Rynchopeterine inhibits the formation of hypertrophic scars by regulating the miR-21/HIF1AN axis.

Hypertrophic scar (HS) is a fibroproliferative skin disease characterized by abnormal wound healing and pathological excessive fibrosis of the skin. Currently, the molecular mechanism of the disease is still largely unknown, and there is no effective drug treatment. In this study, we explored the effect of Rynchopeterine on the formation of HS. HS fibroblasts (HSFs) were isolated from the HS tissues of patients recovering from severe burns. After treating HSFs with different concentrations of Rynchopeterine, CCK-8, EdU, and Annexin V-FITC/PI assays were used to detect the proliferation, apoptosis, and contractile ability of HSFs. RT-qPCR and Western blotting were performed to evaluate the effect of Rynchopeterine on the expression of miR-21 and hypoxia-inducible factor 1-alpha subunit suppressor (HIF1AN). The dual-luciferase reporter gene was used to verify the targeting relationship between miR-21 and HIF1AN. Rynchopeterine reduced the expression of Col1a2, Col3a1, and α-SMA, inhibited proliferation and contraction of HSFs, and increased apoptosis in a dose-dependent manner. miR-21 was highly expressed in HS tissues and HSFs, and Rynchopeterine could inhibit miR-21 expression. Overexpression of miR-21 and knockdown of HIF1AN increased proliferation, activation, contraction, and collagen synthesis of HSFs, and inhibited their apoptosis. In vivo, Rynchopeterine could reduce the collagen content of the dermis and the positive ratio of PCNA and α-SMA. Rynchopeterine is a good therapeutic agent for HS, which up-regulates the expression of HIF1AN by inhibiting miR-21, thereby inhibiting the formation of HS.

Tumor-associated macrophage-derived exosomal miR21-5p promotes tumor angiogenesis by regulating YAP1/HIF-1α axis in head and neck squamous cell carcinoma.

Extracellular vesicles (EVs) have recently received increasing attention as essential mediators of communication between tumor cells and their microenvironments. Tumor-associated macrophages (TAMs) play a proangiogenic role in various tumors, especially head and neck squamous cell carcinoma (HNSCC), and angiogenesis is closely related to tumor growth and metastasis. This research focused on exploring the mechanisms by which EVs derived from TAMs modulate tumor angiogenesis in HNSCC. Our results indicated that TAMs infiltration correlated positively with microvascular density in HNSCC. Then we collected and identified EVs from TAMs. In the microfluidic chip, TAMs derived EVs significantly enhanced the angiogenic potential of pHUVECs and successfully induced the formation of perfusable blood vessels. qPCR and immunofluorescence analyses revealed that EVs from TAMs transferred miR-21-5p to endothelial cells (ECs). And targeting miR-21-5p of TAMs could effectively inhibit TAM-EVs induced angiogenesis. Western blot and tube formation assays showed that miR-21-5p from TAM-EVs downregulated LATS1 and VHL levels but upregulated YAP1 and HIF-1α levels, and the inhibitors of YAP1 and HIF-1α could both reduce the miR-21-5p enhanced angiogenesis in HUVECs. The in vivo experiments further proved that miR-21-5p carried by TAM-EVs promoted the process of tumor angiogenesis via YAP1/HIF-1α axis in HNSCC. Conclusively, TAM-derived EVs transferred miR-21-5p to ECs to target the mRNA of LATS1 and VHL, which inhibited YAP1 phosphorylation and subsequently enhanced YAP1-mediated HIF-1α transcription and reduced VHL-mediated HIF-1α ubiquitination, contributing to angiogenesis in HNSCC. These findings present a novel regulatory mechanism of tumor angiogenesis, and miR-21-5p/YAP1/HIF-1α might be a potential therapeutic target for HNSCC.

The reduction of the m6A methyltransferase METTL3 in granulosa cells is related to the follicular cysts in pigs.

Follicular cysts are a common reproductive disorder in domestic animals that cause considerable economic losses to the farming industry. Effective prevention and treatment methods are lacking because neither the pathogenesis nor formation mechanisms of follicular cysts are well-understood. In this study, we first investigated the granulosa cells (GCs) of cystic follicles isolated from pigs. We observed a significant reduction in the expression of methyltransferase-like 3 (METTL3). Subsequent experiments revealed that METTL3 downregulation in GCs caused a decrease in m6A modification of pri-miR-21. This reduction further inhibited DGCR8 recognition and binding to pri-miR-21, dampening the synthesis of mature miR-21-5p. Additionally, the decrease in miR-21-5p promotes IL-1ß expression in GCs. Elevated IL-1ß activates the NFκB pathway, in turn upregulating apoptotic genes TNFa and BAX/BCL2. The subsequent apoptosis of GCs and inhibition of autophagy causes downregulation of CYP19A1 expression. These processes lower oestrogen secretion and contribute to follicular cyst formation. In conclusion, our findings provide a foundation for understanding and further exploring the mechanisms of follicular-cyst development in farm animals. This work has important implications for treating ovarian disorders in livestock and could potentially be extended to humans.

Glucocorticoid modulates oxidative and thermogenic function of rat brown adipose tissue and human brown adipocytes.

Chronic and excessive glucocorticoid (GC) exposure can cause Cushing's syndrome, resulting in fat accumulation in selected body areas. Particularly in the brown adipose tissue (BAT), GC acts negatively, resulting in whitening of the tissue. We hypothesized that dysregulation of microRNAs by GC could be an additional mechanism to explain its negative actions in BAT. Male Wistar rats were divided into two groups: (1) Control sham and (2) GC group that was administered dexamethasone 6.25 mg/200 µL via osmotic pump implantation over 28 days. After this period, the animals were euthanized and BAT tissue was properly stored. Human fat cells treated with dexamethasone were used to translate the experimental results found in animals to human biology. GC-treated rat BAT presented with large lipid droplets, severely impaired thermogenic activation, and reduced glucose uptake measured by 18F-FDG PET/CT. GC exposure induced a reduction in the mitochondrial OXPHOS system and oxygen consumption. MicroRNA profiling of BAT revealed five top-regulated microRNAs and among them miR-21-5p was the most significantly upregulated in GC-treated rats compared to the control group. Although upregulation of miR-21-5p in the tissue, differentiated primary brown adipocytes from GC-treated rats had decreased miR-21-5p levels compared to the control group. To translate these results to the clinic, human brown adipocytes were treated with dexamethasone and miR-21-5p inhibitor. In human brown cells, inhibition of miR-21-5p increased brown adipocyte differentiation and prevented GC-induced glucose uptake, resulting in a lower glycolysis rate. In conclusion, high-dose GC therapy significantly impacts brown adipose tissue function, with a notable association between glucose uptake and miR-21-5p.

LncRNA AFAP1-AS1 mediates breast cancer cell proliferation and migration through the miR-21/PTEN axis.

LncRNAs play various effects, mostly by sponging with miRNAs. Based on public databases integrating bioinformatics analyses and further validation in breast cancer (BC) tissue and cell lines, the effect of lncRNA AFAP1-AS1 on breast cancer cell proliferation and migration was verified. It might work via the miR-21/PTEN axis. The expression of AFAP1-AS1, which was significantly upregulated in BC tissues and cell lines, was correlated with old age and lymph node metastasis of patients with BC. Knockdown of AFAP1-AS1 inhibited the proliferation and migration of BC cells in vitro and in vivo. And downregulated miR-21 expression and upregulated PTEN expression additionally. Mechanistically, the knockdown of lncRNA AFAP1-AS1 upregulated PTEN expression and consequently attenuated miR-21-mediated enhanced BC cell proliferation and migration. LncRNA AFAP1-AS1 is a potential prognostic biomarker for BC patients.

Nur77 Mediates Anaphylaxis by Regulating miR-21a.

Nur77 belongs to the NR4A subfamily of orphan nuclear hormone receptors. It has been shown to play important roles in metabolism, cancer progression, cellular differentiation, and the regulation of immune process. However, there has yet to be research reporting on the role of Nur77 in allergic inflammations such as anaphylaxis. This study aimed to identify molecules that could mediate allergic inflammations. To this end, we performed RNA sequencing analysis employing bone marrow-derived mast cells (BMMCs). Antigen (DNP-HSA) stimulation increased the expression levels of transcription factors such as Nr4a3 (NOR1), Nr4a1 (Nur77), and Nr4a2 (Nurr1). We focused our study on Nur77. Antigen stimulation increased the expression of Nur77 in a time- and dose-dependent manner in rat basophilic leukemia cells (RBL2H3). The downregulation of Nur77 prevented both antigen-induced increase in ß-hexosaminidase activity as well as hallmarks of allergic reactions such as HDAC3, COX2, and MCP1 in RBL2H3 cells. Nur77 was necessary for both passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA). TargetScan analysis predicted that miR-21a would be a negative regulator of Nur77. miR-21a mimic negatively regulated PCA and PSA by inhibiting the hallmarks of allergic reactions. ChIP assays showed that c-JUN could bind to the promoter sequences of Nur77. Antigen stimulation increased the expression of c-JUN in RBL2H3 cells. Altogether, our findings demonstrate the regulatory role played by Nur77-miR-21a loop in allergic inflammations such as anaphylaxis, making this the first report to present the role played by Nur77 in an allergic inflammation. Our results suggest that Nur77 and miR-21 might serve as targets for developing anti-allergy drugs.

Circular RNA circWNK1 inhibits the progression of gastric cancer via regulating the miR-21-3p/SMAD7 axis.

Gastric cancer (GC) is a highly aggressive malignancy with limited treatment options for advanced-stage patients. Recent studies have highlighted the role of circular RNA (circRNA) as a novel regulator of cancer progression in various malignancies. However, the underlying mechanisms by which circRNA contributes to the development and progression of GC remain poorly understood. In this study, we utilized microarrays and real-time quantitative polymerase chain reaction (qRT-PCR) to identify and validate a downregulated circRNA, hsa_circ_0003251 (referred to as circWNK1), in paired GC and normal tissues. Through a series of in vitro and in vivo gain-of-function and loss-of-function assays, we demonstrated that circWNK1 exerts inhibitory effects on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of GC cells. Additionally, we discovered that circWNK1 acts as a competitive endogenous RNA (ceRNA) for SMAD7 by sequestering miR-21-3p. Our findings were supported by comprehensive biological information analysis, as well as RNA pull-down, luciferase reporter gene, and western blot assays. Notably, the downregulation of circWNK1 in GC cells resulted in reduced SMAD7 expression, subsequently activating the TGF-ß signaling pathway. Collectively, our study reveals that circWNK1 functions as a tumor suppressor in GC by regulating the miR-21-3p/SMAD7-mediated TGF-ß signaling pathway. Furthermore, circWNK1 holds promise as a potential biomarker for the diagnosis and treatment of GC.

Cancer-associated fibroblasts promote stemness maintenance and gemcitabine resistance via HIF-1α/miR-21 axis under hypoxic conditions in pancreatic cancer.

Gemcitabine (GEM) resistance affects chemotherapy efficacy of pancreatic cancer (PC). Cancer-associated fibroblasts (CAFs) possess the ability of regulating chemoresistance. This study probed the mechanism of hypoxia-treated CAFs regulating cell stemness and GEM resistance in PC. Miapaca-2/SW1990 were co-cultured with PC-derived CAFs under normoxic/hypoxic conditions. Cell viability/self-renewal ability was determined by MTT/sphere formation assays, respectively. Protein levels of CD44, CD133, Oct4, and Sox2 were determined by western blot. GEM tumoricidal assay was performed. PC cell GEM resistance was evaluated by MTT assay. CAFs were cultured at normoxia/hypoxia. HIF-1α and miR-21 expression levels were assessed by RT-qPCR and western blot, with their binding sites and binding relationship predicted and verified. CAF-extracellular vesicles (EVs) were incubated with Miapaca-2 cells. The RAS/AKT/ERK pathway activation was detected by western blot. PC xenograft models were established and treated with hypoxic CAF-EVs and GEM. CAFs and PC cell co-culture increased cell stemness maintenance, GEM resistance, cell viability, stem cell sphere number, and protein levels of CD44, CD133, Oct4, and Sox2, and weakened GEM tumoricidal ability to PC cells, with the effects further enhanced by hypoxia. Hypoxia induced HIF-1α and miR-21 overexpression in CAFs. Hypoxia promoted CAFs to secrete high-level miR-21 EVs via the HIF-1α/miR-21 axis, and activated the miR-21/RAS/AKT/ERK pathway. CAF-EVs promoted GEM resistance in PC via the miR-21/RAS/ATK/ERK pathway in vivo. Hypoxia promoted CAFs to secrete high-level miR-21 EVs through the HIF-1α/miR-21 axis, and activated the miR-21/RAS/AKT/ERK pathway via EVs to trigger stemness maintenance and GEM resistance in PC.

Extracellular microRNAs induce dendritic cell-dependent joint inflammation and potentiate osteoclast differentiation via TLR7/8 engagement.

OBJECTIVES: Monocyte-derived dendritic cells (DCs) are key players in the induction of inflammation, autoreactive T cell activation and loss of tolerance in rheumatoid arthritis (RA), but the precise mechanisms underlying their activation remain elusive. Here, we hypothesized that extracellular microRNAs released in RA synovial fluids may represent a novel, physiological stimulus triggering unwanted immune response via TLR8-expressing DC stimulation. METHODS: Human monocyte-derived DCs were stimulated with a mixture of GU-rich miRNAs upregulated in RA tissues and released in synovial fluids (Ex-miRNAs). Activation of DCs was assessed in terms of NF-κB activation by Western blot, cytokine production by ELISA, T cell proliferation and polarization by allogeneic mixed lymphocyte reaction. DC differentiation into osteoclasts was evaluated in terms of tartrate-resistant acid phosphatase production and formation of resorption pits in dentine slices. Induction of joint inflammation in vivo was evaluated using a murine model of DC-induced arthritis. TLR7/8 involvement was assessed by specific inhibitors. RESULTS: Ex-miRNAs activate DCs to secrete TNFα, induce joint inflammation, start an early autoimmune response and potentiate the differentiation of DCs into aggressive osteoclasts. CONCLUSIONS: This work represents a proof of concept that the pool of extracellular miRNAs overexpressed in RA joints can act as a physiological activator of inflammation via the stimulation of TLR8 expressed by human DCs, which in turn exert arthritogenic functions. In this scenario, pharmacological inhibition of TLR8 might offer a new therapeutic option to reduce inflammation and osteoclast-mediated bone destruction in RA.

Role of MicroRNA-21 in Prostate Cancer Progression and Metastasis: Molecular Mechanisms to Therapeutic Targets.

The role of noncoding RNA has made remarkable progress in understanding progression, metastasis, and metastatic castration-resistant prostate cancer (mCRPC). A better understanding of the miRNAs has enhanced our knowledge of their targeting mainly at the therapy level in solid tumors, such as prostate cancer (PCa). microRNAs (miRNAs) belong to a class of endogenous RNA that deficit encoded proteins. Therefore, the role of miRNAs has been well-coined in the progression and development of PCa. miR-21 has a dual nature in its work both as a tumor suppressor and oncogenic role, but most of the recent studies showed that miR-21 is a tumor promoter and also is involved in castration-resistant prostate cancer (CRPC). Upregulation of miR-21 suppresses programmed cell death and inducing metastasis and castration resistant in PCa. miR-21 is involved in the different stages, such as proliferation, angiogenesis, migration, and invasion, and plays an important role in the progression, metastasis, and advanced stages of PCa. Recently, various studies directly linked the role of high levels of miR-21 with a poor therapeutic response in the patient of PCa. In the present review, we have explained the molecular mechanisms/pathways of miR-21 in PCa progression, metastasis, and castration resistant and summarized the role of miR-21 in diagnosis and therapeutic levels in PCa. In addition, we have spotlighted the recent therapeutic strategies for targeting different stages of PCa.

Skin wound repairing effects of adipose mesenchymal stem cells is promoted by the combined application of insulin-like growth factor 1: The key role of miR-21-5p-mediated signaling transduction.

Mesenchymal stem cells (ADMSCs) have been applied to the treatment of skin injuries and the co-administration of cytokines can enhance the effects. In the current study, the promoting effects of insulin-like growth factor 1 (IGF-1) on the skin wound healing effects of adipose-derived MSCs (ADMSCs) were assessed and the associated mechanism was explored by focusing on miR-21-5p mediated pathways. ADMSCs were isolated from epididymis rats, and skin wounded rats were employed as the in vivo model for evaluating the effect of ADMCs on skin healing and secretion of cytokines. Then a microarray assay was employed to select potential miR target of IGF-1 on ADMSCs. The level of the selected miR was modulated in ADMSCs, and the effects on skin injuries were also assessed. Administration of ADMSCs promoted skin wound healing and induced the production of bFGF, IL-1ß, PDGF, SDF-1, IGF-1, and TNF-α. The co-administration of IGF-1 and ADMSCs strengthened the effect of ADMSCs on skin wound by suppressing activity of matrix metalloproteinase-1 (MMP-1). At molecular level, the treatment of IGF-1 up-regulated miR-21-5p level in ADMSCs, which then suppressed the expression of KLF6 in injured skin tissues and promoted wound healing. The inhibition of miR-21-5p counteracted the promoting effects of IGF-1 on the skin healing effects of ADMSCs. Findings outlined in the current study indicated that IGF-1 could promote the wound healing effects of ADMSCs by up-regulating miR-21-5p level.

Bone Marrow Mesenchymal Stem Cells-Derived miR-21-5p Protects Grafted Islets Against Apoptosis by Targeting PDCD4.

The apoptosis of grafted islets is an urgent problem due to the high rate of islet loss soon after transplantation. MicroRNA-21-5p (miR-21-5p) is an essential mediator of bone marrow mesenchymal stem cells-derived exosomes (BMSCs-Exo) during anti-apoptosis, but its effect and the underlying molecular mechanism in islet transplantation remain partially understood. Here, we found that miR-21-5p could be delivered to islet cells via BMSCs-Exo. Subsequently, we demonstrated that miR-21-5p overexpression reduced apoptosis in islets and INS-1 cells, whereas miR-21-5p inhibition enhanced apoptosis. A mechanistic analysis involving RNA sequencing and bioinformatic analysis was performed to determine the interaction between miR-21-5p and its target gene programmed cell death 4 (PDCD4), which was further verified by a dual luciferase assay. In vivo, the grafted islets overexpressing miR-21-5p showed a higher survival rate, better insulin secretion function, and a lower apoptosis rate. In conclusion, these results demonstrated that miR­21­5p from BMSCs-Exo protects against the apoptosis of grafted islets by inhibiting PDCD4 expression. Hence, miR-21-5p can be used as a cell-free therapeutic agent to minimize ß-cell apoptosis at the early stage of islet transplantation.

miR-21 attenuated inflammation targeting MyD88 in human chondrocytes stimulated with Hyaluronan oligosaccharides.

Inflammation is the body's response to injuries, which depends on numerous regulatory factors. Among them, miRNAs have gained much attention for their role in regulating inflammatory gene expression at multiple levels. In particular, miR-21 is up-regulated during the inflammatory response and reported to be involved in the resolution of inflammation by down-regulating pro-inflammatory mediators, including MyD88. Herein, we evaluated the regulatory effects of miR-21 on the TLR-4/MyD88 pathway in an in vitro model of 6-mer HA oligosaccharides-induced inflammation in human chondrocytes. The exposition of chondrocytes to 6-mer HA induced the activation of the TLR4/MyD88 pathway, which culminates in NF-kB activation. Changes in miR-21, TLR-4, MyD88, NLRP3 inflammasome, IL-29, Caspase1, MMP-9, iNOS, and COX-2 mRNA expression of 6-mer HA-stimulated chondrocytes were examined by qRT-PCR. Protein amounts of TLR-4, MyD88, NLRP3 inflammasome, p-ERK1/2, p-AKT, IL-29, caspase1, MMP-9, p-NK-kB p65 subunit, and IKB-a have been evaluated by ELISA kits. NO and PGE2 levels have been assayed by colorimetric and ELISA kits, respectively. HA oligosaccharides induced a significant increase in the expression of the above parameters, including NF-kB activity. The use of a miR-21 mimic attenuated MyD88 expression levels and the downstream effectors. On the contrary, treatment with a miR-21 inhibitor induced opposite effects. Interestingly, the use of a MyD88 siRNA confirmed MyD88 as the target of miR-21 action. Our results suggest that miR-21 expression could increase in an attempt to reduce the inflammatory response, targeting MyD88.

miR-21-5p Modulates Airway Inflammation and Epithelial-Mesenchymal Transition Processes in a Mouse Model of Combined Allergic Rhinitis and Asthma Syndrome.

INTRODUCTION: Combined allergic rhinitis and asthma syndrome (CARAS) is a concurrent allergic symptom of diseases of allergic rhinitis and asthma. However, the mechanism of CARAS remains unclear. The study aimed to investigate the impact of microRNA-21 (miR-21) on CARAS via targeting poly (ADP-ribose) polymerase-1 (PARP-1) and phosphoinositide 3-kinase (PI3K)/AKT pathways. METHODS: The levels of miR-21-5p and PARP-1 in CARAS patients were detected by quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). An ovalbumin-sensitized mouse model of CARAS was established. And knock down of miR-21-5p was constructed by intranasally administering with miR-21-5p shRNA-encoding adeno-associated virus vector. Airway resistance and airway inflammatory response were detected. ELISA was used to evaluate IL-4/IL-5/IL-13 levels in bronchoalveolar lavage fluid (BALF). Expression levels of E-cadherin, fibronectin, and α-SMA were determined using Western blotting. The levels of PARP-1 and the activation of PI3K/AKT were assayed. RESULTS: Downregulation of miR-21-5p relieved pathophysiological symptoms of asthma including airway hyperreactivity and inflammatory cell infiltration. Downregulation of miR-21-5p significantly reduced the levels of IL4, IL-5, and IL-13 in BALF. Additionally, downregulation of miR-21-5p inhibited the epithelial-mesenchymal transition (EMT) process in CARAS mice. Furthermore, miR-21-5p regulated PARP-1 and was involved in PI3K/AKT activation in CARAS mice. CONCLUSION: Downregulation of miR-21-5p ameliorated CARAS-associated lung injury by alleviating airway inflammation, inhibiting the EMT process, and regulating PARP-1/PI3K/AKT in a mouse model of CARAS.

Exploring miR-21 as a key regulator in two distinct approaches of bone marrow stromal cells differentiation into Schwann-like cells.

The differentiation of bone marrow stromal cells (BMSCs) into Schwann-like cells (SCLCs) has the potential to promote the structural and functional restoration of injured axons. However, the optimal induction protocol and its underlying mechanisms remain unclear. This study aimed to compare the effectiveness of different induction protocols in promoting the differentiation of rat BMSCs into SCLCs and to explore their potential mechanisms. BMSCs were induced using two distinct methods: a composite factor induction approach (Protocol-1) and a conditioned culture medium induction approach (Protocol-2). The expression of Schwann cells (SCs) marker proteins and neurotrophic factors (NTFs) in the differentiated cells was assessed. Cell proliferation and apoptosis were also measured. During induction, changes in miR-21 and Sprouty RTK signaling antagonist 2 (SPRY2) mRNA were analyzed. Following the transfection of BMSCs with miR-21 agomir or miR-21 antagomir, induction was carried out using both protocols, and the expression of SPRY2, ERK1/2, and SCs marker proteins was examined. The results revealed that NTFs expression was higher in Protocol-1, whereas SCs marker proteins expression did not significantly differ between the two groups. Compared to Protocol-1, Protocol-2 exhibited enhanced cell proliferation and fewer apoptotic and necrotic cells. Both protocols showed a negative correlation between miR-21 and SPRY2 expression throughout the induction stages. After induction, the miR-21 agomir group exhibited reduced SPRY2 expression, increased ERK1/2 expression, and significantly elevated expression of SCs marker proteins. This study demonstrates that Protocol-1 yields higher NTFs expression, whereas Protocol-2 results in stronger SCLCs proliferation. Upregulating miR-21 suppresses SPRY2 expression, activates the ERK1/2 signaling pathway, and promotes BMSC differentiation into SCLCs.

Adipocyte derived exosomes promote cell invasion and challenge paclitaxel efficacy in ovarian cancer.

BACKGROUND: Epithelial ovarian cancer (EOC) is the deadliest gynaecological cancer with high mortality rates driven by the common development of resistance to chemotherapy. EOC frequently invades the omentum, an adipocyte-rich organ of the peritoneum and omental adipocytes have been implicated in promoting disease progression, metastasis and chemoresistance. The signalling mechanisms underpinning EOC omentum tropism have yet to be elucidated. METHODS: Three-dimensional co-culture models were used to explore adipocyte-EOC interactions. The impact of adipocytes on EOC proliferation, response to therapy and invasive capacity was assessed. Primary adipocytes and omental tissue were isolated from patients with ovarian malignancies and benign ovarian neoplasms. Exosomes were isolated from omentum tissue conditioned media and the effect of omentum-derived exosomes on EOC evaluated. Exosomal microRNA (miRNA) sequencing was used to identify miRNAs abundant in omental exosomes and EOC cells were transfected with highly abundant miRNAs miR-21, let-7b, miR-16 and miR-92a. RESULTS: We demonstrate the capacity of adipocytes to induce an invasive phenotype in EOC populations through driving epithelial-to-mesenchymal transition (EMT). Exosomes secreted by omental tissue of ovarian cancer patients, as well as patients without malignancies, induced proliferation, upregulated EMT markers and reduced response to paclitaxel therapy in EOC cell lines and HGSOC patient samples. Analysis of the omentum-derived exosomes from cancer patients revealed highly abundant miRNAs that included miR-21, let-7b, miR-16 and miR-92a that promoted cancer cell proliferation and protection from chemotherapy when transfected in ovarian cancer cells. CONCLUSIONS: These observations highlight the capacity of omental adipocytes to generate a pro-tumorigenic and chemoprotective microenvironment in ovarian cancer and other adipose-related malignancies.

Apigenin accelerates wound healing in diabetic mice by promoting macrophage M2-type polarization via increasing miR-21 expression.

The alteration of inflammatory phenotype by macrophage polarization plays an important role in diabetic wound repair. Apigenin has been reported to be anti-inflammatory and promote tissue repair; however, whether it regulates macrophage polarization to participate in diabetic wound repair remains to be investigated. We found that apigenin promoted miR-21 expression in LPS-stimulated RAW264.7 cells, inhibited cellular M1-type factor TNF-α and IL-1ß secretion and increased M2-type factor IL-10 and TGF-ß secretion, and accelerated macrophage conversion from M1 type to M2 type, whereas this protective effect of apigenin was counteracted by a miR-21 inhibitor. Moreover, we established a macrophage-HUVECs cell in vitro co-culture system and found that apigenin accelerated the migration, proliferation, and VEGF secretion of HUVECs by promoting macrophage miR-21 expression. Further, mechanistic studies revealed that this was mediated by the TLR4/Myd88/NF-κB axis. In in vivo study, diabetic mice had significantly delayed wound healing compared to non-diabetic mice, accelerated wound healing in apigenin-treated diabetic mice, and decreased M1-type macrophages and increased M2-type macrophages in wound tissues.

miR-21-5p-loaded bone mesenchymal stem cell-derived exosomes repair ovarian function in autoimmune premature ovarian insufficiency by targeting MSX1.

RESEARCH QUESTION: What is the effect of micro-RNA (miR)-21-5p-loaded bone marrow mesenchymal stem cell-derived exosomes (miR-21-Exo) on autoimmune premature ovarian insufficiency (POI)? DESIGN: The Cell Counting Kit 8 (CCK8) assay, fluorescence-activated cell sorting, western blotting, quantitative reverse transcriptase (qRT)-PCR and enzyme-linked immunosorbent assay (ELISA) verified the effect of miR-21-Exo on interferon-γ (IFN-γ)-induced KGN cells. qRT-PCR, western blotting and dual-luciferase reporter gene assays verified that miR-21-Exo mediated Msh homeobox 1 (MSX1) regulation of the Notch signalling pathway and that miR-21 interacted directly with MSX1. The effects of miR-21-Exo on the ovaries were verified by monitoring of the oestrous cycle, haematoxylin and eosin staining, follicle counts, ELISA, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), western blotting and qRT-PCR. RESULTS: The results showed that miR-21-Exo promoted IFN-γ-induced KGN cell proliferation and hormone synthesis, and inhibited apoptosis. Using dual-luciferase reporter gene assays, miR-21 and MSX1 were shown to have direct interactions. Moreover, the findings elucidated that miR-21-Exo inhibited cell apoptosis and promoted hormone synthesis by mediating MSX1 to regulate the Notch signalling pathway. miR-21-Exo restored the ovarian structure in a mouse model of autoimmune POI, promoted endocrine function and proliferation, and inhibited apoptosis and inflammation in vivo. CONCLUSIONS: This study demonstrates that miR-21-Exo regulates the MSX1-mediated Notch signalling pathway to inhibit granulosa cell apoptosis and improve hormone synthesis function, providing insight into a potential mechanism of molecular therapy for the treatment of autoimmune POI.

Decrease of exosomal miR-21-5p and the increase of CD62p+ exosomes are associated with the development of sepsis in polytraumatized patients.

Sepsis as a severe systemic inflammation leads oftentimes to organ dysfunction and subsequently to death. In polytrauma patients, septic complications represent with 45% the predominant cause of late death and are responsible for extremely high costs in the healthcare system. Therefore, clinicians have to detect as early as possible the begin of sepsis to improve the patient's outcome. One new promising diagnostic tool to diagnose septic complications in polytraumatized patients are exosomes. Plasma samples from polytraumatized patients (Injury Severity Score (ISS) ≥16) which developed sepsis (n = 10) and without sepsis (n = 10), were collected at emergency room (ER), 24h and 5 days after trauma. The EVs subpopulations were investigated by a bead-based multiplex flow cytometry measurement of surface epitopes and were compared with plasma EVs from healthy controls (n = 10). Moreover, exosomal cytokine concentrations were measured via high-sensitive ELISA and were correlated with systemic concentrations. For miRNA cargo analysis, we analysed the miRNAs miR-1298-5p, miR-1262, miR-125b-5p, miR-92a-3p, miR-93-5p, miR-155-5p and miR-21-5p and compared their exosomal concentrations by means of RT-qPCR. CD62p + exosomes were significantly increased in septic polytrauma-patients (p ≤ 0.05), while CD40+exosomes, as well as CD49e + exosomes were diminished (p ≤ 0.05). Furthermore, we observed that the exosomal IL-6 concentration reflects the systemic IL-6 concentration (r2 = 0.63) and did not significantly alter between patients with and without sepsis. The exosomal IL-10 concentration seemed to be constant in all patients and healthy controls. We observed that a decrease of miR-21-5p in exosomes was associated with the development of sepsis (p ≤ 0.05), while exosomal miR-93-5p, miR-155-5p and miR-92a-3p were not specifically altered in septic patients. Taken together, the present study in polytraumatized patients demonstrated that the development of sepsis is associated with an increase of CD62p + exosomes. Furthermore, the exosomal cargo was changed in septic patients: miR-21-5p was diminished.

MiR-21-5p knockdown inhibits epithelial to mesenchymal transition in A549 lung adenocarcinoma cells by upregulating RhoB.

BACKGROUND: MiR-21-5p is a highly expressed microRNA that plays an important role in various cancer-promoting processes, including anchorage-independent growth, invasion, migration metastasis, and drug resistance in lung cancer. Studies indicate that miR-21-5p may contribute to these processes by promoting epithelial-mesenchymal transition (EMT). Ras homolog gene family member B (RhoB), a gene downregulated by miR-21-5p, has also been linked to EMT in lung cancer. However, the role of the miR-21-5p/RhoB axis in EMT regulation in lung adenocarcinoma remains unclear. In this study, we aimed to investigate the regulatory role of the miR-21-5p/RhoB axis in EMT and related in vitro functional characteristics such as migration, invasion, cisplatin resistance, and the formation of tumor spheroids. METHODS AND RESULTS: A549 cells were transfected with the miR-21-5p inhibitor, RhoB siRNA, and their corresponding negative controls. Wound healing, transwell invasion, Methyl thiazole tetrazolium (MTT), and sphere formation assays were also performed to evaluate the migration, invasion, cisplatin resistance, and anchorage-independent growth of A549 cells. RT-qPCR was used to determine the mRNA expression levels of EMT markers. MiR-21-5p knockdown inhibited migration, invasion, cisplatin resistance, and sphere formation while upregulating E-cadherin and downregulating Slug. Furthermore, RhoB silencing restored EMT and related in vitro functional characteristics in A549 cells. CONCLUSIONS: Knockdown of miR-21-5p inhibits EMT and related in vitro functional characteristics by upregulating RhoB, suggesting that miR-21-5p may promote EMT through downregulation of RhoB.