MicroRNA-223-3p downregulates the inflammatory response in preeclampsia placenta via targeting NLRP3.

OBJECTIVE: To investigate the regulatory role of miR-223-3p in the inflammatory response of PE placenta. METHODS: PE and normal placental tissues were collected to measure the expression of NLRP3 and miR-223-3p. The targeting relationship between NLRP3 and miR-223-3P was verified by bioinformatics analysis and classical double-luciferase reporter gene assay. Lipopolysaccharide (LPS) was used to induce HTR8/SVneo cells as PE placental cell inflammation model. Then we transfected miR-223-3p overexpression/miR-223-3p negative control plasmid into the LPS-induced HTR8/SVneo cells. Next, the expressions of NLRP3, Caspase-1, GSDMD, IL-1ß and IL-18 were evaluated to elucidate the regulatory effect of miR-223-3p on the inflammatory response mediated by NLRP3 in PE placenta. RESULTS: Compared with normal controls, NLRP3 was significantly up-regulated in PE placenta, while miR-223-3p was down-regulated. In addition, NLRP3 was a direct target of miR-223-3p. Further research revealed that the expression of NLRP3, Caspase-1, GSDMD, IL-1ß and IL-18 could be obviously promoted in HTR8/SVneo cells treated with LPS (500 ng/ml) for 24 h, nevertheless it could be significantly suppressesed under the overexpression of miR-223-3p. CONCLUSION: MiR-223-3p suppressed NLRP3 inflamariomes activation, downstream inflammatory factors secretion and pyroptosis in LPS-induced HTR8/SVneo cells indicating that miR-223-3p could serve as an anti-inflammatory factor in preeclampsia.

Human bone marrow mesenchymal stem cell-derived extracellular vesicles reduce inflammation and pyroptosis in acute kidney injury via miR-223-3p/HDAC2/SNRK.

OBJECTIVE: Bone marrow mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) have been demonstrated as a potential therapeutic agent in acute kidney injury (AKI). However, little is known about the mechanisms of action of BMSC-derived EVs in AKI. Based on this, our research was designed to investigate the mechanism behind BMSC-derived EVs controlling inflammation and pyroptosis during AKI. METHODS: Peripheral blood from AKI patients was used for detection of microRNA (miR)-223-3p, HDAC2, and SNRK expression. An AKI rat model was established, and HK-2 cell injury was induced by lipopolysaccharide (LPS) to establish a cellular model. Co-culture with BMSC-derived EVs and/or gain- and loss-of-function assays were conducted in LPS-treated HK-2 to evaluate the functions of BMSCs-EVs, miR-223-3p, HDAC2, and SNRK. AKI rats were simultaneously injected with EVs and short hairpin RNAs targeting SNRK. The interactions among miR-223-3p, HDAC2, and SNRK were evaluated by RIP, ChIP, and dual-luciferase gene reporter assays. RESULTS: Patients with AKI had low miR-223-3p and SNRK expression and high HDAC2 expression in peripheral blood. Mechanistically, miR-223-3p targeted HDAC2 to accelerate SNRK transcription. In LPS-treated HK-2 cells, BMSCs-EVs overexpressing miR-223-3p increased cell viability and diminished cell apoptosis, KIM-1, LDH, IL-1ß, IL-6, TNF-α, NLRP3, ASC, cleaved caspase-1, and IL-18 expression, and GSDMD cleavage, which was nullified by HDAC2 overexpression or SNRK silencing. In AKI rats, BMSCs-EV-shuttled miR-223-3p reduced CRE and BUN levels, apoptosis, inflammation, and pyroptosis, which was abrogated by SNRK silencing. CONCLUSION: Conclusively, BMSC-derived EV-encapsulated miR-223-3p mitigated AKI-induced inflammation and pyroptosis by targeting HDAC2 and promoting SNRK transcription.

Human umbilical cord mesenchymal stromal cell small extracellular vesicle transfer of microRNA-223-3p to lung epithelial cells attenuates inflammation in acute lung injury in mice.

BACKGROUND: Acute lung injury (ALI), manifested as strong pulmonary inflammation and alveolar epithelial damage, is a life-threatening disease with high morbidity and mortality. Small extracellular vesicles (sEVs), secreted by multiple types of cells, are critical cellular communication mediators and can inhibit inflammation by transferring bioactive molecules, such as microRNAs (miRNAs). Thus, we hypothesized that sEVs derived from mesenchymal stromal cells (MSC sEVs) could transfer miRNAs to attenuate inflammation of lung epithelial cells during ALI. METHODS: C57BL/6 male mice were intratracheally administered LPS (10 mg/kg). Six hours later, the mice were randomly administered with MSC sEVs (40 µg per mouse in 150 µl of saline), which were collected by ultracentrifugation. Control group received saline administration. After 48 h, the mice were sacrificed to evaluate pulmonary microvascular permeability and inflammatory responses. In vitro, A549 cells and primary human small airway epithelial cells (SAECs) were stimulated with LPS with or without MSC sEVs treatment. RESULTS: In vitro, MSC sEVs could also inhibit the inflammation induced by LPS in A549 cells and SAECs (reducing TNF-α, IL-1ß, IL-6 and MCP-1). Moreover, MSC sEV treatment improved the survival rate, alleviated pulmonary microvascular permeability, and inhibited proinflammatory responses (reducing TNF-α, IL-1ß, IL-6 and JE-1) in ALI mice. Notably, miR-223-3p was found to be served as a critical mediator in MSC sEV-induced regulatory effects through inhibition of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) in lung epithelial cells. CONCLUSIONS: Overall, these findings suggest that MSC sEVs may offer a novel promising strategy for ALI.

miR-22-3p ameliorates the symptoms of premature ovarian failure in mice by inhibiting CMKLR1 expression.

Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. This study was aimed at exploring the improving effects of miR-22-3p on the symptoms of POF in mice by inhibiting chemokine-like receptor 1 (CMKLR1) expression. Female mice were intraperitoneally injected with cyclophosphamide to construct POF mice models. Lentiviral vectors containing miR-22-3p, short hairpin RNA (sh)-CMKLR1, and overexpression (oe)-CMKLR1, respectively, or in combination, were injected into the ovaries of both sides of POF mice. miR-22-3p and CMKLR1 expression in ovarian tissues of mice was assessed, and the targeting relationship between miR-22-3p and CMKLR1 was predicted and verified. Serum estradiol (E2), anti-Mullerian hormone, and follicle-stimulating hormone levels were assessed. Ovarian weight was weighed, and pathological changes and the number of primordial follicles, primary follicles, secondary follicles, and atresia follicles were observed. Apoptosis of ovarian tissues was determined. In ovarian tissues of POF mice, miR-22-3p expression was decreased while CMKLR1 expression was increased. miR-22-3p up-regulation or CMKLR1 down-regulation restored sex hormone levels, improved ovarian weight and the number of primordial follicles, primary follicles, and secondary follicles, and reduced the number of atresia follicle and ovarian granulosa cell apoptosis in POF mice. miR-22-3p targeted CMKLR1, and overexpressing CMKLR1 reversed the ameliorative effects of miR-22-3p overexpression on POF mice. Our research highlights that overexpressed miR-22-3p down-regulates CMKLR1 to ameliorate the symptoms of POF in mice. Therefore, the miR-22-3p/CMKLR1 axis could improve the symptoms of POF.

[MicroRNA-22-3p Regulates the Expression of Kruppel-like Factor 6 to Affect the Cardiomyocyte-like Differentiation of Bone Marrow Mesenchymal Stem Cell].

Objective To explore the effect of microRNA-22-3p (miR-22-3p) regulating the expression of Kruppel-like factor 6 (KLF6) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cell (BMSC). Methods Rat BMSC was isolated and cultured,and the third-generation BMSC was divided into a control group,a 5-azacytidine(5-AZA)group,a mimics-NC group,a miR-22-3p mimics group,a miR-22-3p mimics+pcDNA group,and a miR-22-3p mimics+pcDNA-KLF6 group.Real-time fluorescent quantitative PCR (qRT-PCR) was carried out to determine the expression of miR-22-3p and KLF6 in cells.Immunofluorescence staining was employed to detect the expression of Desmin,cardiac troponin T (cTnT),and connexin 43 (Cx43).Western blotting was employed to determine the protein levels of cTnT,Cx43,Desmin,and KLF6,and flow cytometry to detect the apoptosis of BMSC.The targeting relationship between miR-22-3p and KLF6 was analyzed by dual luciferase reporter gene assay. Results Compared with the control group,5-AZA up-regulated the expression of miR-22-3p (q=7.971,P<0.001),Desmin (q=7.876,P<0.001),cTnT (q=10.272,P<0.001),and Cx43 (q=6.256,P<0.001),increased the apoptosis rate of BMSC (q=12.708,P<0.001),and down-regulated the mRNA (q=20.850,P<0.001) and protein (q=11.080,P<0.001) levels of KLF6.Compared with the 5-AZA group and the mimics-NC group,miR-22-3p mimics up-regulated the expression of miR-22-3p (q=3.591,P<0.001;q=11.650,P<0.001),Desmin (q=5.975,P<0.001;q=13.579,P<0.001),cTnT (q=7.133,P<0.001;q=17.548,P<0.001),and Cx43 (q=4.571,P=0.037;q=11.068,P<0.001),and down-regulated the mRNA (q=7.384,P<0.001;q=28.234,P<0.001) and protein (q=4.594,P=0.036;q=15.945,P<0.001) levels of KLF6.The apoptosis rate of miR-22-3p mimics group was lower than that of 5-AZA group (q=8.216,P<0.001).Compared with the miR-22-3p mimics+pcDNA group,miR-22-3p mimics+pcDNA-KLF6 up-regulated the mRNA(q=23.891,P<0.001) and protein(q=13.378,P<0.001)levels of KLF6,down-regulated the expression of Desmin (q=9.505,P<0.001),cTnT (q=10.985,P<0.001),and Cx43 (q=8.301,P<0.001),and increased the apoptosis rate (q=4.713,P=0.029).The dual luciferase reporter gene experiment demonstrated that KLF6 was a potential target gene of miR-22-3p. Conclusion MiR-22-3p promotes cardiomyocyte-like differentiation of BMSC by inhibiting the expression of KLF6.

MiR-223-3p increases resistance of colorectal cancer cells to 5-fluorouracil via targeting SORBS1. / MiR-223-3p通过靶向SORBS1å¢žåŠ ç»“ç›´è‚ ç™Œç»†èƒžå¯¹5-氟尿嘧啶的耐药性.

OBJECTIVES: 5-Fluorouracil (5-FU) is the first-line drug for treating colorectal cancer (CRC), and the resistance of tumor cells to 5-FU is the main cause of chemotherapeutic failure. However, the resistant mechanism is still unclear. This study aims to explore the tumor suppressor genes involved in 5-FU resistance in CRC, and to find the microRNA (miRNA) that regulates these genes. METHODS: CRC data sets GSE28702 and GSE69657 were downloaded from Gene Expression Omnibus (GEO) database, and gene expression profiles of patients in the FOLFOX chemotherapeutic response group and the non-response group were analyzed, and differential expression genes were identified between the 2 groups. Target gene was then selected. Online bioinformatics databases TargetScan, miRwalk, and miRDB were used to predict miRNA targeting the interested gene sorbin and SH3 domain containing 1 (SORBS1). siSORBS1, HA-SORBS1, miR-223-3p mimic, anti-miR-223-3p, and their corresponding negative controls (siNC, HA, miR-NC, and anti-miR-NC) were transfected into CRC cell lines of HCT116 and SW620 by transient transfection technique, respectively. Co-transfection was done with miRNA and plasmid (miR-NC+HA, miR-223-3p mimic+HA, or miR-223-3p mimic+HA-SORBS1) or anti-miRNA and siRNA (anti-miR-NC+siNC, anti-miR-223-3p+siNC, or anti-miR-223-3p+siSORBS1) in HCT116 cells. Real-time reverse transcription PCR (real-time RT-PCR) and/or Western blotting were used to detect the expression levels of SORBS1 and miR-223-3p in cells. After transfection, the cells were treated with different concentrations of 5-FU, and the cell viability was detected by methyl thiazolyl tetrazolium (MTT) method. The targeting relationship between miR-223-3p and SORBS1 was comfirmed by dual luciferase reporter gene assay. RESULTS: There were 409 and 528 highly expressed genes in the FOLFOX chemotherapeutic response group of GSE69657 and GSE28702, respectively. There were 22 overlapping genes in the response group, among which exist 3 tumor suppressor genes might be involved in chemosensitivity in CRC, and SORBS1 was selected as the target gene for further study. Three online bioinformatics databases predicted miRNAs targeting SORBS1 and obtained an intersection molecule miR-223-3p. After treatment with 5-FU (25 µmol/L) for 12-36 h, the levels of miR-223-3p in HCT116 and SW620 cells were significantly down-regulated (all P<0.05). After transfection with siSORBS1 or miR-223-3p mimic, the expression levels of SORBS1 in HCT116 and SW620 cells were down-regulated, and the cell viability was increased (all P<0.05). After transfection with HA-SORBS1 or anti-miR-223-3p, the expression levels of SORBS1 in HCT116 and SW620 cells were up-regulated, and the cell viability was decreased (all P<0.05). The result of dual luciferase reporter gene assay showed that the luciferase activity of cells co-transfected with SORBS1 3'-UTR wild plasmid and miR-223-3p mimic was significantly lower than that of the 3'-UTR wild plasmid and miR-NC cells (P<0.05). Compared with co-transfection with miR-223-3p mimic and HA, the cell viability of cells co-transfected with miR-223-3p mimic and HA-SORBS1 was decreased significantly (P<0.01). Compared with the co-transfected anti-miR-223-3p and siNC, the cell viability of the co-transfected anti-miR-223-3p and siSORBS1 was significantly increased (P<0.05). CONCLUSIONS: MiR-223-3p increases 5-FU resistance in CRC cells by targeting SORBS1,and miR-223-3p is expected to become a new target for clinical treatment of CRC.

MicroRNA-22-3p alleviates spinal cord ischemia/reperfusion injury by modulating M2 macrophage polarization via IRF5.

Cell death after spinal cord ischemia/reperfusion (I/R) can occur through necrosis, apoptosis, and autophagy, resulting in changes to the immune environment. However, the molecular mechanism of this immune regulation is not clear. Accumulating evidence indicates that microRNAs (miRs) play a crucial role in the pathogenesis of spinal cord I/R injury. Here, we hypothesized miR-22-3p may be involved in spinal cord I/R injury by interacting with interferon regulatory factor (IRF) 5. Rat models of spinal cord I/R injury were established by 12-min occlusion of the aortic arch followed by 48-hr reperfusion, with L4-6 segments of spinal cord tissues collected. MiR-22-3p agomir, a lentivirus-delivered siRNA specific for IRF5, or a lentivirus expressing wild-type IRF5 was injected intrathecally to rats with I/R injury to evaluate the effects of miR-22-3p and IRF5 on hindlimb motor function. Macrophages isolated from rats were treated with miR-22-3p mimic or siRNA specific for IRF5 to evaluate their effects on macrophage polarization. The levels of IL-1ß and TNF-α in spinal cord tissues were detected by ELISA. miR-22-3p was down-regulated, whereas IRF5 was up-regulated in rat spinal cord tissues following I/R. IRF5 was a target gene of miR-22-3p and could be negatively regulated by miR-22-3p. Silencing IRF5 or over-expressing miR-22-3p relieved inflammation, elevated Tarlov score, and reduced the degree of severity of spinal cord I/R injury. Increased miR-22-3p facilitated M2 polarization of macrophages and inhibited inflammation in tissues by inhibiting IRF5, thereby attenuating spinal cord I/R injury. Taken together, these results demonstrate that increased miR-22-3p can inhibit the progression of spinal cord I/R injury by repressing IRF5 in macrophages, highlighting the discovery of a promising new target for spinal cord I/R injury treatment.

microRNA-22-3p plays a protective role in a murine asthma model through the inhibition of the NLRP3-caspase-1-IL-1ß axis.

NEW FINDINGS: What is the central question of this study? How does miR-22-3p exert a protective role in asthma? What is the main finding and its importance? Upregulation of miR-22-3p hampered airway inflammation and release of inflammatory cytokines through blocking the activation of the NLRP3-caspase-1-IL-1ß signalling pathway in asthma. ABSTRACT: Asthma, a great public health burden, is triggered by inflammatory responses in the airways and these are not addressed appropriately by current therapies. This study aims to investigate the regulatory mechanism of microRNA-22-3p (miR-22-3p) on the proliferation of bronchial epithelial cells exposed to lipopolysaccharide (LPS) and expression of pro-inflammatory cytokines in a murine asthma model challenged by ovalbumin. We first confirmed the downregulation of miR-22-3p in the murine asthma model and bronchial epithelial cells. miR-22-3p remarkably reversed the decline in bronchial epithelial cell viability, enhancement in apoptosis rate and release of inflammatory factors induced by LPS. miR-22-3p targeted and conversely regulated NACHT, LRR and PYD domains-containing protein 3 (NLRP3). Overexpression of NLRP3 counteracted the inhibitory effect of miR-22-3p on inflammatory damage in bronchial epithelial cells through activation of caspase-1/interleukin (IL)-1ß. In an in vivo model, overexpression of miR-22-3p significantly attenuated airway obstruction and tissue damage in mice. In summary, our study underscores that miR-22-3p serves both as a negative regulator of the NLRP3-caspase-1-IL-1ß axis and as a protective factor against the inflammatory response, suggesting a future therapeutic role in asthma.

Inflammation-induced overexpression of microRNA-223-3p regulates odontoblastic differentiation of human dental pulp stem cells by targeting SMAD3.

AIM: To profile miRNA expression between inflamed and healthy human dental pulp tissues and to investigate how the upregulation of miR-223-3p in the inflamed pulp tissue regulates odontoblast differentiation and regeneration. METHODOLOGY: Microarray analysis was used to identify differences in miRNA expression patterns between healthy and inflamed pulp tissue. The results were validated using quantitative real-time PCR. To determine the effect of miR-223-3p on odontoblast differentiation, miR-223-3p was overexpressed in human dental pulp stem cells (DPSCs), which were cultured in mineralizing induction medium (to induce odontoblast differentiation). To identify the target genes of miR-223-3p, an SABiosciences Human Osteogenesis PCR Array, combined with bioinformatics, was used. Furthermore, a dual-luciferase reporter assay and a small interfering RNA (siRNA) experiment were used to confirm the relationship between miR-223-3p and its target gene. Statistical analysis was performed using the Student's t-test or one-way analysis of variance (anova); P < 0.05 was considered statistically significant. RESULTS: Seventy-nine miRNAs were significantly differentially expressed (fold change >2.0; P < 0.05) between the two tissues. In particular, miR-223-3p was markedly upregulated in inflamed dental pulp. Overexpression of miR-223-3p in DPSCs significantly increased the protein levels of dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP-1) (P < 0.05). However, the SMAD family member 3 (SMAD3) protein level was significantly lower than in control DPSCs (P < 0.05). Bioinformatics and the dual-luciferase assay reporter assay indicated that Smad3 was a potential target of miR-223-3p. Knockdown of Smad3 in DPSCs subjected to mineralization induction resulted in detection of DSPP and DMP-1 earlier than in control DPSCs, and it increased the protein level of alkaline phosphatase (ALP), thereby promoting odontoblast differentiation. CONCLUSIONS: miR-223-3p is implicated in the regulation of odontoblast differentiation, which may be involved in the process of pulpitis repair.

MiR-22-3p Regulates Cell Proliferation and Inhibits Cell Apoptosis through Targeting the eIF4EBP3 Gene in Human Cervical Squamous Carcinoma Cells.

Background: MicroRNAs (miRNAs) are non-coding small RNAs that function as negative regulators of gene expression and are involved in tumour biology. The eIF4E-binding proteins (eIF4EBPs) play essential roles in preventing translation initiation and inhibiting protein synthesis at a global or message-specific level in a variety of tumours. Methods: According to comparative miRNA profiles of clinical cervical cancer and non-cancerous cervical tissue specimens, several miRNAs were aberrantly expressed in the cervical cancer samples. C33a and SiHa cell proliferation and apoptosis were detected using methyl thiazolyl tetrazolium (MTT) and flow cytometry assays, respectively. Results: Among the aberrantly expressed miRNAs, miR-22-3p was significantly differentially expressed in cervical cancer tissues and was highly associated with cervical cancer cell growth regulation. In addition, bioinformatic predictions and experimental validation were used to identify whether eIF4E-binding protein 3 (eIF4EBP3) was a direct target of miR-22-3p; eIF4EBP3 protein levels were generally low in the cervical cancer tissues. Furthermore, functional studies revealed that either a miR-22-3p inhibitor or eIF4EBP3 overexpression could induce apoptosis in cervical cancer cells in vitro. Importantly, we found that eIF4EBP3 accumulation could significantly attenuate cervical cancer cell proliferation triggered by a miR-22-3p mimic as well as enhance apoptosis in cervical cancer cells. Conclusion: Taken together, our data provide primary proof that miR-22-3p can induce cervical cancer cell growth at least in part by up-regulating its expression to decrease eIF4EBP3 expression levels; miR-22-3p thus holds promise as a prognostic biomarker and potential therapeutic target for treating cervical cancer.

The effect of miR-223-3p on endothelial cells in coronary artery disease.

Endothelial cell damage and dysfunction are crucial factors in the development and early stages of coronary artery disease (CAD) and apoptosis plays a significant role in this process. In this study, We aimed to simulate the CAD vascular microenvironment by treating endothelial cells with tumor necrosis factor alpha (TNF-α) to construct an endothelial cell apoptosis model. Our findings revealed that the TNF-α model resulted in increased micro-RNA 223-3p (miR-223-3p) mRNA and Bax protein expression, decreased kruppel-like factor 15 (KLF15) and Bcl-2 protein expression, and decreased cell viability. More importantly, in the TNF-α-induced endothelial cell apoptosis model, transfection with the miR-223-3p inhibitor reversed the effects of TNF-α on Bcl-2, Bax expression. We transfected miRNA-223-3p mimics or inhibitors into endothelial cells and assessed miR-223-3p levels using RT-PCR. Cell viability was detected using CCK8. Western blot technology was used to detect the expression of Bcl-2, Bax, and KLF15. In summary, this study demonstrates the role and possible mechanism of miR-223-3p in endothelial cells during CAD, suggesting that miR-223-3p may serve as a promising therapeutic target in CAD by regulating KLF15.

MicroRNA-223-3p inhibits oxidized low-density lipoprotein-mediated NLRP3 inflammasome activation via directly targeting NLRP3 and FOXO3.

BACKGROUND: MicroRNAs (miRNAs) have emerged as crucial players in the initiation and development of atherosclerosis (AS), and the low miR-223-3p level is observed in AS patients. However, the function and mechanism behind miR-223-3p in AS progression have not been fully elucidated. METHOD: In the present study, THP-1 cells treated with oxidized low-density lipoprotein (ox-LDL) were employed as the cell model of AS. The expression levels of miR-223-3p, NLR family pyrin domain containing 3 (NLRP3), caspase-1, pro-caspase-1, cleaved interleukin 18 (IL-18), cleaved IL-1ß, and forkhead box O3 (FOXO3) were measured by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot (WB) analyses. The relationship between miR-223-3p and FOXO3 or NLRP3 was determined using a dual-luciferase reporter assay. The production of IL-1ß, IL-18, IL-6, and TNF-α was examined by Enzyme-linked immunosorbent assay (ELISA). RESULTS: MiR-223-3p was decreased in AS patients and ox-LDL-induced THP-1 cells, and its upregulation downregulated the abundance of NLRP3, caspase-1, cleaved IL-18, cleaved IL-1ß, IL-1ß, IL-6, and TNF-α in THP-1 cells treated with ox-LDL or not, and the depletion of miR-223-3p revealed an opposite phenomenon. NLPR3 and FOXO3 were identified as two authentic targets of miR-223-3p. Knockdown of NLRP3 or FOXO3 reversed the stimulatory effect of the miR-223-3p inhibitor on the inflammatory responses of THP-1 cells. CONCLUSIONS: Our data indicate that miR-223-3p inhibited ox-LDL-mediated NLRP3 inflammasome activation via directly targeting NLRP3 and FOXO3 in THP-1 cells, which offered a prospective therapeutic target for AS therapy.

Large extracellular vesicles in the left atrial appendage in patients with atrial fibrillation-the missing link?

Atrial fibrillation (AF) is the most frequent arrhythmic disease in humans, which leads to thrombus formation in the left atrial appendage and stroke through peripheral embolization. Depending on their origin, large extracellular vesicles (lEVs) can exert pro-coagulant functions. In the present study, we investigated how different types of AF influence the levels of large EV subtypes in three distinct atrial localizations. Blood samples were collected from the right and left atrium and the left atrial appendage of 58 patients. 49% of the patients had permanent AF, 34% had non-permanent AF, and 17% had no history of AF. Flow cytometric analysis of the origin of the lEVs showed that the proportion of platelet-derived lEVs in the left atrial appendage was significantly higher in permanent AF patients compared to non-permanent AF. When we grouped patients according to their current heart rhythm, we also detected significantly higher levels of platelet-derived lEVs in the left atrial appendage (LAA) in patients with atrial fibrillation. In vitro studies revealed, that platelet activation with lipopolysaccharide (LPS) leads to higher levels of miR-222-3p and miR-223-3p in platelet-derived lEVs. Treatment with lEVs from LPS- or thrombin-activated platelets reduces the migration of endothelial cells in vitro. These results suggest that permanent atrial fibrillation is associated with increased levels of platelet-derived lEVs in the LAA, which are potentially involved in LAA thrombus formation.

Mesenchymal stem cells-derived extracellular vesicles-shuttled microRNA-223-3p suppress lipopolysaccharide-induced cardiac inflammation, pyroptosis, and dysfunction.

INTRODUCTION: Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) possess therapeutical potentials in cardiac disorders. We probed into the mechanisms of MSC-EV-enclosed miR-223-3p in lipopolysaccharide (LPS)-induced cardiac inflammation, pyroptosis, and dysfunction. METHODS: The cardiomyocyte model of cardiac dysfunction was induced by LPS, followed by determination of miR-223-3p expression. Next, we discerned the relation among miR-223-3p, FOXO3, and NLRP3. LPS-exposed cardiomyocytes were co-incubated with EVs from mouse MSCs to detect inflammation and pyroptosis using the gain- or loss-of-function experimentations. LPS-induced myocarditis mouse models were also prepared for further validating the effects of miR-223-3p from MSCs-derived EVs. RESULTS: Reduced miR-223-3p was witnessed in LPS-induced cardiomyocytes. Specifically, miR-223-3p could target and inhibit FOXO3 to reduce NLRP3 expression. MSC-EVs could transfer miR-223-3p into cardiomyocytes to repress LPS-induced cardiomyocyte inflammation and pyroptosis. Additionally, in LPS-induced mice, pyroptosis, immune cell infiltration, inflammatory cytokine secretion, and cardiac dysfunction were alleviated by MSC-EV-loading miR-223-3p. CONCLUSION: Conclusively, miR-223-3p shuttled by MSC-EVs restricted cardiac inflammation, pyroptosis, and dysfunction by disrupting FOXO3/NLRP3 axis.

Adipose-derived mesenchymal stem cell-secreted extracellular vesicles alleviate non-alcoholic fatty liver disease via delivering miR-223-3p.

Increasing studies have identified the potential of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) in non-alcoholic fatty liver disease (NAFLD) treatment. Hence, we further focused on the potential of adipose-derived MSC (ADSC)-EVs in NAFLD by delivering miR-223-3p. The uptake of isolated ADSC-EVs by hepatocytes was assessed, and the expression of miR-223-3p in ADSC-EVs and hepatocytes was characterized. It was established that miR-223-3p, enriched in ADSC-EVs, could be delivered by ADSC-EVs into hepatocytes. Using co-culture system and gain-of-function approach, we evaluated the effect of ADSC-EVs carrying miR-223-3p on lipid accumulation and liver fibrosis in pyrrolizidine alkaloids (PA)-induced hepatocytes and a high-fat diet-induced NAFLD mouse model. Bioinformatics websites and dual-luciferase reporter gene assay were performed to determine the interactions between miR-223-3p and E2F1, which was further validated by rescue experiments. ADSC-EVs containing miR-223-3p displayed suppressive effects on lipid accumulation and liver fibrosis through E2F1 inhibition, since E2F1 was demonstrated as a target gene of miR-223-3p. The protective role of ADSC-EVs by delivering miR-223-3p was then confirmed in the mouse model. Collectively, this study elucidated that ADSC-EVs delayed the progression NAFLD through the delivery of anti-fibrotic miR-223-3p and subsequent E2F1 suppression, which may suggest miR-223-3p-loaded ADSC-EVs to be a potential therapeutic approach for NAFLD.

MicroRNA-22-3p targeted regulating transcription factor 7-like 2 (TCF7L2) constrains the Wnt/ß-catenin pathway and malignant behavior in osteosarcoma.

Various studies have manifested that microRNAs (miRNAs) are involved in the modulation of the occurrence and development of osteosarcoma (OS). However, whether miR-22-3p is associated with OS growth remains unclear. In the study, the potential molecular mechanisms of miR-22-3p in OS was explored. It was affirmed that miR-22-3p was associated with distant metastasis and tumor size in OS patients, and reduced in OS tissues and cells while transcription factor 7-like 2 (TCF7L2) was elevated. Elevated miR-22-3p repressed OS cell progression, and the Wnt/ß-catenin pathway, while elevated TCF7L2 was opposite. MiR-22-3p targeted TCF7L2 in OS. In functional rescue experiments, knockdown of miR-22-3p on OS progression and promotion of Wnt/ß-catenin were reversed by simultaneous knockdown of TCF7L2. Transplantation experiments in nude mice showed that elevated miR-22-3p repressed OS tumor growth and decreased TCF7L2, Wnt and ß-catenin. Shortly, this study suggest that miR-22-3p refrains the Wnt/ß-catenin pathway by targeting TCF7L2 and thereby preventing OS deterioration. MiR-22-3p/TCF7L2 axis is supposed to be a candidate molecular target for future OS treatment.

lncRNA GAS5 promotes pyroptosis in COPD by functioning as a ceRNA to regulate the miR­223­3p/NLRP3 axis.

Chronic obstructive pulmonary disease (COPD) is characterized by irreversible and progressive airflow limitation and encompasses a spectrum of diseases, including chronic obstructive bronchitis and emphysema. Pyroptosis is a unique form of inflammatory cell death mediated by the activation of caspase­1 and inflammasomes. The long non­coding RNA (lncRNA) growth arrest­specific 5 (GAS5) is a well­documented tumor suppressor, which is associated with cell proliferation and death in various diseases. The aim of the present study was to evaluate whether lncRNA GAS5 is associated with the pyroptosis in COPD. To create a COPD cell model, MRC­5 cells were treated with 10 µg/ml lipopolysaccharide (LPS) for 48 h. Then the level of pro­caspase 1, caspase 1, IL­1ß, IL­18, NLRP3 and cleaved gasdermin D (GSDMD) was examined by western blotting. GAS5 mRNA level was detected by qualitative PCR following LPS treatment in MRC­5 cells. Subsequently, IL­2, IL­6, IL­10 and TNF­α in MRC­5 cells was measured by ELISA. Then the proliferation ability of MRC­5 cells was detected by CCK­8. Cell death was detected by TUNEL assay. LDH release was measured using an LDH Cytotoxicity Assay kit. The Magna RIP kit was used to validate the interaction between GAS5 and miR­223­3p. The present study revealed that increased expression levels of caspase­1, IL­1ß, IL­18 and cleaved GSDMD were observed in LPS­treated MRC­5 cells, indicating that pyroptosis is involved in COPD progression. Additionally, LPS induced the increase in GAS5 mRNA expression levels and the release of inflammatory factors (IL­2, IL­6, IL­10 and TNF­α), suggesting that GAS5 is implicated in pyroptosis in COPD. Furthermore, upregulation of GAS5 promoted cell death and inhibited proliferation in the MRC­5 cell line. Additionally, increased GAS5 expression significantly promoted the production of caspase­1, IL­1ß, IL­18, cleaved GSDMD and NLR pyrin domain containing protein 3 (NLRP3). A dual­luciferase assay demonstrated that GAS5 could directly bind to microRNA­223­3p (miR­223­3p), and NLRP3 is a direct target of miR­223­3p. Furthermore, GAS5 reduced the expression levels of miR­223­3p, while it increased the expression levels of NLRP3. The present study concluded that lncRNA GAS5 promoted pyroptosis in COPD by targeting the miR­223­3p/NLRP3 axis, implying that GAS5 could be a potential target for COPD.

MicroRNA-223-3p regulates allergic inflammation by targeting INPP4A.

INTRODUCTION: Emerging evidence indicates that physiological and pathological conditions of the nose are posttranscriptionally regulated by microRNAs, a class of small noncoding RNAs. Recently, microRNA-223-3p has been increasingly implicated in the modulation of allergic rhinitis OBJECTIVE: This study aimed to assess the role and mechanism of microRNA-223-3p in a mouse model of allergic rhinitis. METHODS: The expression level of miR-223-3p was measured in the serum of 41 allergic rhinitis patients and 39 healthy controls using quantitative real time polymerase chain reaction. BALB/c mice were used to establish an allergic rhinitis model by intraperitoneal sensitization and intranasal challenge with ovalbumin. MicroRNA-223-3p agomir/antagomir was then intranasally administered to mice after ovalbumin challenge for another week. The symptoms of nasal rubbing and sneezing were recorded. Serum ovalbumin-specific immunoglobulin E concentration, microRNA-223-3p expression and proinflammatory cytokine (IL-4, IL-5, IFN-γ) levels in nasal mucosa were measured by ELISA and quantitative real time polymerase chain reaction, respectively. Histopathologic changes were evaluated using hematoxylin and eosin staining. RESULTS: MicroRNA-223-3p levels increased significantly in both allergic rhinitis patients and allergic rhinitis mice. In addition, upregulation of microRNA-223-3p levels by nasal administration of microRNA-223-3p agomir also markedly increased the concentration of ovalbumin -specific IgE, the frequencies of nasal rubbing and sneezing, the levels of proinflammatory cytokines (IL-4, IL-5, IFN-γ) and eosinophil infiltration in the nasal mucosa of allergic rhinitis mice. Moreover, microRNA-223-3p antagomir appeared to strongly ameliorate the symptoms and pathology in nasal mucosa. Subsequently, we demonstrated for the first time that microRNA-223-3p negatively regulated INPP4A expression by binding with the 3' untranslated region (3'UTR) of INPP4A. CONCLUSIONS: These findings indicate that microRNA-223-3p plays an important role in regulating the pathology and symptoms of allergic rhinitis by targeting INPP4A.

Regulatory effect of microRNA-223-3p on breast cancer cell processes via the Hippo/Yap signaling pathway.

According to the 2018 global cancer statistics, the incidence and mortality rates of breast cancer are increasing gradually, which seriously threatens the health of women. MicroRNA-223-3p (miR-223-3p) can promote the proliferation and invasion of breast cancer cells. Hippo/Yes-related protein (Yap) signaling pathway activation has been found in a variety of tumors. The present study aimed to investigate the potential mechanism of miR-223-3p in breast cancer. The Cell Counting Kit-8 assay was used to detect cell viability and flow cytometry was used to detect apoptosis. The abilities of cell migration and invasion were detected using scratch and Transwell assays, as well as reverse transcription-quantitative PCR and western blotting to detect gene and protein expression, respectively. The current results demonstrated that miR-223-3p transcription levels were increased in breast cancer cells, and inhibition of miR-223-3p gene expression decreased cell proliferation, migration and invasion. Additionally, inhibition of miR-223-3p expression inhibited epithelial-mesenchymal transition (EMT) in breast cancer cells. miR-223-3p promoted cell proliferation, migration, invasion and EMT, and the western blotting results demonstrated that miR-223-3p inhibition increased the phosphorylation of Yap1 and the protein expression levels of large tumor suppressor kinase 1. In conclusion, results from the present results suggested that miR-223-3p may promote cell proliferation, migration, invasion and EMT through the Hippo/Yap signaling pathway. Therefore, miR-223-3p may be a potential biomarker for breast cancer.

Deregulated microRNA-22-3p in patients with sepsis-induced acute kidney injury serves as a new biomarker to predict disease occurrence and 28-day survival outcomes.

BACKGROUND: Acute kidney injury (AKI) is a common and serious complication of sepsis. MicroRNA-22-3p (miR-22-3p) has been found to be involved in septic AKI progression. The purpose of this study was to analyze both the serum and urinary expression of miR-22-3p in septic AKI patients, and evaluated the clinical value of miR-22-3p in the diagnosis and prognosis of sepsis-induced AKI. METHODS: Serum and urinary expression of miR-22-3p was examined using qRT-PCR. The risk factors related with septic AKI onset were assessed using logistic analysis. A receiver-operating characteristic (ROC) curve was constructed to evaluate the diagnostic performance of miR-22-3p, and the Kaplan-Meier survival curves and Cox regression analysis were used to evaluate the predictive value of miR-22-3p for the 28-day survival of septic AKI patients. RESULTS: Both serum and urinary miR-22-3p expression was decreased and negatively correlated with kidney injury biomarkers in septic AKI patients. MiR-22-3p expression was a risk factor for AKI onset and had diagnostic accuracy in septic AKI patients. The expression of both serum and urinary miR-22-3p was lower in patients who died, and served as a prognostic biomarker to predict 28-day survival in septic AKI patients. CONCLUSION: Serum and urinary miR-22-3p was reduced in sepsis-induced AKI patients, and served as a biomarker to predict AKI occurrence and 28-day survival in sepsis patients.