Long noncoding RNA ANRIL contributes to the development of ulcerative colitis by miR-323b-5p/TLR4/MyD88/NF-κB pathway

The aim of this study was to investigate the role and possible mechanism of long noncoding RNA ANRIL in the development of ulcerative colitis (UC). The expression of ANRIL in colonic mucosa tissues collected from the sigmoid colon of UC patients and healthy control was determined. Subsequently, fetal human cells (FHCs) were treated with lipopolysaccharide (LPS) to stimulate UC-caused inflammatory injury, followed by detection of the effects of suppression of ANRIL on cell viability, apoptosis and cytokines production in LPS-stimulated FHCs. Moreover, the regulatory relationship between ANRIL and miR-323b-5p as well as the target relationship between miR-323b-5p and TLR4 were investigated. Furthermore, the effects of ANRIL/miR-323b-5p axis on the activation of TLR4/MyD88/NF-κB pathway in LPS-stimulated FHCs were investigated. LncRNA ANRIL was highly expressed in colonic mucosa tissues of UC patients. In addition, LPS markedly induced cell injury in FHC cells (inhibited cell viability and promoted cell apoptosis and cytokine production). Suppression of ANRIL alleviated LPS-induced injury in FHC cells, which was achieved by negatively regulating miR-323b-5p. Moreover, miR-323b-5p negatively regulated TLR4 expression and TLR4 was a target of miR-323b-5p. Knockdown of TLR4 reversed the effects of miR-323b-5p suppression on LPS-induced injury in LPS-stimulated FHCs. Furthermore, the effects of ANRIL on LPS-induced cell injury were achieved by TLR4/MyD88/NF-κB pathway. Our data indicate that suppression of ANRIL may inhibit the development of UC by regulating miR-323b-5p/TLR4/MyD88/NF-κB pathway. ANRIL/miR-323b-5p/TLR4/MyD88/NF-κB pathway may provide a new strategy for UC therapy.     

Heterotrimeric Gi/Go proteins modulate endothelial TLR signaling independent of the MyD88-dependent pathway

The innate immune recognition of bacterial lipopolysaccharide (LPS) is mediated by Toll-like receptor 4 (TLR4) and results in activation of proinflammatory signaling including NF-κB and MAPK pathways. Heterotrimeric G proteins have been previously implicated in LPS signaling in macrophages and monocytes. In the present study, we show that pertussis toxin sensitive heterotrimeric G proteins (Gα(i/o)) are involved in the activation of MAPK and Akt downstream of TLR2, TLR3, and TLR4 in endothelial cells. Gα(i/o) are also required for full activation of interferon signaling downstream of TLR3 and TLR4 but are not required for the activation of NF-κB. We find that Gα(i/o)-mediated activation of the MAPK is independent of the canonical MyD88, interleukin-1 receptor-associated kinase, and tumor necrosis factor receptor-associated factor 6 signaling cascade in LPS-stimulated cells. Taken together, the data presented here suggest that heterotrimeric G proteins are widely involved in TLR pathways along a signaling cascade that is distinct from MyD88-TRAF6.     

miR-382-3p suppressed IL-1β induced inflammatory response of chondrocytes via the TLR4/MyD88/NF-κB signaling pathway by directly targeting CX43

miR-382-3p has been reported to be upregulated in synovial membrane in knee osteoarthritis (OA). Nevertheless, its role in OA remains largely unknown. The aim of this study was to investigate the specific function and mechanisms of miR-382-3p in the course of OA. In this study, human OA chondrocytes were pretreated with interleukin-1β (IL-1β) at 5 ng/ml for 12 hr to stimulate inflammatory response and matrix metalloproteinases (MMPs) expression in chondrocytes. Meanwhile, miR-382-3p was downregulated in IL-1β-stimulated chondrocytes. In addition, we found that miR-382-3p directly interacts with connexin 43 (CX43) and attenuates the increase of cytochrome c oxidase polypeptide II, inducible nitric oxide synthase, and MMP-1/13 that is induced by IL-1β. Furthermore, our observations indicated that miR-382-3p inhibited the expression of Toll-like receptor 4 (TLR4), Myeloid differentiation primary response 88 (MyD88) and nuclear factor κB (NF-κB) in IL-1β-stimulated chondrocytes, while CX43 overexpression could partly reverse these decreases. In conclusion, miR-382-3p participated in OA may through the TLR4/MyD88/NF-κB signaling pathway by directly targeting CX43.     

Leishmania donovani exploits host deubiquitinating enzyme A20, a negative regulator of TLR signaling, to subvert host immune response

TLRs, which form an interface between mammalian host and microbe, play a key role in pathogen recognition and initiation of proinflammatory response thus stimulating antimicrobial activity and host survival. However, certain intracellular pathogens such as Leishmania can successfully manipulate the TLR signaling, thus hijacking the defensive strategies of the host. Despite the presence of lipophosphoglycan, a TLR2 ligand capable of eliciting host-defensive cytokine response, on the surface of Leishmania, the strategies adopted by the parasite to silence the TLR2-mediated proinflammatory response is not understood. In this study, we showed that Leishmania donovani modulates the TLR2-mediated pathway in macrophages through inhibition of the IKK-NF-κB cascade and suppression of IL-12 and TNF-α production. This may be due to impairment of the association of TRAF6 with the TAK-TAB complex, thus inhibiting the recruitment of TRAF6 in TLR2 signaling. L. donovani infection drastically reduced Lys 63-linked ubiquitination of TRAF6, and the deubiquitinating enzyme A20 was found to be significantly upregulated in infected macrophages. Small interfering RNA-mediated silencing of A20 restored the Lys 63-linked ubiquitination of TRAF6 as well as IL-12 and TNF-α levels with a concomitant decrease in IL-10 and TGF-β synthesis in infected macrophages. Knockdown of A20 led to lower parasite survival within macrophages. Moreover, in vivo silencing of A20 by short hairpin RNA in BALB/c mice led to increased NF-κB DNA binding and host-protective proinflammatory cytokine response resulting in effective parasite clearance. These results suggest that L. donovani might exploit host A20 to inhibit the TLR2-mediated proinflammatory gene expression, thus escaping the immune responses of the host.     

Regulation of miRNA-21 by reactive oxygen species-activated ERK/NF-κB in arsenite-induced cell transformation

After acute exposure of cells to arsenic, reactive oxygen species mediate changes in cell behavior, including activation of proliferative signaling. For chronic exposure to arsenic, however, the function of reactive oxygen species in cell transformation remains poorly understood. Although microRNA-21 (miR-21) has been implicated in various aspects of carcinogenesis, its functions and molecular mechanisms in carcinogen-induced tumorigenesis are unclear. The purpose of this study was to determine if miR-21 is involved in arsenite-induced malignant transformation and to characterize the associated signaling pathways. During arsenite-induced transformation of human embryo lung fibroblast (HELF) cells, miR-21 was upregulated, and the extracellular signal-regulated kinase (ERK)/nuclear factor-κB (NF-κB) signal pathway was activated. Moreover, superoxide radical dismutase (a scavenger of superoxide) and catalase (a scavenger of hydroperoxides) blocked the arsenite-induced effects in HELF cells and mouse embryonic fibroblasts. Blockage of ERK by the inhibitor U0126 or inhibition of NF-κB p65 by siRNA or Bay 11-7082 prevented the increases in miR-21 and the decreases in Spry1, Pten, and Pdcd4, the target proteins of miR-21, induced by arsenite. As determined by a ChIP-qPCR assay, NF-κB p65 regulated miR-21 expression by binding directly to the promoter of miR-21. Further, anti-miR-21 downregulated miR-21 expression and prevented the arsenite-induced activation of ERK via the increase in Spry1, indicating that miR-21 has a feedback effect in regulating ERK activation. Overexpression of miR-21 with an miR-21 mimic and feedback activation of ERK and NF-κB via the decrease in Spry1 promoted the malignancy of HELF cells exposed to arsenite, but knockdown of miR-21 with anti-miR-21 and feedback blockage of ERK and NF-κB activation through an increase in Spry1 decreased anchorage-independent growth of arsenite-transformed cells. Thus, the transformation of HELF cells induced by chronic exposure to arsenite is mediated by increased miR-21 expression, which, in turn, is mediated by reactive oxygen species activation of the ERK/NF-κB pathway.     

Bacterial lipoprotein-induced tolerance is reversed by overexpression of IRAK-1

Tolerance to bacterial cell wall components including bacterial lipoprotein (BLP) represents an essential regulatory mechanism during bacterial infection. Reduced Toll-like receptor 2 (TLR2) and IL-1 receptor-associated kinase 1 (IRAK-1) expression is a characteristic of the downregulated TLR signaling pathway observed in BLP-tolerised cells. In this study, we attempted to clarify whether TLR2 and/or IRAK-1 are the key molecules responsible for BLP-induced tolerance. Transfection of HEK293 cells and THP-1 cells with the plasmid encoding TLR2 affected neither BLP tolerisation-induced NF-κB deactivation nor BLP tolerisation-attenuated pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) production, indicating that BLP tolerance develops despite overexpression of TLR2 in these cells. In contrast, overexpression of IRAK-1 reversed BLP-induced tolerance, as transfection of IRAK-1 expressing vector resulted in a dose-dependent NF-κB activation and TNF-α release in BLP-tolerised cells. Furthermore, BLP-tolerised cells exhibited markedly repressed NF-κB p65 phosphorylation and impaired binding of p65 to several pro-inflammatory cytokine gene promoters including TNF-α and interleukin-6 (IL-6). Overexpression of IRAK-1 restored the nuclear transactivation of p65 at both TNF-α and IL-6 promoters. These results indicate a crucial role for IRAK-1 in BLP-induced tolerance, and suggest IRAK-1 as a potential target for manipulation of the TLR-mediated inflammatory response during microbial sepsis.     

miR-145-5p inhibits tumor occurrence and metastasis through the NF-κB signaling pathway by targeting TLR4 in malignant melanoma

Compelling evidence shows that deregulated microRNAs (miRNAs) are important regulators in the progression of melanoma. miR-145-5p has been suggested to exhibit antitumorigenic activity in melanoma. However, the molecular mechanism underlying the biological activity of miR-145-5p in melanoma remains to be further understood. Herein, quantitative real-time polymerase chain reaction was used to examine the miR-145-5p expression in malignant melanoma tissues and cells. The interaction between miR-145-5p and toll-like receptor 4 (TLR4) was explored by bioinformatics analyses, luciferase reporter assay, and Western blot. The effects of miR-145-5p or combined with TLR4 on cell proliferation, colony formation, migration, and invasion abilities were investigated by (4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, colony formation, wound healing, and transwell assays, respectively. The melanoma xenograft tumor models were established to determine the biological activity of miR-145-5p in melanoma in vivo. In addition, the changes of the nuclear factor kappa B (NF-κB) pathway were analyzed by detecting the NF-κB activity and the NF-κB p65 protein level. We observed that the miR-145-5p expression was underexpressed in melanoma tissues and cells. miR-145-5p suppressed the TLR4 expression by binding to its 3′untranslated region in melanoma cells. Moreover, TLR4 overexpression abolished the inhibition of cell proliferation, colony formation, migration, and invasion abilities induced by miR-145-5p in melanoma cells. Meanwhile, miR-145-5p was confirmed to restrain melanoma tumor growth in vivo by targeting TLR4. Furthermore, miR-145-5p overexpression inactivated the NF-κB pathway in melanoma in vitro and in vivo, which was reversed by TLR4 overexpression. We concluded that miR-145-5p hindered the occurrence and metastasis of melanoma cells in vitro and in vivo by targeting TLR4 via inactivation of the NF-κB pathway.     

Atorvastatin suppresses LPS-induced rapid upregulation of Toll-like receptor 4 and its signaling pathway in endothelial cells

In the present study, we tested our hypothesis that atorvastatin exerts its anti-inflammation effect via suppressing LPS-induced rapid upregulation of Toll-like receptor 4 (TLR4) mRNA and its downstream p38, ERK, and NF-κB signaling pathways in human umbilical-vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). TLR4 mRNA expression and its downstream kinase activities induced by LPS alone or atorvastatin + LPS in endothelial cells were quantified using quantitative real-time PCR and enzyme-linked immunosorbent assay. Preincubation of LPS-stimulated endothelial cells with TLR4 siRNA was conducted to identify the target of the anti-inflammatory effects of atorvastatin. Atorvastatin incubation resulted in the reduction of LPS-induced TLR4 mRNA expression, ERK1/2 and P38 MAPK phosphorylation, and NF-κB binding activity. Pretreatment with MEK/ERK1/2 inhibitor PD98059 attenuated atorvastatin + LPS-induced NF-κB activity but had no effect on P38 MAPK phosphorylation. In contrast, pretreatment with P38 MAPK inhibitor SB203580 resulted in upregulation of atorvastatin + LPS-induced ERK1/2 phosphorylation but had no significant effects on NF-κB activity. On the other hand, blocking NF-κB with SN50 produced no effects on atorvastatin + LPS-induced ERK1/2 and P38 MAPK phosphorylation. Moreover, TLR4 gene silencing produced the same effects as the atorvastatin treatment. In conclusion, atorvastatin downregulated TLR4 mRNA expression by two distinct signaling pathways. First, atorvastatin stabilized Iκ-Bα, which directly inhibited NF-κB activation. Second, atorvastatin inactivated ERK phosphorylation, which indirectly inhibited NF-κB activation. Suppression of p38 MAPK by atorvastatin upregulates ERK but exerts no effect on NF-κB.     

E3 ubiquitin ligase tripartite motif 38 negatively regulates TLR-mediated immune responses by proteasomal degradation of TNF receptor-associated factor 6 in macrophages

Activation of TLR signaling in the innate immune cells is critical for the elimination of invading microorganisms. However, uncontrolled activation may lead to autoimmune and inflammatory diseases. In this article, we report the identification of tripartite motif (TRIM) 38 as a negative feedback regulator in TLR signaling by targeting TNFR-associated factor 6 (TRAF6). TRIM38 was induced by TLR stimulation in an NF-κB-dependent manner in macrophages. Knockdown of TRIM38 expression by small interfering RNA resulted in augmented activation of NF-κB and MAPKs, and enhanced expression of proinflammatory cytokines, whereas overexpression of TRIM38 has an opposite effect. As an E3 ligase, TRIM38 bound to TRAF6 and promoted K48-linked polyubiquitination, which led to the proteasomal degradation of TRAF6. Consistently, knockdown of TRIM38 expression resulted in higher protein level of TRAF6 in primary macrophages. Our findings defined a novel function for TRIM38 to prevent excessive TLR-induced inflammatory responses through proteasomal degradation of TRAF6.     

过表达miR-31对结肠炎模型小鼠TLR4/NF-κB信号通路及凋亡蛋白的调控

目的: 探讨miR-31对DSS诱发结肠炎小鼠TLR4/NF-κB信号通路和凋亡相关蛋白的影响。方法: ①小鼠结肠炎实验:用1%葡聚糖硫酸钠(DSS)诱发小鼠溃疡性结肠炎(UC)。14只FVB非转基因小鼠随机分为control组(n=6),DSS组(n=8),16只FVB miR-31转基因小鼠随机分为miR-31过表达组(n=8),miR-31过表达+DSS 组(n=8),DSS溶于水后通过饮水给予小鼠。DSS组和miR-31+DSS组第一周饮用1%DSS水,第二周饮用正常无菌水,第三周饮用1%DSS水,如此5周后造模完成,之后留取小鼠的结肠组织,通过Western blot和IHC检测小鼠结肠组织NF-κB p65、TLR4、Bax、Bcl-2蛋白的表达;TUNEL检测小鼠结肠组织细胞凋亡。②细胞培养实验:在人结肠上皮细胞系HCT 116细胞中通过脂质体转染的方法转染miR-31 mimic和inhibitor,使miR-31过表达或敲低,每组均进行三次重复,48 h后收取细胞,通过Western blot检测NF-κB p65、TLR4蛋白的表达。结果: ①动物实验中,与control组相比,小鼠结肠组织中DSS组和miR-31过表达组NF-κB p65、TLR4蛋白表达水平和凋亡细胞指数均显著升高(P＜0.05或P＜0.01),Bcl-2/Bax比值显著降低(P＜0.05或P＜0.01);且与DSS组相比,miR-31+DSS组NF-κB p65、TLR4蛋白表达水平和凋亡细胞指数也显著升高(P＜0.01),Bcl-2/Bax比值显著降低(P＜0.01)。②细胞实验中,与control组相比, HCT 116细胞过表达miR-31组的NF-κB p65、TLR4蛋白表达水平均显著升高(P＜0.05或P＜0.01),敲低miR-31组的NF-κB p65、TLR4蛋白表达水平下降(P＜0.05)。结论: miR-31通过促进TLR4/NF-κB信号通路和介导肠上皮细胞凋亡促进结肠炎的发展。     

TRAF6 is functional in inhibition of TLR4-mediated NF-κB activation by resveratrol

Resveratrol was suggested to inhibit Toll-like receptor (TLR)4-mediated activation of nuclear factor-κB (NF-κB) and Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-β (TRIF)–(TANK)-binding kinase 1, but the myeloid differentiation primary response gene 88–tumor necrosis factor receptor-associated factor 6 (TRAF6) pathway is not involved in this effect. However, involvement of TRAF6 in this process is still elusive since cross talk between TRIF and TRAF6 has been reported in lipopolysaccharide (LPS)-induced signaling. Using RAW 264.7 macrophages, we determined the effect of resveratrol on LPS-induced TRAF6 expression, ubiquitination as well as activation of mitogen-activated protein (MAP) kinases and Akt in order to elucidate its involvement in TLR4 signaling. LPS-induced transient elevation in TRAF6 mRNA and protein expressions is suppressed by resveratrol. LPS induces the ubiquitination of TRAF6, which has been reported to be essential for Akt activation and for transforming growth factor-β activated kinase-1–NAP kinase kinase 6 (MKK6)-mediated p38 and c-Jun N-terminal kinase (JNK) activation. We found that resveratrol diminishes the effect of LPS on TRAF6 ubiquitination and activation of JNK and p38 MAP kinases, while it has no effect on the activation of extracellular-signal-regulated kinase (ERK)1/2. The effect of resveratrol on MAP kinase inhibition is significant since TRAF6 activation was reported to induce activation of JNK and p38 MAP kinase while not affecting ERK1/2. Moreover, Akt was identified previously as a direct target of TRAF6, and we found that, similarly to MAPKs, phosphorylation pattern of Akt followed the activation of TRAF6, and it was inhibited by resveratrol at all time points. Here, we provide the first evidence that resveratrol, by suppressing LPS-induced TRAF6 expression and ubiquitination, attenuates the LPS-induced TLR4–TRAF6, MAP kinase and Akt pathways that can be significant in its anti-inflammatory effects.     

LncRNA OIP5-AS1 facilitates ox-LDL-induced endothelial cell injury through the miR-98-5p/HMGB1 axis

Cerebrovascular diseases have a high mortality and disability rate in developed countries. Endothelial cell injury is the main cause of atherosclerosis and cerebrovascular disease. Long non-coding RNA (lncRNA) has been proved to participate in the progression of endothelial cell. Our study aimed to develop the function of lncRNA opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in oxidative low-density lipoprotein (ox-LDL)-induced endothelial cell injury. The expression of OIP5-AS1, miR-98-5p and High-mobility group protein box-1 (HMGB1) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry were used to detect the cell proliferation and apoptosis. The levels of cyclinD1, Bcl-2 Associated X Protein (Bax), Cleaved-caspase-3, Toll like receptors 4 (TLR4), phosphorylation of p65 (p-P65), phosphorylation of nuclear factor-kappa B inhibitor α (p-IκB-α) and HMGB1 were measured by Western blot. The concentrations of Interleukin-6 (IL-6), Interleukin-1β (IL-1β) and Tumor necrosis factor-α (TNF-α) were detected by Enzyme-linked immunosorbent assay (ELISA). The production of Reactive oxygen species (ROS), Superoxide Dismutase (SOD) and malondialdehyde (MDA) was detected by the corresponding kit. The targets of OIP5-AS and miR-98-5p were predicted by starBase 3.0 and TargetScan and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The expression of OIP5-AS1 was upregulated, while miR-98-5p was downregulated in ox-LDL-induced human umbilical vein endothelial cells (HUVECs). Functionally, knockdown of OIP5-AS1 induced proliferation and inhibited apoptosis, inflammatory injury and oxidative stress injury in ox-LDL-induced HUVEC cells. Interestingly, miR-98-5p was a target of OIP5-AS1 and miR-98-5p inhibition abolished the effects of OIP5-AS1 downregulation on ox-LDL-induced HUVECs injury. More importantly, miR-98-5p directly targeted HMGB1, and OIP5-AS1 regulated the expression of HMGB1 by sponging miR-98-5p. Finally, OIP5-AS1 regulated the TLR4/nuclear factor-kappa B (NF-κB) signaling pathway through miR-98-5p/HMGB1 axis. LncRNA OIP5-AS1 accelerates ox-LDL-induced endothelial cell injury through regulating HMGB1 mediated by miR-98-5p via the TLR4/NF-κB signaling pathway.

     

miR-140-5p is negatively correlated with proliferation,invasion, and tumorigenesis in malignant melanoma by targeting SOX4 via the Wnt/β-catenin and NF-κB cascades

MicroRNAs (miRNAs) have been validated as critical regulators in the development of melanoma. miR-140 was abnormally downregulated in uveal melanoma samples. However, the expression level and roles of miR-140-5p remain unclear in melanoma for now. We speculate that miR-140-5p is abnormally expressed and may play an important role in melanoma. The expressions of miR-140-5p and SOX4 messenger RNA were determined by quantitative real-time polymerase chain reaction assays. Western blot assays were employed to detect the expression levels of SOX4, Ki67, MMP-2, MMP-7, p-β-catenin, c-Myc, cyclin D1, p65, and IκBα. Luciferase reporter assays were employed to elucidate the interaction between SOX4 and miR-140-5p. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) and transwell invasion assays were applied to evaluate capabilities of cell proliferation and invasion, respectively. Xenograft models of melanoma were established to verify the role and molecular basis of miR-140-5p. Immunohistochemical (IHC) assays were employed to measure the Ki67 and SOX4 at the protein level in xenografted melanoma tissues. Herein, these observations showed that, miR-140-5p was abnormally downregulated in melanoma tissues and cells, while SOX4 was upregulated. miR-140-5p directly targeted SOX4 and inhibited its expression in melanoma cells. miR-140-5p overexpression repressed melanoma cell proliferation and invasion and its effects were partially restored SOX4 overexpression. Moreover, miR-140-5p hindered melanoma growth in vivo by downregulating SOX4. Mechanistically, miR-140-5p suppressed activation of the Wnt/β-catenin and NF-κB pathways by targeting SOX4. Our study concluded that miR-140-5p hindered cell proliferation, invasion, and tumorigenesis by targeting SOX4 via inactivation of the Wnt/β-catenin and NF-κB signaling pathways in malignant melanoma, which provides an underlying molecular mechanism for the treatment for melanoma with miRNAs.     

尼罗罗非鱼traf6基因表达谱及信号转导

肿瘤坏死因子受体相关因子6(TRAF6)是一种重要的接头蛋白,在Toll样受体/白细胞介素-1受体(TLR/IL-1R)超家族所触发的信号通路中起重要作用,与先天性免疫密切相关.文章研究了尼罗罗非鱼(Oreo-chromis niloticus)traf6的表达模式和初步的功能.在健康鱼中,traf6转录本广泛表达于所...