in hADSCs functions, miR-103a-3p mimics were transfected into hADSCs in order to overexpress miR-103a-3p. Osteogenic differentiation was induced for 14 days in an osetogenic differentiation medium and assessed by using an Alizarin Red S stain. The regulation of the expression of CDK6 (cyclin-dependent kinase 6), a predicted target of miR-103a-3p, was determined by western blot, real-time PCR and luciferase reporter assays. Overexpression of miR-103a-3p inhibited the proliferation and osteogenic differentiation of hADSCs. In addition, it downregulated protein and mRNA levels of predicted target of miR-103a-3p (CDK6 and DICER1). In contrast, inhibition of miR-103a-3p with 2′O methyl antisense RNA increased the proliferation and osteogenic differentiation of hADSCs. The luciferase reporter activity of the construct containing the miR-103a-3p target site within the CDK6 and DICER1 3′-untranslated regions was lower in miR-103a-3p-transfected hADSCs than in control miRNA-transfected hADSCs. RNA interference-mediated downregulation of CDK6 and DICER1 in hADSCs inhibited their proliferation and osteogenic differentiation. The results of the current study indicate that miR-103a-3p regulates the osteogenic differentiation of hADSCs and proliferation of hADSCs by direct targeting of CDK6 and DICER1 partly. These findings further elucidate the molecular mechanisms governing the differentiation and proliferation of hADSCs.     

Development,Validation, and Application of the UPLC-DAD Methodology for the Evaluation of the Qualitative and Quantitative Composition of Phenolic Compounds in the Fruit of American Cranberry (Vaccinium macrocarpon Aiton)

Phenolic compounds in the fruit of American cranberry (Vaccinium macrocarpon Aiton) determine the antioxidant, anti-inflammatory, anticancer, and other biological effects. The berries are used in the production of medicinal preparations and food supplements, which highlights the importance of qualitative and quantitative analysis of phenolic compounds in cranberry fruit raw material. The aim of our study was to develop and validate an efficient, cost-effective, reproducible, and fast UPLC-DAD methodology for the evaluation of the qualitative and quantitative composition of phenolic compounds in raw material and preparations of American cranberry fruit. During the development of the methodology, chlorogenic acid and the following flavonols were identified in cranberry fruit samples: myricetin-3-galactoside, quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-α-L-arabinopyranoside, quercetin-3-α-L-arabinofuranoside, quercetin-3-rhamnoside, myricetin, and quercetin. The developed and optimized UPLC-DAD methodology was validated according to the guidelines of the International Council for Harmonization (ICH), evaluating the following parameters: range, specificity, linearity (R² > 0.999), precision (%RSD < 2%), LOD (0.38–1.01 µg/mL), LOQ (0.54–3.06 µg/mL), and recovery (80–110%). The developed methodology was applied to evaluate the qualitative and quantitative composition of phenolic compounds in fruit samples of cranberry cultivars ‘Baifay’, ‘Bergman’, ‘Prolific’, and ‘Searles’, as well as ‘Bain-MC’ and ‘BL-12′ clones. In the tested samples, the majority (about 70%) of the identified flavonols were quercetin derivatives. The greatest amount of quercetin-3-galactoside (1035.35 ± 4.26 µg/g DW) was found in fruit samples of the ‘Searles’ cultivar, and the greatest amount of myricetin-3-galactoside (940.06 ± 24.91 µg/g DW) was detected in fruit samples of the ‘Woolman’ cultivar.     

MiodesinTM Positively Modulates the Immune Response in Endometrial and Vaginal Cells

Endometriosis presents high prevalence and its physiopathology involves hyperactivation of endometrial and vaginal cells, especially by bacteria. The disease has no cure and therapies aiming to inhibit its development are highly desirable. Therefore, this study investigated whether Miodesin^TM (10 µg/mL = IC₈₀; 200 µg/mL = IC₅₀), a natural compound constituted by Uncaria tomentosa, Endopleura uchi, and astaxanthin, could exert anti-inflammatory and anti-proliferative effects against Lipopolysaccharides (LPS) stimulation in endometrial and Candida albicans vaginal cell lines. VK2 E6/E7 (vaginal) and KLE (epithelial) cell lines were stimulated with Candida albicans (1 × 10⁷ to 5 × 10⁷/mL) and LPS (1 μg/mL), respectively. Miodesin^TM inhibited mRNA expression for Nuclear factor kappa B (NF-κB), ciclo-oxigenase 1 (COX-1), and phospholipase A2 (PLA2), beyond the C–C motif chemokine ligand 2 (CCL2), CCL3, and CCL5 in VK2 E6/E7 cells (p < 0.05). In addition, the inhibitory effects of both doses of Miodesin^TM (10 µg/mL and 200 µg/mL) resulted in reduced secretion of interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor α (TNF-α) (24 h, 48 h, and 72 h) and CCL2, CCL3, and CLL5 (p < 0.05) by VK2 E6/E7 cells. In the same way, COX-1 Miodesin^TM inhibited LPS-induced hyperactivation of KLE cells, as demonstrated by reduced secretion of IL-1β, IL-6, IL-8, TNF-α (24 h, 48 h, and 72 h) and CCL2, CCL3, and CLL5 (p < 0.05). Furthermore, Miodesin^TM also inhibited mRNA expression and secretion of matrix metalloproteinase-2 (MMP-2), MMP-9, and vascular endothelial growth factor (VEGF), which are key regulators of invasion of endometrial cells. Thus, the study concludes that Miodesin^TM presents beneficial effects in the context of endometriosis, positively affecting the inflammatory and proliferative response.     

Anti-arthritic and cartilage damage prevention via regulation of Nrf2/HO-1 signaling by glabridin on osteoarthritis

Osteoarthritis is a common degenerative disease linked with inflammatory disorders and oxidative stress. Glabridin is an isoflavonoid and major active constituent of licorice. This study aims to investigate the anti-arthritic effects of glabridin on nuclear factor erythroid 2 – related factor 2 (Nrf2) signaling pathway, inflammatory responses, and cartilage degeneration in in-vitro and in-vivo models. Studies on IL-1β-induced chondrocyte model was performed to evaluate matrix metalloproteinase (MMP), Nrf2 signaling pathway. Glabridin was orally administered in monosodium-iodoacetate (MIA)-induced osteoarthritic (OA) rats for in-vivo evaluation. Pain and swelling of limbs were observed, oxidative stress markers and inflammatory cytokines were measured. Histomorphological changes in the joint cartilage were analyzed. Glabridin significantly reduced the arthritic score and paw swelling along with improved body weight, and organ index of rats. Histopathological score showed significant prevention of joint cartilage degeneration by glabridin. Expressions of TNF-α, IL-6, IL-10, and IL-1β were attenuated by glabridin (p < 0.05) in MIA-induced rats. Protein expressions of iNOS, COX-2, ADAMTS5, MMP-3, and MMP-13 were suppressed (p < 0.05) whereas the Nrf2/HO-1 signaling was activated by glabridin (p < 0.05) in osteoarthritic chondrocytes. Therefore, anti-arthritic and chondroprotective activity of glabridin is suggested by the inhibition of MMP expression and regulation of Nrf2/HO-1 signaling.