miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6

It has been suggested that microRNAs (miRs) are involved in the immune regulation of periodontitis. However, it is unclear whether and how miRs regulate the function of B cells in the context of periodontitis. This study is to explore the role of miR-146a on the inflammatory cytokine production of B cells challenged by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). Primary B cells were harvested from mouse spleen. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of inflammatory cytokines in B cells in the presence or absence of P. gingivalis LPS and/or miR-146a. Bioinformatics, luciferase reporter assay and overexpression assay were used to explore the binding target of miR-146a. Our results showed that miR-146a level in B cells was elevated by P. gingivalis LPS stimulation, and the mRNA expressions of interleukin (IL)-1β, 6 and 10, and IL-1 receptor associated kinase-1 (IRAK1), but not TNF receptor associated factor 6 (TRAF6), were also upregulated. The expression levels of IL-1β, 6, 10 and IRAK1 were reduced in the presence of miR-146a mimic, but were elevated by the addition of miR-146a inhibitor. MiR-146a could bind with IRAK1 3′ untranslated region (UTR) but not TRAF6 3′-UTR. Overexpression of IRAK1 reversed the inhibitory effects of miR-146a on IL-1β, 6 and 10. In summary, miR-146a inhibits inflammatory cytokine production in B cells through directly targeting IRAK1, suggesting a regulatory role of miR-146a in B cell-mediated periodontal inflammation.     

Downregulated microRNA-27b attenuates lipopolysaccharide-induced acute lung injury via activation of NF-E2-related factor 2 and inhibition of nuclear factor κB signaling pathway

Acute lung injury (ALI) is a life-threatening, diffuse heterogeneous lung injury characterized by acute onset, pulmonary edema, and respiratory failure. Lipopolysaccharide (LPS) is a leading cause for ALI and when administered to a mouse it induces a lung phenotype exhibiting some of the clinical characteristics of human ALI. This study focused on investigating whether microRNA-27b (miR-27b) affects ALI in a mouse model established by LPS-induction and to further explore the underlying mechanism. After model establishment, the mice were treated with miR-27b agomir, miR-27b antagomir, or D-ribofuranosylbenzimidazole (an inhibitor of nuclear factor-E2-related factor 2 [Nrf2]) to determine levels of miR-27b, Nrf2, nuclear factor kappa-light-chain-enhancer of activated B cells nuclear factor κB (NF-κB), p-NF-κB, and heme oxygenase-1 (HO-1). The levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid (BALF) were determined. The results of luciferase activity suggested that Nrf2 was a target gene of miR-27b. It was indicated that the Nrf2 level decreased in lung tissues from ALI mice. The downregulation of miR-27b decreased the levels of IL-1β, IL-6, and TNF-α in BALF of ALI mice. Downregulated miR-27b increased Nrf2 level, thus enhancing HO-1 level along with reduction of NF-κB level as well as the extent of NF-κB phosphorylation in the lung tissues of the transfected mice. Pathological changes were ameliorated in LPS-reduced mice elicited by miR-27b inhibition. The results of this study demonstrate that downregulated miR-27b couldenhance Nrf2 and HO-1 expressions, inhibit NF-κB signaling pathway, which exerts a protective effect on LPS-induced ALI in mice.     

Mechanistic role of microRNA-146a in endotoxin-induced differential cross-regulation of TLR signaling

Human TLRs are critical sensors for microbial components leading to the production of proinflammatory cytokines that are controlled by various mechanisms. Monocytes pretreated with LPS exhibit a state of hyporesponsiveness, referred to as cross-tolerance, to both homologous and heterologous ligands, which play a broader role in innate immunity. To date, LPS-induced cross-tolerance has not been examined regarding microRNA expression kinetics. In this study, THP-1 monocytes treated with various inflammatory ligands showed a continuous amplification of microRNA (miR)-146a over 24 h that is inversely correlated to TNF-α production. In contrast, inhibition of miR-146a showed a reciprocal effect. Thus, the characteristic upregulation of miR-146a in LPS-exposed THP-1 monocytes was studied for cross-tolerance. Strikingly, in LPS-tolerized THP-1 monocytes, only miR-146a showed a continuous overexpression, suggesting its crucial role in cross-tolerance. Similarly, peptidoglycan-primed THP-1 cells showed homologous tolerance associated with miR-146a upregulation. Subsequently, interchangeable differential cross-regulation was observed among non-LPS ligands. TLR2 and TLR5 ligands showed both homologous and heterologous tolerance correlated to miR-146a overexpression. More importantly, inflammatory responses to TLR4, TLR2, and TLR5 ligands were reduced due to knockdown of miR-146a targets IL-1R-associated kinase 1 or TNFR-associated factor 6, suggesting the regulatory effect of miR-146a on these TLRs signaling. Transfection of miR-146a into THP-1 cells caused reduction of TNF-α production, mimicking LPS-induced cross-tolerance. Aside from individual ligands, a whole bacterial challenge in LPS-primed THP-1 monocytes was accompanied by less TNF-α production, which is conversely correlated to miR-146a expression. Our studies have thus demonstrated that miR-146a plays a crucial role for in vitro monocytic cell-based endotoxin-induced cross-tolerance.     

Mitochondrial DNA plays an important role in lung injury induced by sepsis

The effects and mechanisms of mitochondrial DNA (mtDNA) in the development of sepsis-induced lung injury is not well understood. In our present study, we studied the mtDNA effects in sepsis-induced lung injury model, in vitro and in vivo. Compared with the Normal group, the lung histopathological score, the number of positive apoptosis cell, wet/dry (W/D) ratio and TNF-α, IL-1β, and IL-6 concentrations of lipopolysaccharides (LPSs) and mtDNA groups were significantly increased (P < 0.001, respectively). Meanwhile, the lung histopathological score, positive W/D ratio, number of apoptosis cell and tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 concentrations of LPS + mtDNA and small interfering RNA (siRNA)-NC + LPS + mtDNA groups were significantly upregulated compared with those of LPS group (P < 0.05, respectively). However, the lung histopathological score, the number of positive apoptosis cell, W/D ratio and TNF-α, IL-1β, and IL-6 concentrations were significantly improved within the toll-like receptor (TLR9)siRNA + LPS + mtDNA group compared with the LPS group (P < 0.01, respectively). The TLR9, MyD88, and NF-κB proteins or gene expressions of the LPS group and mtDNA group were significantly upregulated compared with those of Normal group by Western blot analysis or immunohistochemistry assay (P < 0.01, respectively), and the TLR9, MyD88, and NF-κB proteins or gene expressions of LPS + mtDNA and siRNA-NC + LPS + mtDNA groups were significantly enhanced compared with those of LPS group (P < 0.05, respectively). However, the TLR9, MyD88, and NF-κB proteins or gene expressions of TLR9siRNA + LPS + mtDNA group were significantly suppressed compared with those of the LPS group (P < 0.01, respectively). In conclusion, mtDNA could provoke lung injury induced by sepsis via regulation of TLR9/MyD88/NF-κB pathway in vitro and in vivo.     

Angiotensin II type-1 receptor antagonist attenuates LPS-induced acute lung injury

Angiotensin II is able to trigger inflammatory responses through an angiotensin II type 1 (AT1) receptor. The role of AT1 receptor in acute lung injury (ALI) is poorly understood. Mice were randomly divided into three groups (n = 40 each groups): NS group; LPS group (2 mg/kg LPS intratracheally); and LPS + ZD 7155 group, 10 mg/kg ZD 7155 (an AT1 receptor antagonist) intraperitoneally 30 min prior to LPS exposure. Samples from the lung were isolated and assayed for histopathology analyses or proinflammatory gene expressions, angiotensin II receptors expressions and nuclear factors activities. LPS exposure resulted in severe ALI, elevated levels of TNF-α and IL-1β mRNA expressions, and increased activities of NF-κB and activated protein (AP)-1. Upregulation of AT1 receptor and down-regulation of AT2 receptor were also observed after LPS challenge. Pretreatment with ZD 7155 significantly inhibited the increase of AT1 receptor expression and upregulated AT2 receptor expression. ZD 7155 also reduced the mRNA expression of TNF-α and IL-1β, inhibited the activation of NF-κB and AP-1, and improved lung histopathology. These findings suggest that antagonism of AT1 receptor inhibits the activation of NF-κB and AP-1 in the lung, which may mediate the release of TNF-α and IL-1β and contribute to LPS-induced ALI.     

幽门螺杆菌感染诱导microRNA-146a表达的机制研究

目的：探讨幽门螺杆菌（H.pylori）感染引起人胃上皮细胞microRNA-146a（miR-146a）上调的分子机制。方法：分别用H.pylori重组蛋白、全菌蛋白、培养上清、感染相关炎性因子（IL-8、TNF-α、IL-1β）以及TLR配体刺激人胃上皮细胞,检测细胞miR-146a的表达;通过生物信息学软件预测和荧光素酶实验鉴定miR-146a启动子,分析诱导表达的相关信号通路。结果：除H.pylori感染相关炎性因子IL-8、TNF-α、IL-1β能够明显诱导miR-146a表达上调（P〈0.01）外,其他刺激因素均不能诱导miR-146a的显著表达;当采用RNAi技术将IL-8、TNF-α、IL-1β分别沉默,检测H.pylori诱导miR-146a表达时,各沉默组与对照组均无显著差异。软件预测显示miR-146a启动子序列中含有多个NF-κB结合位点;H.pylori能够显著增加miR-146a启动子荧光素酶报告载体的相对荧光素酶值;当启动子序列中的NF-κB结合位点发生突变,其相对荧光素酶比值显著降低（P〈0.05）。结论：H.pylori感染相关炎性因子IL-8、TNF-α、IL-1β能够诱导miR-146a表达明显上调;NF-κB信号通路在H.pylori感染诱导miR-146a的表达中发挥关键作用。     

Protective effect of miR-138-5p inhibition modified human mesenchymal stem cell on ovalbumin-induced allergic rhinitis and asthma syndrome

The objective of the study is to evaluate the protective effects of human mesenchymal stem cells (hMSCs) modified with miR-138-5p inhibitor against the allergic rhinitis and asthma syndrome (ARAS). MiR-138-5p or negative control was transfected into hMSCs, and fluorescence-activated cell sorting was used to evaluate hMSC surface markers. Quantitative real-time PCR (qRT-PCR) was used to evaluate miR-138-5p, SIRT1, caspase-3, IL-6, IL-1β and TNF-α levels after TNF-α and IL-6 stimulations. hMSCs with or without miR-138-5p inhibition was intranasally administered into ARAS mice (n = 10 each group), followed by monitoring sneezing and nasal rubbing events to evaluate the allergic symptoms. Histamine, ovalbumin-specific IgE, IgG2a, IgG1 and LTC4 release were monitored in the serum and nasal lavage fluid using enzyme-linked immunosorbent assay. Expression of SIRT1 and HMGB1/TLR4 pathway in nasal mucosa was assessed. After miR-138-5p inhibitor transfection, the hMSC lineage was preserved. Binding between SIRT1 and miR-138-4p was observed, and miR-138-5p inhibition led to upregulation of SIRT1. Inhibition of miR-138-5p led to attenuated inflammatory responses of hMSCs upon TNF-α and IL-6 stimulation, and allergic symptoms in mice, as well as histamine and ovalbumin-specific IgG release. hMSCs with miR-138-5p inhibition showed characteristics of activated SIRT1 and inhibited HMGB1/TLR4 pathway. Inhibition of miR-138-5p in hMSCs enhanced its effects in attenuating inflammatory responses and allergic reaction in the ARAS model, which is presumably regulated by SIRT1 and the HMGB1/TLR4 pathway.     

Salmosan,a β-galactomannan-rich product,in combination with Lactobacillus plantarum contributes to restore intestinal epithelial barrier function by modulation of cytokine production

Mannan-oligosaccharides (MOSs) are mannose-rich substrates with several intestinal health-promoting properties. The aim of this study was to investigate the potential capacity of Salmosan (S-βGM), a β-galactomannan-rich MOS product, to restore epithelial barrier function independently from its capacity to reduce bacterial invasion. In addition, the combination of S-βGM with the proven probiotic Lactobacillus plantarum (LP) was also tested. Paracellular permeability was assessed by transepithelial electrical resistance (TER) in co-cultures of Caco-2 cells and macrophages (differentiated from THP-1 cells) stimulated with LPS of Salmonella Enteritidis and in Caco-2 cell cultures stimulated with TNF-α in the absence or presence of 500 μg/ml S-βGM, LP (MOI 10) or a combination of both. In both culture models, TER was significantly reduced up to 25% by LPS or TNF-α stimulation, and the addition of S-βGM or LP alone did not modify TER, whereas the combination of both restored TER to values of nonstimulated cells. Under LPS stimulation, TNF-α production was significantly increased by 10-fold, whereas IL-10 and IL-6 levels were not modified. The combination of S-βGM and LP reduced TNF-α production to nonstimulated cell values and significantly increased IL-10 and IL-6 levels (5- and 7.5-fold, respectively). Moreover, S-βGM has the capacity to induce an increase of fivefold in LP growth. In conclusion, we have demonstrated that S-βGM in combination with LP protects epithelial barrier function by modulation of cytokine secretion, thus giving an additional value to this MOS as a potential symbiotic.     

Retracted: Notoginsenoside R1 protects WI-38 cells against lipopolysaccharide-triggered injury via adjusting the miR-181a/TLR4 axis

Notoginsenoside R1 (NGR1) is a neoteric phytoestrogen extracted from Panax notoginseng, and possesses comprehensive pharmacological functions in multitudinous ailments. But, whether NGR1 is utilized in neonatal pneumonia is not clear. This research study aspired to disclose the protective activity of NGR1 in neonatal pneumonia. WI-38 cells were co-stimulated with NGR1 and lipopolysaccharide (LPS, 10 ng/mL), CCK-8 and flow cytometry assays were implemented for cell viability and apoptosis assessment. Real-time quantitative plymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blot analysis were executed for inflammatory cytokine determination. MicroRNA-181a (miR-181a) expression was evaluated through RT-qPCR, simultaneously, the impact of miR-181a was estimated in NGR1 and LPS co-managed cells. Dual luciferase report assay was performed to disclose the relation between miR-181a and Toll-like receptor 4 (TLR4). The nuclear factor-κB (NF-κB) and TAK1/JNK pathways were ultimately appraised. We found that NGR1 decreased cell viability, evoked apoptosis and impeded interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) expression and secretions in LPS-managed WI-38 cells. MiR-181a expression was enhanced by NGR1, and miR-181a inhibition inverted the impacts of NGR1 in LPS-managed WI-38 cells. Besides, TLR4 was predicted to be a firsthand direct target of miR-181a. Furthermore, NGR1 hindered NF-κB and TAK1/JNK pathways through modulating TLR4. These discoveries disclosed the fact that NGR1 protected WI-38 cells against LPS-triggered injury via adjusting the miR-181a/TLR4 and NF-κB and TAK1/JNK pathways.     

One functional variant in the 3′-untranslated region of TLR4 is associated with the elevated risk of ventilator-associated pneumonia in the patients with chronic obstructive pulmonary disease

The aim of this study was to identify the association polymorphism (rs11536889) in the 3′-untranslated region (3′-UTR) of Toll-like receptors 4 (TLR4) and the risk for ventilator-associated pneumonia (VAP). miRNA database online and luciferase assays were used to validate TLR4 as the target gene of miR-1236. Enzyme-linked immunosorbent assay analysis and western blot were used to analyze the level of TLR4 in different genotype groups. In the present study, miR-1236 was predicted to bind to the rs11536889 G allele rather than the rs11536889 C allele, which was further confirmed by the luciferase activity suppressed by a fragment of 3′-UTR containing the rs11536889 G allele induced by lipopolysaccharide (LPS) and interleukin-6 (IL-6). Bronchial epithelial cells isolated from participants genotyped as GG, GC, and CC, with no remarkable difference in TLR4 messenger RNA (mRNA) levels were observed among these genotype groups. After stimulating by LPS, a TLR4 ligand, the CC-genotyped cells expressed higher levels of IL-8, IL-6, and tumor necrosis factor alpha (TNF-α) on their surfaces than cells with the other genotypes. Finally, the western blot analysis results showed that the expression level of IL-8, IL-6, and TNF-α protein was much higher in the CC group than the GC and GG groups subsequent to stimulation by LPS, and the IL-8, IL-6, and TNF-α protein levels in the GC were grouped much lower compared with the GG group. These findings indicated the regulatory association of miR-1236 with TLR4 and the abnormal expression of TLR4 caused by the presence of rs11536889 in the 3′-UTR of mRNA, which interfere with its interaction with the miR-1236, contributing to the risk of VAP.     

Upregulation of microRNA-146a was not accompanied by downregulation of pro-inflammatory markers in diabetic kidney

The present study was designed to evaluate whether microRNA-146a and its adapter proteins (TRAF6 and IRAK1) are involved in the pathogenesis of diabetes-induced kidney damage. Male Sprague–Dawley rats were divided into control and diabetic groups (n = 6 in each). Diabetes was induced by injection of streptozotocin (55 mg/kg; i.p.) in 12 h fasted rats. Diabetic kidney damage was diagnosed by renal hypertrophy, thickened glomerular basement membrane, widened filtration slits, mesangial expansion, as well as by elevated levels of blood urea and creatinine in diabetic rats 2 months after induction of diabetes. While the expression of NF-κB mRNA and miR-146a were increased in diabetic kidney compared to the sham controls (p < 0.01 for both comparisons), the mRNA levels of IRAK1 and TRAF6 did not statistically reduce. The NF-κB activity and the concentrations of TNF-α, IL-6 and IL-1β in the kidney of diabetic rats were higher than the kidney of controls (p < 0.05 for TNF-α and NF-κB; p < 0.01 for IL-6 and IL-1β). Our results indicate that the upregulation of miR-146a was not accompanied by downregulation of inflammatory mediators in diabetic kidney. It is possible that a defect in the miR-146a-mediated negative loop provides a situation for sustained activation of NF-κB and its targets to promote cells toward abnormalities.     

MicroRNA-27a Negatively Modulates the Inflammatory Response in Lipopolysaccharide-Stimulated Microglia by Targeting TLR4 and IRAK4

microRNA, a family of small non-coding RNA, plays significant roles in regulating gene expression, mainly via binding to the 3′-untranslated region of target genes. Although the role of miRNA in regulating neuroinflammation via the innate immune pathway has been studied, its role in the production of inflammatory mediators during microglial activation is poorly understood. In this study, we investigated the effect of miR-27a on lipopolysaccharide (LPS)-induced microglial inflammation. miR-27a expression was found to be rapidly decreased in microglia by real-time polymerase chain reaction (real-time PCR) after LPS stimulation. Over-expression of miR-27a significantly decreased the production of inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO), whereas knockdown of miR-27a increased the expression of these inflammatory factors. We also demonstrated by loss- and gain-of-function studies that miR-27a directly suppressed the expression of toll-like receptor 4 (TLR4) and interleukin-1 receptor-associated kinase 4 (IRAK4)—a pivotal adaptor kinase in the TLR4/MyD88 signaling pathway—by directly binding their 3′-UTRs: knocking down TLR4 or IRAK4 in microglia significantly decreased TLR4 or IRAK4 expression and inhibited the downstream production of inflammatory mediators. Moreover, the inflammatory cytokines IL-6 and IL-1β were regulated by IRAK4, whereas TNF-α and NO were more dependent on TLR4 activation. Thus, miR-27a might regulate the LPS-induced production of inflammatory cytokines in microglia independently of TLR4 and IRAK4. Taken together, our results suggest that miR-27a is associated with microglial activation and the inflammatory response.     

Long non-coding RNA HCG18 promotes M1 macrophage polarization through regulating the miR-146a/TRAF6 axis,facilitating the progression of diabetic peripheral neuropathy

Diabetic peripheral neuropathy (DPN) is one of the most important complications in diabetes mellitus (DM), which has been reported to be modulated by long non-coding RNAs (lncRNAs). The purpose of the current study is to explore the regulatory mechanism of lncRNA HCG18 on DPN in vitro. The expression of lncRNA HCG18, miR-146a, TRAF6, CD11c, and iNOS was detected by qRT-PCR. Through Enzyme-linked immunosorbent assay, the levels of inflammatory factors (TNF-α, IL-1β, and IL-6) were determined. M1 macrophage polarization was measured by flow cytometry analysis. The interactions between miR-146a and HCG18/TRAF6 were predicted by Starbase/Targetscan software and verified by the dual luciferase reporter assay. Western blot assay was performed to determine the protein expression of TRAF6. LncRNA HCG18 was highly expressed in DPN model and HG-induced macrophages. The levels of inflammatory factors (TNF-α, IL-1β, and IL-6) were elevated in DPN model. The expression of M1 markers (CD11c and iNOS) was visibly up-regulated in DPN model and was positively correlated with HCG18 expression. LncRNA HCG18 facilitated M1 macrophage polarization. In addition, miR-146a was identified as a target of lncRNA HCG18. Overexpression of miR-146a reversed the promoting effect of HCG18 on M1 macrophage polarization. Simultaneously, TRAF6 was a target gene of miR-146a TRAF6 expression was positively modulated by HCG18 and was negatively modulated by miR-146a. Down-regulation of TRAF6 reversed the promoting effect of HCG18 on M1 macrophage polarization. LncRNA HCG18 promotes M1 macrophage polarization via regulating the miR-146a/TRAF6 axis, facilitating the progression of DPN. This study provides a possible therapeutic strategy for DPN.

     

Anti-inflammatory effects of miRNA-146a induced in adipose and periodontal tissues

MicroRNA (miRNA) plays an important role in diverse cellular biological processes such as inflammatory response, differentiation and proliferation, and carcinogenesis. miR-146a has been suggested as a negative regulator of the inflammatory reaction. Although, it has been reported as expressed in inflamed adipose and periodontal tissues, however, miR-146a's inhibitory effects against inflammatory response in both the tissues, are not well understood. Therefore, in this study, the inhibitory effects of miR-146a on both adipose and periodontal inflammation, was investigated. In vitro study has revealed that miR-146a transfection into either adipocytes or gingival fibroblasts, has resulted in a reduced cytokine gene expression, observed on co-culturing the cells with macrophages in the presence of lipopolysaccharides (LPS), in comparison to the control miRNA transfected. Similarly, miR-146a transfection into macrophages resulted in a reduced expression of TNF-α gene and protein in response to LPS stimulation. In vivo study revealed that a continuous intravenous miR-146a administration into mice via tail vein, protected the mice from developing high-fat diet-induced obesity and the inflammatory cytokine gene expression was down-regulated in both adipose and periodontal tissues. miR-146a appeared to be induced by macrophage-derived inflammatory signals such as TNF-α by negative feed-back mechanism, and it suppressed inflammatory reaction in both adipose and periodontal tissues. Therefore, miR-146a could be suggested as a potential therapeutic molecule and as a common inflammatory regulator for both obesity-induced diabetes and related periodontal diseases.     

miR-146b overexpression ameliorates lipopolysaccharide-induced acute lung injury in vivo and in vitro

Acute respiratory distress syndrome (ARDS) is a type of acute lung injury (ALI), which causes high morbidity and mortality. So far, effective clinical treatment of ARDS is still limited. Recently, miR-146b has been reported to play a key role in inflammation. In the present study, we evaluated the functional role of miR-146b in ARDS using the murine model of lipopolysaccharide (LPS)-induced ALI. The miR-146b expression could be induced by LPS stimulation, and miR-146b overexpression was required in the maintenance of body weight and survival of ALI mice; after miR-146b overexpression, LPS-induced lung injury, pulmonary inflammation, total cell and neutrophil counts, proinflammatory cytokines, and chemokines in bronchial alveolar lavage (BAL) fluid were significantly reduced. The promotive effect of LPS on lung permeability through increasing total protein, albumin and IgM in BAL fluid could be partially reversed by miR-146b overexpression. Moreover, in murine alveolar macrophages, miR-146b overexpression reduced LPS-induced TNF-α and interleukin (IL)-1β releasing. Taken together, we demonstrated that miR-146b overexpression could effectively improve the LPS-induced ALI; miR-146b is a promising target in ARDS treatment.     

MiR-375-3p alleviates the severity of inflammation through targeting YAP1/LEKTI pathway in HaCaT cells

ABSTRACT

Atopic dermatitis (AD) is a relapsing inflammatory skin disease with a complicated pathogenesis. This study aimed to investigate whether miR-375-3p could regulate AD through the Yes-associated protein 1 (YAP1) pathway. In this study, inflammatory response was induced by TNF-α and IFN-γ administration in HaCaT cells. We found that viability and inflammatory factor release, including interleukin-1β (IL-1β) and IL-6, were negatively related to miR-375-3p expression in HaCaT cells. We also found that YAP1 overexpression down-regulated lympho-epithelial Kazal type inhibitor (LEKTI) levels and aggravated viability and inflammation in TNF-α and IFN-γ-treated HaCaT cells. Dual-luciferase reporter assay proved the targeted binding of miR-375-3p and YAP1 3?-UTR. Additionally, the protective effect of miR-375-3p on inflammatory response in TNF-α and IFN-γ-treated HaCaT cells could be impeded by YAP1 overexpression. Collectively, our results suggested that miR-375-3p could modulate HaCaT cell viability and inflammation through the YAP1/LEKTI pathway.     

Divergent intracellular pathways regulate interleukin-1β-induced miR-146a and miR-146b expression and chemokine release in human alveolar epithelial cells

We have previously reported that IL-β-induced miR-146a and miR-146b expression negatively regulates IL-8 and RANTES release in human alveolar A549 epithelial cells. To determine the intracellular pathways that regulate this response, we demonstrate IL-1β-induced activation of the nuclear factor (NF)-κB, extracellular regulated kinase (ERK)-1/2, c-jun N-terminal kinase (JNK)-1/2 and p38 mitogen activated kinase (MAP) kinase pathways. Subsequent pharmacological studies show that IL-1β-induced miR-146a, IL-8 and RANTES production was regulated via NF-κB and JNK-1/2 whilst miR-146b expression was mediated via MEK-1/2 and JNK-1/2. These divergent intracellular pathways likely explain the differential expression and biological action of the miR-146 isoforms.     

MicroRNA-513a-5p mediates TNF-α and LPS induced apoptosis via downregulation of X-linked inhibitor of apoptotic protein in endothelial cells

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that have emerged as one of the central players of gene expression regulation. Endothelial cell apoptosis plays a fundamental role in the development of atherosclerosis. This study was designed to determine the effect of miR-513a-5p on apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs were treated with tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) and miR-513a-5p expression levels were determined. MiR-513a-5p target gene indentification, validation, and signalling pathways were investigated. Treatment of HUVECs with TNF-α and LPS up-regulated miR-513a-5p expressions more than 2-fold compared to control (P < 0.05). Inhibition of miR-513a-5p by antisense (AS) miR-513a-5p reversed TNF-α and LPS induced apoptosis (P < 0.01). Transfection of HUVECs with miR-513a-5p mimics also induced apoptosis (P < 0.01). Treatment of HUVECs with TNF-α and LPS attenuated X-linked inhibitor of apoptosis (XIAP) while increased caspase-3 expression, poly ADP-ribose polymerase (PARP) cleavage, and p53 expression. These effects were reversed by inhibition of miR-513a-5p. Of those miR-513a-5p candidate target genes, we identified and validated XIAP as a miR-513a-5p target gene. Targeting of the XIAP 3′-untranslated region by miR-513a-5p using luciferase reporter assay resulted in attenuated luciferase activity. Transfection of HUVECs with AS miR-513a-5p increased XIAP protein expression while miR-513a-5p mimics attenuated XIAP expression. These results together suggest that miR-513a-5p mediates TNF-α and LPS induced apoptosis via downregulation of XIAP in HUVECs.