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Air pollution poses a significant global concern, notably impacting pregnancy outcomes through mechanisms such as DNA damage, oxidative stress, inflammation, and altered miRNA expression, all of which can adversely affect trophoblast functions. Cleistocalyx nervosum var. paniala, known for its abundance of anthocyanins with diverse biological activities including anti-mutagenic, antioxidant, and anti-inflammatory properties, is the focus of this study examining its effect on Particulate Matter 10 (PM10) soluble extract-induced trophoblast cell dysfunction via miRNA expression. The study involved the extraction of C. nervosum fruit using 70% ethanol, followed by fractionation with hexane, dichloromethane, and ethyl acetate. Subsequent testing for total phenolics, flavonoids, anthocyanins, and antioxidant activity revealed the ethyl acetate fraction (CN-EtOAcF) as possessing the highest phenolic and anthocyanin content along with potent antioxidant activity, prompting its selection for further investigation. In vitro studies on HTR-8/SVneo cells demonstrated that 5–10 µg/mL PM10 soluble extract exposure inhibited cell proliferation, migration, invasion, and induced apoptosis. However, pretreatment with 20–80 µg/mL CN-EtOAcF followed by 5 µg/mL PM10 soluble extract exposure exhibited protective effects against PM10 soluble extract-induced damage, including inflammation inhibition and intracellular ROS suppression. Notably, CN-EtOAcF down-regulated PM10-induced miR-146a-5p expression, with SOX5 identified as a potential target. Overall, CN-EtOAcF demonstrated the potential to protect against PM10-induced harm in trophoblast cells, suggesting its possible application in future therapeutic approaches. [ABSTRACT FROM AUTHOR]

Aims: The study aimed to investigate the role of miR‐146a‐5p in osteogenesis of hPDLSCs irradiated with low‐energy red LEDs. Methods: After irradiation with 5 J/cm2 red LED, miR‐146a‐5p expression was detected by real‐time quantitative polymerase chain reaction (RT‐qPCR), and osteogenic markers expression was determined by RT‐qPCR and Western blotting. Alkaline phosphatase (ALP) activity was assessed by ALP staining, and mineralization was assessed by Alizarin Red staining, respectively. Lentiviral vectors were designed to regulate miR‐146a‐5p expression. Dual‐luciferase reporter assay was performed to confirm the targeted relationship between miR‐146a‐5p and MAPK1. Short hairpin RNA (shRNA) was used to regulate MAPK1 expression. Results: RT‐qPCR and western blotting revealed that 5 J/cm2 irradiation elevated the levels of the osteogenic markers osterix (OSX) and bone sialoprotein (BSP) in hPDLSCs. miR‐146a‐5p is downregulated in hPDLSCs under the low‐energy red LED light irradiation. miR‐146a‐5p underexpression markedly promoted the osteogenic potential of hPDLSCs. miR‐146a‐5p targeted MAPK1. 5 J/cm2 red LED irradiation rescued the inhibitory effects of upregulated miR‐146a‐5p on osteogenic differentiation, and the positive influence of red LED irradiation could be reversed by downregulated MAPK1. Conclusion: These findings confirm that miR‐146a‐5p is involved in the effect of LED irradiation on the osteogenic differentiation of hPDLSCs by targeting MAPK1. Red LED irradiation may be a potential clinical adjunct therapy for periodontal regeneration. [ABSTRACT FROM AUTHOR]

Introduction: Allergic asthma is prevalent in children, with Dermatophagoides farinae as a common indoor allergen. Current treatments for allergic airway inflammation are limited and carry risks. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) show promise as a cell-free therapeutic approach. However, the use of engineered MSC-EVs for D. farinae-induced allergic airway epithelial cell inflammation remains unexplored. Methods: We generated miR-146a-5p-engineered EVs from human umbilical cord mesenchymal stem cells (hucMSCs) and established D. farinae-induced mouse and human bronchial epithelial cell allergic models. Levels of IL-1b, IL-18, IL-4, IL-5, IL-6, IL-10, IL-33, TNF-α and IgE were detected using ELISA. The relative TRAF6 and IRAK1 mRNA expression was quantified using qPCR assay and the NLRP3, NF-κB, IRAK1 and TRAF6 protein expression was determined using Western blotting. The regulatory effect of IRAK1 and TRAF6 by miR-146a-5p was examined using a dual luciferase reporter assay, and the nuclear translocation of NF-κB p65 into 16-HBE cells was evaluated using immunofluorescence assay. Results: Treatment with hucMSC-EVs effectively reduced allergic inflammation, while miR-146a-5p engineered hucMSC-EVs showed greater efficacy. The enhanced efficacy in alleviating allergic airway inflammation was attributed to the downregulation of IRAK1 and TRAF6 expression, facilitated by miR-146a-5p. This downregulation subsequently led to a decrease in NF-κB nuclear translocation, which in turn resulted in reduced activation of the NLRP3 inflammasome and diminished production of inflammatory cytokines, including IL-6, TNF-α, IL-1β and IL-18. Conclusion: Our study underscores the potential of miR-146a-5p engineered hucMSC-EVs as a cell-free therapeutic strategy for D. farinae-induced allergic airway inflammation, offering a promising avenue for boosting antiinflammatory responses. [ABSTRACT FROM AUTHOR]

Abstract Air pollution poses a significant global concern, notably impacting pregnancy outcomes through mechanisms such as DNA damage, oxidative stress, inflammation, and altered miRNA expression, all of which can adversely affect trophoblast functions. Cleistocalyx nervosum var. paniala, known for its abundance of anthocyanins with diverse biological activities including anti-mutagenic, antioxidant, and anti-inflammatory properties, is the focus of this study examining its effect on Particulate Matter 10 (PM10) soluble extract-induced trophoblast cell dysfunction via miRNA expression. The study involved the extraction of C. nervosum fruit using 70% ethanol, followed by fractionation with hexane, dichloromethane, and ethyl acetate. Subsequent testing for total phenolics, flavonoids, anthocyanins, and antioxidant activity revealed the ethyl acetate fraction (CN-EtOAcF) as possessing the highest phenolic and anthocyanin content along with potent antioxidant activity, prompting its selection for further investigation. In vitro studies on HTR-8/SVneo cells demonstrated that 5–10 µg/mL PM10 soluble extract exposure inhibited cell proliferation, migration, invasion, and induced apoptosis. However, pretreatment with 20–80 µg/mL CN-EtOAcF followed by 5 µg/mL PM10 soluble extract exposure exhibited protective effects against PM10 soluble extract-induced damage, including inflammation inhibition and intracellular ROS suppression. Notably, CN-EtOAcF down-regulated PM10-induced miR-146a-5p expression, with SOX5 identified as a potential target. Overall, CN-EtOAcF demonstrated the potential to protect against PM10-induced harm in trophoblast cells, suggesting its possible application in future therapeutic approaches.

Objective: To investigate the relationship between the expression of microRNA (mi R)-146a-5p and mi R-146b-5p in villus tissue of patients with unexplained recurrent spontaneous abortion (URSA) and angiogenesis factors and microvessel density (MVD). Methods: 119 URSA patients who admitted to our hospital from January 2020 to December 2023 were selected as URSA group, and 90 women with normal early pregnancy who voluntarily requested abortion to terminate pregnancy were selected as control group.The expressions of mi R-146a-5p, mi R-146b-5p, angiogenesis factors [hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF)] and MVD in villus tissues were compared between two groups. The correlation between mi R-146a-5p, mi R-146b-5p expression and HIF-1α, VEGF expression and MVD in villus tissue were analyzed by Pearson test. Results: Compared with control group, the expression level of mi R-146a-5p was decreased in villus tissue of URSA group (P＜0.05), and the expression level of mi R-146b-5p was increased (P＜0.05). Compared with control group, the MVD of villi in URSA group was decreased (P＜0.05). The expression levels of HIF-1α and VEGF in villus tissues of URSA group were lower than those in control group (P＜0.05). Pearson test showed that, the expression of mi R-146a-5p in villus tissue of URSA patients was positively correlated with HIF-1α, VEGF and MVD (P＜0.05), and the expression of mi R-146b-5p was negatively correlated with HIF-1α, VEGF and MVD (P＜0.05). Conclusion: The low expression of mi R-146a-5p and the high expression of mi R-146b-5p in villous tissues of URSA patients are related to the down-regulation of angiogenesis factors HIF-1α and VEGF and the decrease of MVD. [ABSTRACT FROM AUTHOR]

BACKGROUND: Currently, there have been studies on the regulatory mechanism of lncRNA\miRNA\mRNA co-expression network on the occurrence and development of osteoarthritis. Our research group has screened qualified NONHSAT248596.1 and miR-146a-5p through the database in the previous stage, but the corresponding in vivo experiments to verify the above regulatory mechanisms are still lacking. OBJECTIVE: To explore the role of NONHSAT248596.1 in regulating competitive endogenous RNA of miR-146a-5p in cartilage degeneration mediated by stromal cell derived factor type 1/chemokine receptor 4 axis in vivo. METHODS: The models of osteoarthritis were established in 36 New Zealand rabbits by injecting stromal cell derived factor 1 solution into the knee joint of the right hind limb. According to the random number table method, they were divided into four groups. lncRNA group, miRNA group, ceRNA group and control group were injected with lentivirus vector overexpressing NONHSAT248596.1, lentivirus vector overexpressing miR-146a-5p, lentivirus vector overexpressing miR-146a-5p+NONHSAT248596.1 and empty lentivirus vector into the molded knee joint, respectively. At 4, 8 and 12 weeks of modeling, cartilage tissues and subchondral bone tissues of the knee joint were taken for relevant detection. RESULTS AND CONCLUSION: Hematoxylin-eosin staining and safranin fast green staining showed different degrees of degeneration in the four groups. At 4 weeks, the cartilage tissue of the lncRNA group showed swelling of chondrocytes, loss of cell polarity, destruction of extracellular matrix, surface erosion, fracture formation and partial or full layer loss of cartilage tissue. The degree of cartilage injury was gradually aggravated with time. The progression of articular cartilage inflammation in the miRNA group was the slowest among the four groups. qRT-PCR showed that at the same time point, mRNA expression levels of NONHSAT248596.1, chemokine receptor 4, matrix metalloproteinase 3, matrix metalloproteinase 9 and matrix metalloproteinase 13 in cartilage tissue of the lncRNA group were higher than those of the other three groups (P < 0.05). The mRNA expression levels of miR-146a-5p, aggrecan and type II collagen were lower than those of the other three groups (P < 0.05). The mRNA expression levels of NONHSAT248596.1, chemokine receptor 4, matrix metalloproteinase 3, matrix metalloproteinase 9 and matrix metalloproteinase 13 in the miRNA group were lower than those in the ceRNA group and control group at 8 and 12 weeks after the model construction (P < 0.05). The mRNA expressions of miR-146a-5p, aggrecan and type II collagen were higher than those of the ceRNA group and control group (P < 0.05). Western blot assay showed that at the same time point, the expression levels of aggrecan and type II collagen in cartilage tissue of the lncRNA group were always lower than those of the other three groups (P < 0.05). The expression levels of aggrecan and type II collagen in cartilage tissue of the miRNA group at 8 and 12 weeks after modeling were higher than those of the ceRNA group and control group (P < 0.05). The results showed that miR-146a-5p, as the target of NONHSAT248596.1, could be inhibited by the effect of its ceRNA. After acting on miR-146a-5p, NONHSAT248596.1 regulates the stromal cell derived factor type 1/chemokine receptor 4 axis to affect the expression of matrix metalloprotein, type II collagen, and aggrecan in osteoarthritis chondrocytes, resulting in the degradation of extracellular matrix and the loss of proteoglycan. [ABSTRACT FROM AUTHOR]

Circular E3 ubiquitin-protein ligase (circ-ITCH), a novel circRNA, is generated from several exons of itchy E3 ubiquitin protein ligase. Reports on circ-ITCH have discussed its pathogenic performance in human diseases. Based on this, this study determines whether and how circ-ITCH is involved in the pathogenesis of chronic glomerulonephritis (CGN). First, a rat model of CGN induced by cationic bovine serum albumin was established. Then, CGN rats were injected with lentiviruses interfering with the expression of circ-ITCH, miR-146a-5p or tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG). Then, blood urea nitrogen and serum creatinine levels were measured to evaluate renal function; inflammatory factor content and fibrosis marker expression in kidney tissue were detected; renal pathological damage was analyzed by hematoxylin-eosin staining and periodic acid-Schiff staining. Finally, the binding relationship between miR-146a-5p and circ-ITCH or YWHAG was verified. Elevating circ-ITCH or depleting miR-146a-5p improved renal function (both P<0.05), reduced inflammatory factor content and fibrosis marker expression (all P<0.05) and alleviated renal pathological damage in CGN rats. Circ-ITCH negatively regulated miR-146a-5p expression by adsorbing miR-146a-5p (P<0.05), and miR-146a-5p inhibited YWHAG expression by binding to the 3'-UTR of YWHAG (P<0.05). Loss of YWHAG reversed the protective effect of upregulated circ-ITCH in CGN rats (all P<0.05). We conclude that circ-ITCH improves renal function and attenuates inflammation and renal injury in rats with CGN via the miR-146a-5p/YWHAG axis. [ABSTRACT FROM AUTHOR]

Abstract Background Hypoxia/reoxygenation (H/R) induces cardiomyocyte ferroptosis, a core remodeling event in myocardial ischemia/reperfusion injury. Methyltransferase-like 14 (METTL14) emerges as a writer of N6-methyladenosine (m6A) modification. This study was conducted to decipher the role of METTL14 in H/R-induced cardiomyocyte ferroptosis. Methods Mouse cardiomyocytes HL-1 were cultured and underwent H/R treatment. The degree of ferroptosis after H/R treatment was appraised by the cell counting kit-8 assay, assay kits (ROS/GSH/Fe2+), and Western blotting (GPX4/ACSL4). The intracellular expressions of METTL14, pri-miR-146a-5p, miR-146a-5p, or adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) were examined by real-time quantitative polymerase chain reaction or Western blotting, with m6A quantification analysis and RNA immunoprecipitation to determine the total m6A level and the expression of pri-miR-146a-5p bound to DiGeorge critical region 8 (DGCR8) and m6A-modified pri-miR-146a-5p. The binding of miR-146a-5p to APPL1 was testified by the dual-luciferase assay. Results H/R treatment induced cardiomyocyte ferroptosis (increased ROS, Fe2+, and ACSL4 and decreased GSH and GPX4) and upregulated METTL14 expression. METTL14 knockdown attenuated H/R-induced cardiomyocyte ferroptosis. METTL14 induced the recognition of pri-miR-146a-5p by DGCR8 by increasing m6A modification on pri-miR-146a-5p, which promoted the conversion of pri-miR-146a-5p into miR-146a-5p and further repressed APPL1 transcription. miR-146a-5p upregulation or APPL1 downregulation limited the inhibitory effect of METTL14 downregulation on H/R-induced cardiomyocyte ferroptosis. Conclusion METTL14 promoted miR-146a-5p expression through the recognition and processing of pri-miR-146a-5p by DGCR8, which repressed APPL1 transcription and triggered H/R-induced cardiomyocyte ferroptosis.

Abstract Aim To elaborate the underlying mechanisms by which IL-1β promote progression of Dry eye disease(DED) through effect on pyroptosis and apoptosis of corneal epithelial cells(CECs). Methods 400 mOsM solutions were used to establish the DED model (hCECs- DED). RT-qPCR was performed to measure IL-1β mRNA and miR-146a-5p in CECs. Western blotting was performed to measure STAT3, GSDMD, NLRP3, and Caspase-1 levels. Cell counting kit-8 assay was adopted to check cell viability. Apoptosis was detected by flow cytometry. ELISAs were performed to determine IL-18, IL-33 and LDH. The luciferase test detects targeting relationships. Results After treatment with 400 mOsM solution, cell viability decreased and apoptosis increased. Compared with hCECs, IL-1β was increased and miR-146a-5p was decreased in hCECs-DED. At the same time, GSDMD, NLRP3, Caspase-1, IL-18, IL-33 and LDH were significantly higher in hCECs-DED than in hCECs, while IL-1β silencing reversed this effect. In addition, IL-1β negatively regulated miR-146a-5p. MiR-146a-5p mimics eliminated the inhibition of hCECs-DED pyroptosis and apoptosis caused by IL-1β silencing. At the same time, miR-146a-5p reduced STAT3 levels in hCECs. Conclusion Highly expressed IL-1β promoted pyroptosis and apoptosis of hCECs- DED through downregulated miR-146a-5p and inhibited STAT3.

Introduction: Inflammatory bowel disease (IBD) is a chronic disease involving multiple genes, and the current available targeted drugs for IBD only deliver moderate efficacy. Whether there is a single gene that systematically regulates IBD is not yet known. MiR-146a plays a pivotal role in repression of innate immunity, but its function in the intestinal inflammation is sort of controversy, and the genetic regulatory networks regulated by miR-146a in IBD has not been revealed. Methods: RT-qPCR was employed to detect the expression of miR-146a in IBD patients and in a mouse IBD model induced by dextran sulfate sodium (DSS), and then we generated a miR-146a knock-out mouse line with C57/Bl6N background. The disease activity index was scored in DSS-treated miR-146a deficiency mice and their wild type (WT) littermates. Bulk RNA-sequencing, RTqPCR and immunostaining were done to illustrate the downstream genetic regulatory networks of miR-146a in flamed colon. Finally, the modified miR-146a mimics were used to treat DSS-induced IBD in miR-146a knock-out and WT IBD mice. Results: We showed that the expression of miR-146a in the colon was elevated in dextran sulfate sodium (DSS)-induced IBD mice and patients with IBD. DSS induced dramatic body weight loss and more significant rectal bleeding, shorter colon length, and colitis in miR-146a knock-out mice than WT mice. The miR-146a mimics alleviated DSS-induced symptoms in both miR-146a-/- and WT mice. Further RNA sequencing illustrated that the deficiency of miR-146a de-repressed majority of DSS-induced IBD-related genes that cover multiple genetic regulatory networks in IBD, and supplementation with miR-146a mimics inhibited the expression of many IBD-related genes. Quantitative RT-PCR or immunostaining confirmed that Ccl3, Saa3, Csf3, Lcn2, Serpine1, Serpine2, MMP3, MMP8, MMP10, IL1A, IL1B, IL6, CXCL2, CXCL3, S100A8, S100A9, TRAF6, P65, p-P65, and IRAK1 were regulated by miR-146a in DSS induced IBD. Among them, MMP3, MMP10, IL6, IL1B, S100A8, S100A9, SERPINE1, CSF3, and IL1A were involved in the active stage of IBD in humans. Discussion: Our date demonstrated that miR-146a acts as a top regulator in C57/BL6N mice to systematically repress multiple genetic regulatory networks involved in immune response of intestine to environment factors, and combinatory treatment with miR-146a-5p and miR-146a-3p mimics attenuates DSS-induced IBD in mice through down-regulating multiple genetic regulatory networks which were increased in colon tissue from IBD patients. Our findings suggests that miR-146a is a top inhibitor of IBD, and that miR-146a-5p and miR-146a-3p mimics might be potential drug for IBD. [ABSTRACT FROM AUTHOR]

Background/Objectives: The balance between regulatory and Th17 cells plays an important role in maintaining the immune tolerance after kidney transplantation (KTx) which is essential for transplantation success, defined as a long graft survival and an absence of organ rejection. The present study aimed to assess whether the pretransplant characteristics of IL-17A and IL-17F, their receptors, as well as miR-146a-5p, an miRNA associated with IL-17A/F regulation, can predict KTx outcomes. Methods: A group of 108 pre-KTx dialysis patients and 125 healthy controls were investigated for single nucleotide substitutions within genes coding for IL-17A, IL-17F, their IL-17RA/RC receptors, and miR-146a-5p. Genotyping was performed using LightSNiP assays. In addition, IL17-A/F serum concentrations were determined using ELISA while miR-146a-5p expression was analyzed by RT-PCR. Results: The IL-17F (rs763780) G allele prevailed in KTx recipients as compared to healthy individuals (OR = 23.59, p < 0.0001) and was associated with a higher IL-17F serum level (p = 0.0381) prior to transplantation. Higher miR-146a-5p expression before KTx was more frequently detected in recipients with an increased IL-17A serum concentration (p = 0.0177). Moreover, IL-17A (rs2275913) GG homozygosity was found to be associated with an increased incidence of deaths before KTx (OR = 4.17, p = 0.0307). T-cell or acute rejection episodes were more frequently observed among patients with the C allele of miR-146a-5p (rs2910164) (OR = 5.38, p = 0.0531). IL17-RA/-RC genetic variants (p < 0.05) seem to be associated with eGFR values. Conclusions: These results imply that IL-17F (rs763780) polymorphism is associated with the serum level of this cytokine and may be related to the risk of renal disease and transplant rejection together with miR-146a-5p (rs2910164), while the IL-17A (rs2275913) genotype may affect patients' survival before KTx. [ABSTRACT FROM AUTHOR]

Chronic trauma in diabetes is a leading cause of disability and mortality. Exosomes show promise in tissue regeneration. This study investigates the role of exosomes derived from adipose stem cells (ADSC-Exos) in angiogenesis. MiRNA-seq analysis revealed significant changes in 47 genes in human umbilical vein endothelial cells (HUVECs) treated with ADSC-Exos, with miR-146a-5p highly expressed. MiR-146a-5p mimics enhanced the pro-angiogenic effects of ADSC-Exos, while inhibitors had the opposite effect. JAZF1 was identified as a direct downstream target of miR-146a-5p through bioinformatics, qRT-PCR, and dual luciferase assay. Overexpress of JAZF1 resulted in decreased proliferation, migration, and angiogenic capacity of HUVECs, and reduced VEGFA expression. This study proposes that ADSC-Exos regulate angiogenesis partly via the miR-146a-5p/JAZF1 axis. [ABSTRACT FROM AUTHOR]

Background: Hypoxia/reoxygenation (H/R) induces cardiomyocyte ferroptosis, a core remodeling event in myocardial ischemia/reperfusion injury. Methyltransferase-like 14 (METTL14) emerges as a writer of N6-methyladenosine (m6A) modification. This study was conducted to decipher the role of METTL14 in H/R-induced cardiomyocyte ferroptosis. Methods: Mouse cardiomyocytes HL-1 were cultured and underwent H/R treatment. The degree of ferroptosis after H/R treatment was appraised by the cell counting kit-8 assay, assay kits (ROS/GSH/Fe2+), and Western blotting (GPX4/ACSL4). The intracellular expressions of METTL14, pri-miR-146a-5p, miR-146a-5p, or adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) were examined by real-time quantitative polymerase chain reaction or Western blotting, with m6A quantification analysis and RNA immunoprecipitation to determine the total m6A level and the expression of pri-miR-146a-5p bound to DiGeorge critical region 8 (DGCR8) and m6A-modified pri-miR-146a-5p. The binding of miR-146a-5p to APPL1 was testified by the dual-luciferase assay. Results: H/R treatment induced cardiomyocyte ferroptosis (increased ROS, Fe2+, and ACSL4 and decreased GSH and GPX4) and upregulated METTL14 expression. METTL14 knockdown attenuated H/R-induced cardiomyocyte ferroptosis. METTL14 induced the recognition of pri-miR-146a-5p by DGCR8 by increasing m6A modification on pri-miR-146a-5p, which promoted the conversion of pri-miR-146a-5p into miR-146a-5p and further repressed APPL1 transcription. miR-146a-5p upregulation or APPL1 downregulation limited the inhibitory effect of METTL14 downregulation on H/R-induced cardiomyocyte ferroptosis. Conclusion: METTL14 promoted miR-146a-5p expression through the recognition and processing of pri-miR-146a-5p by DGCR8, which repressed APPL1 transcription and triggered H/R-induced cardiomyocyte ferroptosis. [ABSTRACT FROM AUTHOR]

Aim: To elaborate the underlying mechanisms by which IL-1β promote progression of Dry eye disease(DED) through effect on pyroptosis and apoptosis of corneal epithelial cells(CECs). Methods: 400 mOsM solutions were used to establish the DED model (hCECs- DED). RT-qPCR was performed to measure IL-1β mRNA and miR-146a-5p in CECs. Western blotting was performed to measure STAT3, GSDMD, NLRP3, and Caspase-1 levels. Cell counting kit-8 assay was adopted to check cell viability. Apoptosis was detected by flow cytometry. ELISAs were performed to determine IL-18, IL-33 and LDH. The luciferase test detects targeting relationships. Results: After treatment with 400 mOsM solution, cell viability decreased and apoptosis increased. Compared with hCECs, IL-1β was increased and miR-146a-5p was decreased in hCECs-DED. At the same time, GSDMD, NLRP3, Caspase-1, IL-18, IL-33 and LDH were significantly higher in hCECs-DED than in hCECs, while IL-1β silencing reversed this effect. In addition, IL-1β negatively regulated miR-146a-5p. MiR-146a-5p mimics eliminated the inhibition of hCECs-DED pyroptosis and apoptosis caused by IL-1β silencing. At the same time, miR-146a-5p reduced STAT3 levels in hCECs. Conclusion: Highly expressed IL-1β promoted pyroptosis and apoptosis of hCECs- DED through downregulated miR-146a-5p and inhibited STAT3. [ABSTRACT FROM AUTHOR]

Introduction: The skin is the largest organ of the human body and fulfills protective, immune, and metabolic functions. Skin function and barrier integrity are actively regulated through circadian rhythm--associated genes and epigenetic mechanisms including DNA methylation/demethylation, histone acetylation/deacetylation, and microRNAs. MicroRNA-146a-5p (miR-146a) has been associated with immune activation and skin inflammation; however, the role of miR-146a in regulating skin aging is an open question. This study investigated the role of miR-146a in fibroblasts obtained from different donors in the context of aging, and a potential association of this miRNA with circadian rhythm. Methods: Normal human dermal fibroblasts (NHDFs) from 19y, 27y, 40y, and 62y old donors were used to analyze for miR-146a expression. Expression of miR-146a was downregulated with the hsa-mirVana miR-146a inhibitor, and upregulated with an extract from Adansonia digitata. Effects on markers of skin aging, including cell proliferation, production of Collagen-1 and inflammatory cytokines were assessed. Results: We show that the expression of miR-146a decreases with age in dermal fibroblasts and inhibition of miR-146a in 19y and 62y old NHDFs induced significant changes in essential clock genes indicating an association with circadian rhythm control. Furthermore, downregulation of miR-146a results in a reduction of cellular proliferation, Collagen-1 production, as well as an increase in DNA damage and pro-inflammatory markers. Activation of miR-146a with the Adansonia digitata extract reduced the deleterious effects seen during miR-146a inhibition and increased miR-146a transport through exosome transfer. Conclusion: miR-146a interacts with multiple biological pathways related to skin aging, including circadian rhythm machinery, cell-to-cell communication, cell damage repair, cell proliferation, and collagen production and represents a promising target to fight skin aging. Adansonia digitata extract can promote miR-146a expression and therefore support skin cells' health. [ABSTRACT FROM AUTHOR]

Background: Exosomes are involved in cell-to-cell communication in numerous diseases including cardiovascular diseases, neurological diseases. Little attention has been dedicated to exosomal circular RNAs in obstructive sleep apnea (OSA)-related cardiovascular diseases. The aim of this study was to explore the role of exosomal circular RNA ZNF292 (circZNF292) on AC16 cells exposure to intermittent hypoxia (IH). Methods: Exosome release inhibitor GW4869 was used to examine the effect of exosomes on IH-induced AC16 cells apoptosis. The expression of exosomal circZNF292 was detected by qRT-PCR in AC16 cells exposure to IH, and a luciferase reporter assay was conducted to confirm the connection between circZNF292 and miR-146a-5p. Exosomal circZNF292 was stably transfected with short hairpin RNAs (shRNAs) against circZNF292 and co-cultured with AC16 cells. The expression of miR-146a-5p and apoptosis-related protein was then measured to evaluate the effect of exosomal circZNF292. Results: We found that IH contributed to the AC16 cells apoptosis, and the administration of GW4869 increased the apoptosis of cardiomyocytes when exposed to IH. The expression of exosomal circZNF292 decreased and miR-146a-5p increased significantly in AC16 cells exposed to IH compared to normoxic conditions. Bioinformatics analysis predicted a circZNF292/miR-146a-5p axis in IH-induced cardiomyocytes apoptosis. The dual-luciferase reporter system validated the direct interaction of circZNF292 and miR-146a-5p. Knockdown of circZNF292 increased the expressions of miR-146a-5p and accelerated the AC16 cardiomyocytes apoptosis. Conclusions: The findings of this study suggested a novel mechanism by which exosomes transmit intrinsic regulatory signals to the myocardium through the exosomal circZNF292/miR-146a-5p axis. This finding highlights the potential of targeting this pathway as a therapeutic approach for treating cardiovascular diseases associated with OSA. [ABSTRACT FROM AUTHOR]

Abstract Backgrounds The intestinal development in early life is profoundly influenced by multiple biological components of breast milk, in which milk-derived extracellular vesicles (mEVs) contain a large amount of vertically transmitted signal from the mother. However, little is known about how maternal fiber-rich diet regulates offspring intestinal development by influencing the mEVs. Results In this study, we found that maternal resistant starch (RS) consumption during late gestation and lactation improved the growth and intestinal health of offspring. The mEVs in breast milk are the primary factor driving these beneficial effects, especially enhancing intestinal cell proliferation and migration. To be specific, administration of mEVs after maternal RS intake enhanced intestinal cell proliferation and migration in vivo (performed in mice model and indicated by intestinal histological observation, EdU assay, and the quantification of cyclin proteins) and in vitro (indicated by CCK8, MTT, EdU, and wound healing experiments). Noteworthily, miR-146a-5p was found to be highly expressed in the mEVs from maternal RS group, which also promotes intestinal cell proliferation in cells and mice models. Mechanically, miR-146a-5p target to silence the expression of ubiquitin ligase 3 gene NEDD4L, thereby inhibiting DVL2 ubiquitination, activating the Wnt pathway, and promoting intestinal development. Conclusion These findings demonstrated the beneficial role of mEVs in the connection between maternal fiber rich diet and offspring intestinal growth. In addition, we identified a novel miRNA-146a-5p-NEDD4L-β-catenin/Wnt signaling axis in regulating early intestinal development. This work provided a new perspective for studying the influence of maternal diet on offspring development. Graphical Abstract

Abstract Background Circular RNAs (circRNAs) have been reported to be involved in the initiation and development of cancers. The aim of this study was to determine the role of a circRNA, circ_0020123, in the development of non‐small cell lung cancer (NSCLC). Methods The expression of circ_0020123, microRNA‐146a‐5p (miR‐146a‐5p), and eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) mRNA was detected by quantitative real‐time PCR (qPCR). Western blot was used to determine the protein levels of cyclin D1, Bax, MMP‐9, and EIF4G2. Cell proliferation was assessed by cell counting kit‐8 (CCK‐8) assay and colony formation assay. Flow cytometry assay was applied to determine cell cycle apoptosis. Cell migration and invasion were assessed using transwell assay. The potential relationship between miR‐146a‐5p and circ_0020123 or EIF4G2 was ascertained by dual‐luciferase reporter assay and RIP assay. The role of circ_0020123 in vivo was explored by xenograft assay. Results Circ_0020123 was upregulated in NSCLC, and circ_0020123 knockdown repressed proliferation, migration, and invasion of NSCLC cells. Circ_0020123 targeted miR‐146a‐5p, and miR‐146a‐5p inhibitor reversed the effects of circ_0020123 knockdown on NSCLC cells. In addition, miR‐146a‐5p suppressed cell proliferation, migration, and invasion by targeting EIF4G2. Moreover, the antitumor role of circ_0020123 knockdown was verified in vivo. Conclusion Knockdown of circ_0020123 inhibited NSCLC cell progression and tumor growth by targeting the miR‐146a‐5p/EIF4G2 axis.

Backgrounds: The intestinal development in early life is profoundly influenced by multiple biological components of breast milk, in which milk-derived extracellular vesicles (mEVs) contain a large amount of vertically transmitted signal from the mother. However, little is known about how maternal fiber-rich diet regulates offspring intestinal development by influencing the mEVs. Results: In this study, we found that maternal resistant starch (RS) consumption during late gestation and lactation improved the growth and intestinal health of offspring. The mEVs in breast milk are the primary factor driving these beneficial effects, especially enhancing intestinal cell proliferation and migration. To be specific, administration of mEVs after maternal RS intake enhanced intestinal cell proliferation and migration in vivo (performed in mice model and indicated by intestinal histological observation, EdU assay, and the quantification of cyclin proteins) and in vitro (indicated by CCK8, MTT, EdU, and wound healing experiments). Noteworthily, miR-146a-5p was found to be highly expressed in the mEVs from maternal RS group, which also promotes intestinal cell proliferation in cells and mice models. Mechanically, miR-146a-5p target to silence the expression of ubiquitin ligase 3 gene NEDD4L, thereby inhibiting DVL2 ubiquitination, activating the Wnt pathway, and promoting intestinal development. Conclusion: These findings demonstrated the beneficial role of mEVs in the connection between maternal fiber rich diet and offspring intestinal growth. In addition, we identified a novel miRNA-146a-5p-NEDD4L-β-catenin/Wnt signaling axis in regulating early intestinal development. This work provided a new perspective for studying the influence of maternal diet on offspring development. [ABSTRACT FROM AUTHOR]

Cardiac Vascular disease particularly myocardial infarction (MI) is a threat to health worldwide. microRNAs (miRNAs) have been shown to regulate myocardial fibrosis. Therefore, it is potential to investigate the mechanism of miRNA and fibrosis following myocardial infarction. Hypoxia human cardiac microvascular endothelial cells (HCMECs) were selected for the vitro experimental model. The miR-146a-5p expression was tested via RT-qPCR. The level of endothelial-to-mesenchymal transition (EndMT) and fibrosis markers were detected by Western blotting and immunofluorescence. Then, the inflammation, cell viability and apoptosis were investigated. The target was predicted by an online database and verified by a dual-luciferase activity assay. An MI mouse model was created to validate that miR-146a-5p regulates cardiac fibrosis in vivo. MI mouse was transfected with miR-146a-5p lentivirus. Subsequently, its effect on cardiac fibrosis of infarcted hearts was assessed by In situ hybridization (ISH), Immunohistochemistry (IHC), Triphenylterazolium chloride (TTC) staining and Masson staining. Herein, we confirmed that miR-146a-5p was down-regulated in hypoxia HCMECs. Overexpression of miR-146a-5p inhibited hypoxia-induced cardiac fibrosis following myocardial infarction by inhibiting EndMT in HCMECs. Thioredoxin-interacting protein (TXNIP) was a target that was negatively regulated by miR-146a-5p. Up-regulation of miR-146a-5p inhibited cardiac fibrosis via regulating EndMT by targeting TXNIP, and it also regulated EndMT to inhibit cardiac fibrosis in vivo. [ABSTRACT FROM AUTHOR]

Steroid hormones play a crucial role in the growth and maturation of poultry ovarian follicles, with progesterone secretion by granulosa cells (GC) being essential. According to our previous transcriptome analysis, it apparented that miR-146a-5p expressions were upregulated in the follicles undergoing atresia. In this study, we delved the depth to explore the underlying mechanisms by miR-146a-5p in the regulation of follicle functions in chicken. The study demonstrated that miR-146a-5p suppressed cell growth, lipids accumulation, and progesterone biosynthesis in chicken GC. Through targeting association validations, we identified delta 4-desaturase, sphingolipid 1 (DEGS1) as capable of interacting with miR-146a-5p. Co-transfection experiments further confirmed that DEGS1 reversed the impairment of GC functions by miR-146a-5p. Moreover, we discovered that miR-146a-5p suppressed AKT phosphorylation, while DEGS1 enhanced AKT phosphorylation. Phosphatidylinositol-3 kinase (PI3K) inhibitor (LY294002) studies showed that miR-146a-5p would inhibit AKT phosphorylation by governing the DEGS1/AKT pathway, which in turn regulates GC function. In summary, the findings revealed that miR-146a-5p suppressed cell growth, lipid deposition, and progesterone biosynthesis via the DEGS1 / AKT pathway. These results may further enrich our understandings of how non-coding RNA regulates productive performance in chickens. • miRNA regulates granulosa cells functions by targeting gene expressions in the DEGS1/AKT signaling pathway. • The DEGS1/AKT pathway is associated with lipid synthesis and progesterone production. • The inhibitory effect of miR-146a-5p on the DEGS1/AKT pathway was validated by pharmacological inhibitors. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is a highly metastatic subtype of breast cancer. Due to the absence of obvious therapeutic targets, microRNAs (miRNAs) provide possible hope to treat TNBC. Withaferin A (WA), a steroidal lactone, possesses potential anticancer activity with lesser side effects. The present study identifies hub genes (CDKN3, TRAF6, CCND1, JAK1, MET, AXIN2, JAG1, VEGFA, BRCA1, E2F3, WNT1, CDK6, KRAS, MYB, MYCN, TGFβR2, NOTCH1, SIRT1, MYCN, NOTCH2, WNT3A) from the list of predicted targets of the differentially expressed miRNAs (DEMs) in WA-treated MDAMB-231 cells using in silico protein–protein interaction network analysis. CCND1, CDK6, and TRAF6 hub genes were predicted as targets of miR-34a-5p and miR-146a-5p, respectively. The study found the lower expression of miR-34a-5p and miR-146a-5p in MDA-MB-231 cells, and further, it was observed that WA treatment effectively restored the lost expression of miR-34a-5p and miR-146a-5p in MDA-MB-231 cells. An anti-correlation expression pattern was found among the miR-34a-5p and miR-146a-5p and the respective target hub genes in WA-treated TNBC cells. In conclusion, WA might exert anti-cancer effect in TNBC cells by inducing miR-34a-5p and miR-146a-5p expressions and decreasing CCND1, CDK6, and TARF6 target hub genes in TNBC cells. [ABSTRACT FROM AUTHOR]

Background: Circular RNAs (circRNAs) have been reported to be involved in the initiation and development of cancers. The aim of this study was to determine the role of a circRNA, circ_0020123, in the development of non‐small cell lung cancer (NSCLC). Methods: The expression of circ_0020123, microRNA‐146a‐5p (miR‐146a‐5p), and eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) mRNA was detected by quantitative real‐time PCR (qPCR). Western blot was used to determine the protein levels of cyclin D1, Bax, MMP‐9, and EIF4G2. Cell proliferation was assessed by cell counting kit‐8 (CCK‐8) assay and colony formation assay. Flow cytometry assay was applied to determine cell cycle apoptosis. Cell migration and invasion were assessed using transwell assay. The potential relationship between miR‐146a‐5p and circ_0020123 or EIF4G2 was ascertained by dual‐luciferase reporter assay and RIP assay. The role of circ_0020123 in vivo was explored by xenograft assay. Results: Circ_0020123 was upregulated in NSCLC, and circ_0020123 knockdown repressed proliferation, migration, and invasion of NSCLC cells. Circ_0020123 targeted miR‐146a‐5p, and miR‐146a‐5p inhibitor reversed the effects of circ_0020123 knockdown on NSCLC cells. In addition, miR‐146a‐5p suppressed cell proliferation, migration, and invasion by targeting EIF4G2. Moreover, the antitumor role of circ_0020123 knockdown was verified in vivo. Conclusion: Knockdown of circ_0020123 inhibited NSCLC cell progression and tumor growth by targeting the miR‐146a‐5p/EIF4G2 axis. [ABSTRACT FROM AUTHOR]

Abstract Exosomes originating from human umbilical cord mesenchymal stem cells (hucMSC-exos) have become a novel strategy for treating various diseases owing to their ability to regulate intercellular signal communication. However, the potential of hucMSC-exos to improve placental injury in obstetric antiphospholipid syndrome and its underlying mechanism remain unclear. Our objective was to explore the potential application of hucMSC-exos in the treatment of obstetric antiphospholipid syndrome and elucidate its underlying mechanism. In our study, hucMSC-exos ameliorated the functional impairment of trophoblasts caused by antiphospholipid antibodies in vitro and attenuated placental dysfunction in mice with obstetric antiphospholipid syndrome by delivering miR-146a-5p. Exosomal miR-146a-5p suppressed the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibited the activation of NF-κB signaling, leading to the down-regulation of IL-1β and IL-18 to rescue inflammation and modulation of Cleaved-CASP3, BAX, and BCL2 to inhibit apoptosis in HTR8/SVneo cells and mice placenta. This study identified the potential molecular basis of how hucMSC-exos improved antiphospholipid antibody-induced placental injury and highlighted the functional importance of the miR-146a-5p/TRAF6 axis in the progression of obstetric antiphospholipid syndrome. More importantly, this study provided a fresh outlook on the promising use of hucMSC-exos as a novel and effective treatment approach in obstetric antiphospholipid syndrome. Graphical Abstract

Objective To explore the inhibition effect and mechanism of miR-146a-5p on proliferation and invasion of prostate cancer (PCa) cell line PC-3 by targeting SMAD4. Methods RT-qPCR was used to detect the expression of miR-146a-5p in PCa tissues and cell lines. The relevance of miR-146a-5p expression with Gleason score was also analyzed. MTT, BrdU experiment, cell colony formation experiment, scratch experiment, Transwell assay and nude mouse xenograft model experiment were conducted to detect the effect of miR-146a-5p on cell proliferation, tumorigenicity, migration and invasion. The expression of SMAD4 in PCa tissues was detected by RT-qPCR, and the targeting relationship of SMAD4 and miR-146a-5p was confirmed by double luciferase reporter gene assay and rescue experiment. Western blot was used to detect the expression of SMAD2/SMAD3 complex in nucleus affected by miR-146a-5p and SMAD4. Finally, double luciferase reporter gene assay and ChIP experiment were performed to examine the targeting regulation of TIM3 by miR-146a-5p/SMAD4/SMAD2/SMAD3 signaling axis. Results miR-146a-5p was low expressed in PCa tissues and cell lines; its expression was negatively correlated to Gleason score and had the lowest expression in PC-3 cells. miR-146a-5p inhibited the proliferation and invasion of PC-3 cells by targeting SMAD4. SMAD2/SMAD3/TIM3 axis seemed to be the downstream mechanism of miR-146a-5p/SMAD4 signaling pathway. Conclusions miR-146a-5p can inhibit the proliferation and invasion of PC-3 cells by targeting SMAD4, and the downstream mechanism might be related to the SMAD2/SMAD3/TIM3 signaling pathway.

Abstract Intermittent hypoxia is the best predictor of developing cognitive decline and Alzheimer’s disease progression in patients with obstructive sleep apnea. The nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome has been poorly studied as a regulator of neuroinflammation in cognitive impairment caused by intermittent hypoxia. As critical inflammatory cells, exosomes secreted by microglia have been found to affect the spread of pathologic proteins and neuropathology in neurodegenerative diseases. However, the effects of microglial exosomes on neuroinflammation and cognitive outcomes after intermittent hypoxia remain unclear. In this study, the role of miRNAs in microglial exosomes in improving cognitive deficits in mice exposed to intermittent hypoxia was investigated. We demonstrated that miR-146a-5p fluctuated over time in microglial exosomes of mice exposed to intermittent hypoxia for different periods of time, which could regulate neuronal NLRP3 inflammasome and neuroinflammation. In primary neurons, we found that miR-146a-5p regulated mitochondrial reactive oxygen species by targeting HIF1α, thus affecting the NLRP3 inflammasome and secretion of inflammatory factors. Similarly, further studies showed that inhibition of NLRP3 by administering overexpressed miR-146a-5p in microglial exosomes and MCC950 has improved neuroinflammation and cognitive dysfunction in mice after intermittent hypoxia. In conclusion, NLRP3 inflammasome may be a regulatory target for ameliorating cognitive impairment caused by intermittent hypoxia, and microglial exosomal miR-146a-5p may be a promising therapeutic strategy.

Introduction: The skin is the largest organ of the human body and fulfills protective, immune, and metabolic functions. Skin function and barrier integrity are actively regulated through circadian rhythm–associated genes and epigenetic mechanisms including DNA methylation/demethylation, histone acetylation/deacetylation, and microRNAs. MicroRNA-146a-5p (miR-146a) has been associated with immune activation and skin inflammation; however, the role of miR-146a in regulating skin aging is an open question. This study investigated the role of miR-146a in fibroblasts obtained from different donors in the context of aging, and a potential association of this miRNA with circadian rhythm.Methods: Normal human dermal fibroblasts (NHDFs) from 19y, 27y, 40y, and 62y old donors were used to analyze for miR-146a expression. Expression of miR-146a was downregulated with the hsa-mirVana miR-146a inhibitor, and upregulated with an extract from Adansonia digitata. Effects on markers of skin aging, including cell proliferation, production of Collagen-1 and inflammatory cytokines were assessed.Results: We show that the expression of miR-146a decreases with age in dermal fibroblasts and inhibition of miR-146a in 19y and 62y old NHDFs induced significant changes in essential clock genes indicating an association with circadian rhythm control. Furthermore, downregulation of miR-146a results in a reduction of cellular proliferation, Collagen-1 production, as well as an increase in DNA damage and pro-inflammatory markers. Activation of miR-146a with the Adansonia digitata extract reduced the deleterious effects seen during miR-146a inhibition and increased miR-146a transport through exosome transfer.Conclusion: miR-146a interacts with multiple biological pathways related to skin aging, including circadian rhythm machinery, cell-to-cell communication, cell damage repair, cell proliferation, and collagen production and represents a promising target to fight skin aging. Adansonia digitata extract can promote miR-146a expression and therefore support skin cells’ health.

Exosomes originating from human umbilical cord mesenchymal stem cells (hucMSC-exos) have become a novel strategy for treating various diseases owing to their ability to regulate intercellular signal communication. However, the potential of hucMSC-exos to improve placental injury in obstetric antiphospholipid syndrome and its underlying mechanism remain unclear. Our objective was to explore the potential application of hucMSC-exos in the treatment of obstetric antiphospholipid syndrome and elucidate its underlying mechanism. In our study, hucMSC-exos ameliorated the functional impairment of trophoblasts caused by antiphospholipid antibodies in vitro and attenuated placental dysfunction in mice with obstetric antiphospholipid syndrome by delivering miR-146a-5p. Exosomal miR-146a-5p suppressed the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibited the activation of NF-κB signaling, leading to the down-regulation of IL-1β and IL-18 to rescue inflammation and modulation of Cleaved-CASP3, BAX, and BCL2 to inhibit apoptosis in HTR8/SVneo cells and mice placenta. This study identified the potential molecular basis of how hucMSC-exos improved antiphospholipid antibody-induced placental injury and highlighted the functional importance of the miR-146a-5p/TRAF6 axis in the progression of obstetric antiphospholipid syndrome. More importantly, this study provided a fresh outlook on the promising use of hucMSC-exos as a novel and effective treatment approach in obstetric antiphospholipid syndrome. [ABSTRACT FROM AUTHOR]

Objective. This study aimed to explore the effects of bone marrow mesenchymal stem cell (BMSC)-derived exosomal miR-146a-5p on microglial polarization and the potential underlying mechanisms in oxygen-glucose deprivation (OGD)-exposed microglial cells. Methods. Exosomes were isolated from BMSCs, and their characteristics were examined. The effects of BMSC-derived exosomes on microglial polarization were investigated in OGD-exposed BV-2 cells. Differentially expressed miRNAs were identified and their biological function was explored using enrichment analyses. The regulatory role of miR-146a-5p in microglial polarization was studied via flow cytometry. Finally, the downstream target gene Traf6 was validated, and the role of the miR-146a-5p/Traf6 axis in modulating microglial polarization was investigated in OGD-exposed BV-2 cells. Results. BMSC-derived exosomes were successfully isolated and characterized. A total of 10 upregulated and 33 downregulated miRNAs were identified. Exosomal treatment resulted in significant changes in microglial polarization markers. miR-146a-5p was found to be significantly downregulated in OGD-exposed microglial cells treated with exosomes. Manipulation of miR-146a-5p expression modulated microglial polarization. Moreover, the miR-146a-5p/Traf6 axis regulated microglial polarization. Conclusion. Our findings demonstrate that BMSC-derived exosomal via miR-146a-5p modulates microglial polarization by targeting Traf6, providing a potential thermal target for the treatment of neurological diseases involving microglial activation. [ABSTRACT FROM AUTHOR]

Melanoma has the highest propensity of all cancers to metastasize to the brain with a large percentage of late‐stage patients developing metastases in the central nervous system (CNS). It is well known that metastasis establishment, cell survival, and progression are affected by tumour‐host cell interactions where changes in the host cellular compartments likely play an important role. In this context, miRNAs transferred by tumour derived extracellular vesicles (EVs) have previously been shown to create a favourable tumour microenvironment. Here, we show that miR‐146a‐5p is highly expressed in human melanoma brain metastasis (MBM) EVs, both in MBM cell lines as well as in biopsies, thereby modulating the brain metastatic niche. Mechanistically, miR‐146a‐5p was transferred to astrocytes via EV delivery and inhibited NUMB in the Notch signalling pathway. This resulted in activation of tumour‐promoting cytokines (IL‐6, IL‐8, MCP‐1 and CXCL1). Brain metastases were significantly reduced following miR‐146a‐5p knockdown. Corroborating these findings, miR‐146a‐5p inhibition led to a reduction of IL‐6, IL‐8, MCP‐1 and CXCL1 in astrocytes. Following molecular docking analysis, deserpidine was identified as a functional miR‐146a‐5p inhibitor, both in vitro and in vivo. Our results highlight the pro‐metastatic function of miR‐146a‐5p in EVs and identifies deserpidine for targeted adjuvant treatment. [ABSTRACT FROM AUTHOR]

Psoriasis is nowadays recognized as a multifactorial systemic disease with complex and not fully understood pathogenesis. In psoriatic patients, the increased cardiovascular disease (CVD) risk and frequent comorbidities like obesity are observed. The aim of this study was to investigate differences in miRNA (miR-22-3p, miR-133a-3p, miR-146a-5p, miR-369-3p, and Let-7b-5p) involved in CVD risk among psoriatic patients with overweight/obesity and with normal weight. The study comprised 28 male psoriatic patients and 16 male healthy controls. miRNA isolated from peripheral blood mononuclear cells was reverse-transcribed and RT-qPCR was performed. We have found decreased levels of miR-22, miR-133a, miR-146a, and miR-369 among the psoriatic patients. There was a statistically significant difference in miR-22 and miR-146a levels between psoriatic patients with overweight/obesity and with normal weight. There were positive correlations between miR-22 and miR-146a levels and psoriatic arthritis (PsA) in psoriatic patients with normal weight and between the miR-133a level and PsA in the overweight/obese patients. The decreased levels of selected miRNA are consistent with the levels observed in CVD indicating their impact on the CVD risk in psoriatic patients. miR-22 and miR-146 may be recognized as one of the contributing factors in the obesity-CVD-psoriasis network. [ABSTRACT FROM AUTHOR]

Abstract Background Sulfur mustard (SM) is a highly toxic chemical warfare agent that has caused numerous casualties during wars and conflicts in the past century. Specific antidotes or therapeutic strategies are rare due to the complicated mechanism of toxicity, which still awaits elucidation. Clinical data show that acute lung injury (ALI) is responsible for most mortality and morbidity after SM exposure. Extracellular vesicles are natural materials that participate in intercellular communication by delivering various substances and can be modified. In this study, we aim to show that extracellular vesicles derived from human umbilical cord mesenchymal stromal cells (hucMSC-EVs) could exert therapeutic effects on SM-induced ALI, and to explain the underlying mechanism of effects. Methods MiR-146a-5p contained in hucMSC-EVs may be involved in the process of hucMSC-EVs modulating the inflammatory response to SM-induced ALI. We utilized miR-146a-5p delivered by extracellular vesicles and further modified hucMSCs with a miR-146a-5p mimic or inhibitor to collect miR-146a-5p-overexpressing extracellular vesicles (miR-146a-5p+-EVs) or miR-146a-5p-underexpressing extracellular vesicles (miR-146a-5p−-EVs), respectively. Through in vivo and in vitro experiments, we investigated the mechanism. Results The effect of miR-146a-5p+-EVs on improving the inflammatory reaction tied to SM injury was better than that of hucMSC-EVs. We demonstrated that miR-146a-5p delivered by hucMSC-EVs targeted TRAF6 to negatively regulate inflammation in SM-induced ALI models in vitro and in vivo. Conclusion In summary, miR-146a-5p delivered by hucMSC-EVs targeted TRAF6, causing hucMSC-EVs to exert anti-inflammatory effects in SM-induced ALI; thus, hucMSC-EVs treatment may be a promising clinical therapeutic after SM exposure. Graphical Abstract

Abstract Resveratrol (RSV) has been shown to have a neuroprotective effect in various central nervous system disorders, although the role of RSV in diabetes‐induced cognitive dysfunction is still not fully elucidated. Here, we investigated whether RSV improved diabetes‐related cognitive dysfunction in vivo and in vitro. We induced a rat diabetic model with a high‐fat and high‐sucrose diet followed by intraperitoneal injection of streptozotocin and a diabetic neuron cell model by stimulation with high levels of glucose. We observed that RSV improved impairment in spatial learning and memory in the Morris water maze test (MWM) and novel object recognition test (ORT) in diabetic rats. RSV reversed the reduced miR‐146a‐5p and upregulated thioredoxin‐interacting protein (TXNIP) and inhibited the diabetes‐induced increase in interleukin (IL)‐1β and tumor necrosis factor (TNF)‐α levels in vivo and in vitro. RSV also inhibited diabetes‐induced endoplasmic reticulum stress (ESR) by reducing ESR‐related protein expression in vivo and in vitro. Moreover, inhibition of miR‐146a‐5p partially abolished the protective effects of RSV in HG‐treated primary neurons. Additionally, we used starBase to predict that miR‐146a‐5p interacts with TXNIP, which we then verified using a luciferase reporter gene assay. We further observed that miR‐146a‐5p regulates the mRNA and protein expression of TXNIP in vitro, indicating that the miR‐146a‐5p/TXNIP axis is involved in the regulation of cognitive dysfunction in a rat diabetic model. Collectively, these results demonstrate that RSV plays a neuroprotective role in diabetes‐associated cognitive dysfunction at least in part through regulation of the miR‐146a‐5p/TXNIP axis.

Abstract Melanoma has the highest propensity of all cancers to metastasize to the brain with a large percentage of late‐stage patients developing metastases in the central nervous system (CNS). It is well known that metastasis establishment, cell survival, and progression are affected by tumour‐host cell interactions where changes in the host cellular compartments likely play an important role. In this context, miRNAs transferred by tumour derived extracellular vesicles (EVs) have previously been shown to create a favourable tumour microenvironment. Here, we show that miR‐146a‐5p is highly expressed in human melanoma brain metastasis (MBM) EVs, both in MBM cell lines as well as in biopsies, thereby modulating the brain metastatic niche. Mechanistically, miR‐146a‐5p was transferred to astrocytes via EV delivery and inhibited NUMB in the Notch signalling pathway. This resulted in activation of tumour‐promoting cytokines (IL‐6, IL‐8, MCP‐1 and CXCL1). Brain metastases were significantly reduced following miR‐146a‐5p knockdown. Corroborating these findings, miR‐146a‐5p inhibition led to a reduction of IL‐6, IL‐8, MCP‐1 and CXCL1 in astrocytes. Following molecular docking analysis, deserpidine was identified as a functional miR‐146a‐5p inhibitor, both in vitro and in vivo. Our results highlight the pro‐metastatic function of miR‐146a‐5p in EVs and identifies deserpidine for targeted adjuvant treatment.

• AHN and pattern separation are impaired in APP/PS1 mice at early stage. • miR-146a/Klf4/p-Stat3 signaling is abnormal in hippocampus of APP/PS1 mice. • Correcting miR-146a/Klf4/p-Stat3 pathway rescued AHN and pattern separation. Alzheimer's disease (AD) is the most common neurodegenerative disease, and currently, no effective treatment strategies exist for this condition. MicroRNAs (miRNAs) have emerged as promising therapeutic targets of AD. Previous studies have highlighted the significant role of miR-146a-5p in regulating adult hippocampal neurogenesis (AHN). Here, we aimed to investigate whether miR-146a-5p plays a role in the mechanisms of AD. We employed quantitative real-time PCR (qRT-PCR) to assess the expression of miR-146a-5p. Additionally, we examined the expression of Krüppel-like factor 4 (Klf4), Signal transducer and activator of transcription 3 (Stat3), and phosphorylated Stat3 (p-Stat3) using western blot analysis. Furthermore, we validated the interaction between miR-146a-5p and Klf4 using a dual-luciferase reporter assay. Immunofluorescence staining was employed to evaluate AHN. And Contextual fear conditioning discrimination learning (CFC-DL) experiment was used to detect pattern separation. Our findings in the hippocampus of APP/PS1 mice revealed upregulated levels of miR-146a-5p and p-Stat3, while Klf4 levels were downregulated. Interestingly, both miR-146a-5p antagomir and p-Stat3 inhibitor obviously rescued neurogenesis and pattern separation in APP/PS1 mice. Moreover, application of miR-146a-5p agomir reversed the protective effects of Klf4 upregulation. These findings open new avenues for protection against AD through the modulation of neurogenesis and cognitive decline via the miR-146a-5p/Klf4/p-Stat3 pathway. [ABSTRACT FROM AUTHOR]

Objective To explore the inhibition effect and mechanism of miR-146a-5p on proliferation and invasion of prostate cancer（PCa） cell line PC-3 by targeting SMAD4. Methods RT-qPCR was used to detect the expression of miR-146a-5p in PCa tissues and cell lines. The relevance of miR-146a-5p expression with Gleason score was also analyzed. MTT, BrdU experiment, cell colony formation experiment, scratch experiment, Transwell assay and nude mouse xenograft model experiment were conducted to detect the effect of miR-146a-5p on cell proliferation, tumorigenicity, migration and invasion. The expression of SMAD4 in PCa tissues was detected by RT-qPCR, and the targeting relationship of SMAD4 and miR-146a-5p was confirmed by double luciferase reporter gene assay and rescue experiment. Western blot was used to detect the expression of SMAD2/SMAD3 complex in nucleus affected by miR-146a-5p and SMAD4. Finally, double luciferase reporter gene assay and ChIP experiment were performed to examine the targeting regulation of TIM3 by miR-146a-5p/SMAD4/SMAD2/SMAD3 signaling axis. Results miR-146a-5p was low expressed in PCa tissues and cell lines; its expression was negatively correlated to Gleason score and had the lowest expression in PC-3 cells. miR-146a-5p inhibited the proliferation and invasion of PC-3 cells by targeting SMAD4. SMAD2/SMAD3/TIM3 axis seemed to be the downstream mechanism of miR-146a-5p/SMAD4 signaling pathway. Conclusions miR-146a-5p can inhibit the proliferation and invasion of PC-3 cells by targeting SMAD4, and the downstream mechanism might be related to the SMAD2/SMAD3/TIM3 signaling pathway. [ABSTRACT FROM AUTHOR]

Intermittent hypoxia is the best predictor of developing cognitive decline and Alzheimer's disease progression in patients with obstructive sleep apnea. The nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome has been poorly studied as a regulator of neuroinflammation in cognitive impairment caused by intermittent hypoxia. As critical inflammatory cells, exosomes secreted by microglia have been found to affect the spread of pathologic proteins and neuropathology in neurodegenerative diseases. However, the effects of microglial exosomes on neuroinflammation and cognitive outcomes after intermittent hypoxia remain unclear. In this study, the role of miRNAs in microglial exosomes in improving cognitive deficits in mice exposed to intermittent hypoxia was investigated. We demonstrated that miR-146a-5p fluctuated over time in microglial exosomes of mice exposed to intermittent hypoxia for different periods of time, which could regulate neuronal NLRP3 inflammasome and neuroinflammation. In primary neurons, we found that miR-146a-5p regulated mitochondrial reactive oxygen species by targeting HIF1α, thus affecting the NLRP3 inflammasome and secretion of inflammatory factors. Similarly, further studies showed that inhibition of NLRP3 by administering overexpressed miR-146a-5p in microglial exosomes and MCC950 has improved neuroinflammation and cognitive dysfunction in mice after intermittent hypoxia. In conclusion, NLRP3 inflammasome may be a regulatory target for ameliorating cognitive impairment caused by intermittent hypoxia, and microglial exosomal miR-146a-5p may be a promising therapeutic strategy. [ABSTRACT FROM AUTHOR]

M1/M2 polarization transitions of microglial phenotypes determine the states of neuroinflammation, which is critical in the pathophysiology of diabetic encephalopathy. This study aims to investigate the effects of advanced glycation end products (AGEs) on the microglial polarization state, the role of miR-146a-5p in the regulation of microglial polarization, and the underlying signaling pathways. BV-2 cells were incubated with N-ε-carboxymethyl lysine (CML), one kind of Advanced Glycation End Products (AGEs), to induce polarization. CD11b and iNOS and CD206 and Arg-1 were used to evaluate M1 and M2 microglia, respectively. The mRNA and protein expression levels of miR-146a-5p, transcription factor NF-κB, and inflammasome NLRP3 were measured. High and low expression of miR-146a-5p in the BV-2 cell line was generated by lentivirus transfection technology. RAGE, TLR-4, and NF-κB antagonists were applied to evaluate the underlying signaling pathways. Compared with the control group, CML upregulated the M1 phenotype and downregulated the M2 phenotype. These effects were reversed by overexpression of miR-146a. Furthermore, the expression of inflammasome NLRP3 and NF-κB was upregulated in the CML group and was reduced after miR-146a overexpression. And then overexpression of miR-146a effects was reversed by inhibition miR-146a expression. An NF-κB antagonist (PDTC), a RAGE antagonist (FPS-ZMI), and a TLR-4 antagonist (TLI-095) all reversed the polarization state induced by CML. In summary, CML induced polarization transitions to M1 phenotype and promoted inflammasome NLRP3 expression in BV-2 cells. The RAGE or TLR-4/miR-146a/NLRP3/NF-кB pathway might participate in the regulation of CML-induced BV-2 polarization. [ABSTRACT FROM AUTHOR]

Abstract Panax notoginseng saponins (PNSs) have been found as the major active ingredient of Panax notoginseng (Burkill) F.H.Chen (PN) leaves, which has the effect of reducing inflammatory response, facilitating fibroblast proliferation, as well as promoting angiogenesis. This study aimed to investigate the molecular basis of PNS combined with bone mesenchymal stem cells (BMSCs) for treating diabetic cutaneous ulcers (DCU) and its mechanism of action. Methods. A total of 75 SD rats were selected to make diabetic cutaneous ulcers model. According random number table method, the rats were randomly divided into a control group, a DCU group, a BMSCs group, a PNS group and BMSCs + PNS group. Five groups of rats were given without treatment. After being treated for 7 days, the rats were anesthetized with pentobarbital, and granulation tissue was collected from the central point of the wound. They were used for pathological analysis, Western blot (WB) and polymerase chain reaction (PCR) assays. Results. The wound healing area was the largest in the BMSCs + PNS group. HE staining results showed that the PNS + BMSCs group could promote the formation of new epidermis and reduce the infiltration of inflammatory cells. Immunohistochemistry (IHC) results showed that the PNS + BMSCs group could up-regulate the expression of Ki67 protein and cell proliferation. In addition, PNS combined with BMSCs up-regulated the expression of miR-146-5p and down-regulated the expression of IL-1β, IL-6 and TNF-α, IRAK1, TRAF6 and p65 in the NF-κB signaling pathway (p

Mona A Eldosoky,1 Reham Hammad,1 Areej Rushdi,2 Hanan F Ibrahim,2 Amany M Tawfeik,3 Ahmed Mora,4 Sarah F Fahmy,5 Hossam El-Ashmawy,6 Elham Ali,7 Dina H Hamed,8 Amena Rezk Mohammed,9 Alya Mashaal,10 Hanan Mohsen8 1Clinical Pathology Department, Faculty of Medicine (for Girls), Al-Azhar University, Cairo, Egypt; 2Microbiology and Immunology Department, Faculty of Medicine (for Girls), Al-Azhar University, Cairo, Egypt; 3Microbiology and Immunology Department, Faculty of Medicine, Badr University in Cairo (BUC), Cairo, Egypt; 4Chemistry Department, Faculty of Science (for Boys), Al-Azhar University, Cairo, Egypt; 5Clinical Pharmacy Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt; 6Clinical Pathology Department, Faculty of Medicine (for Boys), Al-Azhar University, Assuit, Egypt; 7Molecular Biology, Zoology and Entomology Department, Faculty of Science (For Girls), Al-Azhar University, Cairo, Egypt; 8Pediatric Department, Pediatric Allergy and Pulmonology Unit, Children’s Hospital, Cairo University, Cairo, Egypt; 9Biochemistry Department, Faculty of Medicine (for Girls), Al-Azhar University, Cairo, Egypt; 10Immunology, Zoology and Entomology Department, Faculty of Science (For Girls), Al-Azhar University, Cairo, EgyptCorrespondence: Dina H Hamed, Email dina.alkhtaib@cu.edu.egBackground: Severe bronchial asthma (BA) affects 5– 10% of children, which imposes socioeconomic burden. Therefore, it is crucial to identify biomarkers for risk stratification in children with BA. T regulatory cells (Tregs) play a balancing role in allergic response regulation. We aimed to investigate the relationship between Treg, miR-210-3p, and miR-146a-5p in relation to asthma phenotypes in search of novel biomarkers of disease severity.Methods: This study included 50 children with BA classified into Group 1 (n = 25) children with mild to moderate asthma and Group 2 (n = 25) children with severe asthma. In addition to 26 control subjects. Flow cytometry was used to detect Tregs. Plasma miR-210-3p and miR-146a levels were determined using quantitative real-time PCR. Patients’ FEV1 (Forced Expiratory Volume in the first second) was measured.Results: miR-210-3p level correlated negatively with Treg frequency (r = − 0.828, P < 0.001) and FEV1 (r = − 0.621, P < 0.001). The level of miR-146a-5p positively correlated positively with Treg% (r = 0.303, P = 0.032). ROC curve analysis revealed that miR-210-3p was the most sensitive biomarker of severity, with the area under curve (AUC) = 0.923, 96% sensitivity, and 60% specificity. According to multivariate analysis, miR-210-3p is an independent risk factor for BA severity [OR =3.119, P = 0.030], while miR-146a-5p is a protective factor [OR =0.811, P = 0.049].Conclusion: Treg frequency is linked to FEV1, miR-146a-5p and miR-210-3p in childhood BA. Upregulation of miR-210-3p is a sensitive biomarker and an independent risk factor for BA severity in Egyptian children.Keywords: T reg, miR-210-3p, miR-146a-5p, childhood asthma, asthma classifications

Ligamentum flavum (LF) hypertrophy (LFH) has been recognised as one of the key contributors to lumbar spinal stenosis. Currently, no effective methods are available to ameliorate this hypertrophy. In this study, human umbilical cord mesenchymal stromal cell-derived extracellular vesicles (hUCMSC-EVs) were introduced for the first time as promising vehicles for drug delivery to treat LFH. The downregulation of miR-146a-5p and miR-221-3p expressions in human LF tissues negatively correlated with increased LF thickness. The hUCMSC-EVs enriched with these two miRNAs significantly suppressed LFH in vivo and notably ameliorated the progression of transforming growth factor β1(TGF-β1)-induced fibrosis in vitro after delivering these two miRNAs to mouse LF cells. The results further demonstrated that miR-146a-5p and miR-221-3p directly bonded to the 3′-UTR regions of SMAD4 mRNA, thereby inhibiting the TGF-β/SMAD4 signalling pathway. Therefore, this translational study determined the effectiveness of a hUCMSC-EVs-based approach for the treatment of LFH and revealed the critical target of miR-146a-5p and miR-221-3p. Our findings provide new insights into promising therapeutics using a hUCMSC-EVs-based delivery system for patients with lumbar spinal stenosis.

Abstract Background MicroRNAs (miRNAs) play an important role in gene regulation that controls stem cells differentiation. Periodontal ligament stem cells (PDLSCs) could differentiate into osteo-/cementoblast-like cells that secretes cementum-like matrix both in vitro and in vivo. Whether miRNAs play key roles in osteoblastic differentiation of PDLSCs triggered by a special microenviroment remains elusive. In this study, we aimed to investigate potential miRNA expression changes in osteoblastic differentiation of PDLSCs by the induction of apical tooth germ cell-conditioned medium (APTG-CM). Methods and results First, we analyzed the ability of APTG-CM to osteogenically differentiate PDLSCs. The results exhibited an enhanced mineralization ability, higher ALP activity and increased expression of osteogenic genes in APTG-CM-induced PDLSCs. Second, we used miRNA sequencing to analyze the miRNA expression profile of PDLSCs derived from three donors under 21-day induction or non-induction of APTG-CM. MiR-146a-5p was found to be up-regulated miRNA in induced PDLSCs and validated by RT-qPCR. Third, we used lentivirus-up/down system to verify the role of miR-146a-5p in the regulation of osteoblastic differentiation of PDLSCs. Conclusions In conclusion, our results demonstrated that miR-146a-5p was involved in the promotion effect of APTG-CM on osteoblastic differentiation of PDLSCs, and suggested that miR-146a-5p might be a novel way in deciding the direction of PDLSCs differentiation.

Background: Sulfur mustard (SM) is a highly toxic chemical warfare agent that has caused numerous casualties during wars and conflicts in the past century. Specific antidotes or therapeutic strategies are rare due to the complicated mechanism of toxicity, which still awaits elucidation. Clinical data show that acute lung injury (ALI) is responsible for most mortality and morbidity after SM exposure. Extracellular vesicles are natural materials that participate in intercellular communication by delivering various substances and can be modified. In this study, we aim to show that extracellular vesicles derived from human umbilical cord mesenchymal stromal cells (hucMSC-EVs) could exert therapeutic effects on SM-induced ALI, and to explain the underlying mechanism of effects. Methods: MiR-146a-5p contained in hucMSC-EVs may be involved in the process of hucMSC-EVs modulating the inflammatory response to SM-induced ALI. We utilized miR-146a-5p delivered by extracellular vesicles and further modified hucMSCs with a miR-146a-5p mimic or inhibitor to collect miR-146a-5p-overexpressing extracellular vesicles (miR-146a-5p+-EVs) or miR-146a-5p-underexpressing extracellular vesicles (miR-146a-5p−-EVs), respectively. Through in vivo and in vitro experiments, we investigated the mechanism. Results: The effect of miR-146a-5p+-EVs on improving the inflammatory reaction tied to SM injury was better than that of hucMSC-EVs. We demonstrated that miR-146a-5p delivered by hucMSC-EVs targeted TRAF6 to negatively regulate inflammation in SM-induced ALI models in vitro and in vivo. Conclusion: In summary, miR-146a-5p delivered by hucMSC-EVs targeted TRAF6, causing hucMSC-EVs to exert anti-inflammatory effects in SM-induced ALI; thus, hucMSC-EVs treatment may be a promising clinical therapeutic after SM exposure. [ABSTRACT FROM AUTHOR]

Background. A frequent complication of excimer laser correction (ELC) for myopia is dry eye syndrome (DES), which occurs in 60 % of patients in the first month after surgery, with a gradual regression of symptoms to 20 % over the next 6 months. Objective: to investigate the prognostic role of miRNA and transforming growth factor β2 (TGF-β2) in dry eye syndrome after excimer laser myopia correction. Materials and methods. The study included data from 68 patients (136 eyes) diagnosed with myopia, aged 20 to 44 years, who were divided according to the presence of DES 6 months after ELC using LASIK or Femto-LASIK. As a control group, 15 people (30 eyes) without visual problems were enrolled. The relative expression of miRNA-146a-5p and miRNA450b-5p to U6 in tears was determined by the polymerase chain reaction (Thermo Fisher Scientific, USA), the content of TGF-β2 in tears was measured by the enzyme immunoassay (eBioscience, Austria). SPSS 61 11.0, MedStat and MedCalc v.15.1 software packages (MedCalc Software bvba) were used for statistical analysis. Results. The expression of miRNA-146a-5p in tears before ELC of myopia in patients with DES after 6 months of treatment was significantly reduced (3.6fold; p < 0.001) compared to controls and patients without DES. The expression of miRNA-450b-5p was increased in the presence of DES by 2.5 times compared to controls and by 2.0 times compared to patients without DES (p < 0.001). The TGF-β2 content was significantly lower in DES (1.6-fold; p < 0.001) compared to patients without DES. The determining value of miRNA expression for the occurrence of DES was proven in the receiver operating characteristic analysis, the critical threshold for the occurrence of DES according to the relative expression of miRNA-146a-5p was ≤ 0.85, for miRNA-450b-5, it was > 1.95 (p < 0.001). Conclusions. Divergent changes in the expression of miRNA-146a-5p (suppression) and miRNA-450b-5p (increase) when TGF-β2 content is reduced in the tears of patients with DES after ELC of myopia substantiated the possibility of using these indicators to predict the DES occurrence. [ABSTRACT FROM AUTHOR]

As evidenced by ongoing clinical trials and increased activity in the commercial sector, extracellular vesicle (EV)-based therapies have begun the transition from bench to bedside. As this progression continues, one critical aspect of EV clinical translation is understanding the effects of storage and transport conditions. Several studies have assessed the impact of storage on EV characteristics such as morphology, uptake and component content, but effects of storage duration and temperature on EV functional bioactivity and, especially, loaded cargo are rarely reported. The authors assessed EV outcomes following storage at different temperatures (room temperature, 4°C, –20°C, –80°C) for various durations as well as after lyophilization. Mesenchymal stromal cell (MSC) EVs were observed to retain key aspects of their bioactivity (pro-vascularization, anti-inflammation) for up to 4–6 weeks at –20°C and –80°C and after lyophilization. Furthermore, via in vitro assays and an in vivo wound healing model, these same storage conditions were also demonstrated to enable preservation of the functionality of loaded microRNA and long non-coding RNA cargo in MSC EVs. These findings extend the current understanding of how EV therapeutic potential is impacted by storage conditions and may inform best practices for handling and storing MSC EVs for both basic research and translational purposes. [ABSTRACT FROM AUTHOR]

Simple Summary: Resistance to anti-HER2+ therapy remains the main clinical challenge in the management of the HER2+ breast cancer subtype. The objective of our study was to analyze the involvement of microRNAs in the resistance to trastuzumab. We identified miR-146a-5p as the major dysregulated microRNA among parental and trastuzumab-resistant HER2+ breast cancer cells. The gain- and loss-of-function of this miRNA modulates resistance to trastuzumab in vitro, and elevated levels of miR-146a-5p in the primary tumor have been associated with a poor prognosis. In addition, exosomes from trastuzumab-resistant cells contain high levels of miR-146a-5p, and may reduce the effect of trastuzumab on sensitive cancer cells, increasing the expression of epithelial-to-mesenchymal transition markers and the capacities for migration and angiogenesis. The results of this study demonstrate for the first time the involvement of miR-146a-5p in resistance to trastuzumab, and suggest that exosomes play an important role in this process. Trastuzumab treatment has significantly improved the prognosis of HER2-positive breast cancer patients. Despite this, resistance to therapy still remains the main clinical challenge. In order to evaluate the implication of microRNAs in the trastuzumab response, we performed a microRNA array in parental and acquired trastuzumab-resistant HER2-positive breast cancer cell lines. Our results identified miR-146a-5p as the main dysregulated microRNA. Interestingly, high miR-146a-5p expression in primary tumor tissue significantly correlated with shorter disease-free survival in HER2-positive breast cancer patients. The gain- and loss-of-function of miR-146a-5p modulated the response to trastuzumab. Furthermore, the overexpression of miR-146a-5p increased migration and angiogenesis, and promoted cell cycle progression by reducing CDKN1A expression. Exosomes from trastuzumab-resistant cells showed a high level of miR-146a-5p expression compared with the parental cells. In addition, the co-culture with resistant cells' exosomes was able to decrease in sensitivity and increase the migration capacities in trastuzumab-sensitive cells, as well as angiogenesis in HUVEC-2 cells. Collectively, these data support the role of miR-146a-5p in resistance to trastuzumab, and demonstrate that it can be transferred by exosomes conferring resistance properties to other cells. [ABSTRACT FROM AUTHOR]

Resveratrol (RSV) has been shown to have a neuroprotective effect in various central nervous system disorders, although the role of RSV in diabetes‐induced cognitive dysfunction is still not fully elucidated. Here, we investigated whether RSV improved diabetes‐related cognitive dysfunction in vivo and in vitro. We induced a rat diabetic model with a high‐fat and high‐sucrose diet followed by intraperitoneal injection of streptozotocin and a diabetic neuron cell model by stimulation with high levels of glucose. We observed that RSV improved impairment in spatial learning and memory in the Morris water maze test (MWM) and novel object recognition test (ORT) in diabetic rats. RSV reversed the reduced miR‐146a‐5p and upregulated thioredoxin‐interacting protein (TXNIP) and inhibited the diabetes‐induced increase in interleukin (IL)‐1β and tumor necrosis factor (TNF)‐α levels in vivo and in vitro. RSV also inhibited diabetes‐induced endoplasmic reticulum stress (ESR) by reducing ESR‐related protein expression in vivo and in vitro. Moreover, inhibition of miR‐146a‐5p partially abolished the protective effects of RSV in HG‐treated primary neurons. Additionally, we used starBase to predict that miR‐146a‐5p interacts with TXNIP, which we then verified using a luciferase reporter gene assay. We further observed that miR‐146a‐5p regulates the mRNA and protein expression of TXNIP in vitro, indicating that the miR‐146a‐5p/TXNIP axis is involved in the regulation of cognitive dysfunction in a rat diabetic model. Collectively, these results demonstrate that RSV plays a neuroprotective role in diabetes‐associated cognitive dysfunction at least in part through regulation of the miR‐146a‐5p/TXNIP axis. [ABSTRACT FROM AUTHOR]