IL-1β promotes hypoxic vascular endothelial cell proliferation through the miR-24-3p/NKAP/NF-κB axis

Purpose: Our previous data indicated that miR-24-3p is involved in the regulation of vascular endothelial cell (EC) proliferation and migration/invasion. However, whether IL-1β affects hypoxic HUVECs by miR-24-3p is still unclear. Therefore, the present study aimed to investigate the role and underlying mechanism of interleukin 1β (IL-1β) in hypoxic HUVECs.Methods: We assessed the mRNA expression levels of miR-24-3p, hypoxia-inducible factor-1α (HIF1A) and NF-κB-activating protein (NKAP) by quantitative real-time polymerase chain reaction (RT-qPCR). ELISA measured the expression level of IL-1β. Cell counting kit-8 (CCK-8) assays evaluated the effect of miR-24-3p or si-NKAP+miR-24 on cell proliferation (with or without IL-1β). Transwell migration and invasion assays were used to examine the effects of miR-24-3p or si-NKAP+miR-24-3p on cell migration and invasion (with or without IL-1β). Luciferase reporter assays were used to identify the target of miR-24-3p.Results: We demonstrated that in acute myocardial infarction (AMI) patient blood samples, the expression of miR-24-3p is down-regulated, the expression of IL-1β or NKAP is up-regulated, and IL-1β or NKAP is negatively correlated with miR-24-3p. Furthermore, IL-1β promotes hypoxic HUVECs proliferation by down-regulating miR-24-3p. In addition, IL-1β also significantly promotes the migration and invasion of hypoxic HUVECs; overexpression of miR-24-3p can partially rescue hypoxic HUVECs migration and invasion. Furthermore, we discovered that NKAP is a novel target of miR-24-3p in hypoxic HUVECs. Moreover, both the overexpression of miR-24-3p and the suppression of NKAP can inhibit the NF-κB/pro-IL-1β signaling pathway. However, IL-1β mediates suppression of miR-24-3p activity, leading to activation of the NKAP/NF-κB pathway. In conclusion, our results reveal a new function of IL-1β in suppressing miR-24-3p up-regulation of the NKAP/NF-κB pathway.     

Cantharidin inhibits osteosarcoma proliferation and metastasis by directly targeting miR-214-3p/DKK3 axis to inactivate β-catenin nuclear translocation and LEF1 translation

Background: As the leading primary bone cancer in adolescents and children, osteosarcoma patients with metastasis show a five-year-survival-rate of 20-30%, without improvement over the past 30 years. Wnt/β-catenin is important in promoting osteosarcoma development. DKK3 is a Wnt/β-catenin antagonist and predicted to have the specific binding site in 3′-UTR with miR-214-3p.Methods: miR-214-3p and DKK3 levels were investigated in human osteosarcoma tissues and cells by RT-qPCR; the prognostic importance of DKK3 level in osteosarcoma patients was determined with Log-rank test; direct binding between DKK3 with miR-214-3p was identified with targetscan; anti-osteosarcoma mechanism of cantharidin was investigated by miR-214-3p silence/over-expression with or without cantharidin treatment, and nuclear/cytoplasmic protein assay in osteosarcoma cells.Results: Down-regulated DKK3 indicated poor prognosis of osteosarcoma patients. Up-regulated miR-214-3p promoted proliferation and migration, while suppressed apoptosis of osteosarcoma cells by increasing β-catenin nuclear translocation and LEF1 translation via degradation of DKK3. Cantharidin suppressed viabilities, migration and invasion, while promoted cell cycle arrest and apoptosis in 143B and U-2 OS cells via down-regulating miR-214-3p to up-regulate DKK3, thus inhibited p-GSK-3β expression, β-catenin nuclear translocation and LEF1 translation. Meanwhile, cantharidin inhibited tumor growth in xenograft-bearing mice with 143B cell injection in tibia.Conclusion: miR-214-3p mediated Wnt/β-catenin/LEF1 signaling activation by targeting DKK3 to promote oncogenesis of osteosarcoma; cantharidin inhibited proliferation and metastasis of osteosarcoma cells via down-regulating miR-214-3p to up-regulate DKK3 and decrease β-catenin nuclear translocation, indicating that cantharidin may be a prospective candidate for osteosarcoma treatment by targeting miR-214-3p/DKK3/β-catenin signaling.     

Study on the Mechanism of BMSCs in Regulating NF-κB Signal Pathway by Targeting miR-449a to Improve the Inflammatory Response to Peripheral Nerve Injury

Objective:To evaluate the mechanism of Bone Marrow Mesenchymal Stem Cells (BMSCs) in regulating NF-κB signal pathway by targeting miR-449a.Methods:Stem cells were transfected by over-expressing and inhibiting miR-449a to detect the levels and viability of miR-449a in stem cells after transfection. Stem cells and neurons were co-cultured in vitro to evaluate the in vitro mechanism of stem cells over-expressing miR-449a on neurons.Results:After the addition of neurons, the neuronal activity of miR-449a over-expression group increased significantly, the expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, and the inflammatory cytokines (TNF-α and IL-1β) decreased significantly (P<0.05). In vivo experiments in rats also showed that rats were unresponsive, did not chirp or elude after being stimulated. After stem cell therapy, the weight and response of rats gradually returned to normal levels. miR-449a expression significantly increased in the stem cell + miR-449a over-expression group, expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, inflammatory cytokines (TNF-α and IL-1β) significantly decreased, and cell activity significantly increased (P<0.05).Conclusions:BMSCs can modulate NF-κB signaling pathway by targeting miR-449a, so as to reduce the inflammatory response to peripheral nerve injury and repair nerve injury.     

Inhibition of Kruppel-like factor 7 attenuates cell proliferation and inflammation of fibroblast-like synoviocytes in rheumatoid arthritis through nuclear factor κB and mitogen-activated protein kinase signaling pathway

Rheumatoid arthritis (RA) is an autoimmune disease, which can lead to joint inflammation and progressive joint destruction. Kruppel-like factor 7 (KLF7) is the member of KLF family and plays an important role in multiple biological progresses. However, its precise roles in RA have not been described. Present study aimed to investigate the role of KLF7 in RA-fibroblast-like synoviocytes (FLSs). Data showed that KLF7 expression was obviously upregulated in synovial tissues of rats with adjuvant-induced arthritis. Functional studies demonstrated that the loss of KLF7 may suppress cell proliferation and the expression of pro-inflammatory factors (IL-6, IL-1β, IL-17A) and matrix metalloproteinase (MMP-1, MMP-3, MMP-13) in FLSs through the inhibition of phosphorylation of nuclear factor κB (NF-κB) p65 and JNK. We further showed that miR-9a-5p specifically interacts with KLF7 to negatively regulate the expression of KLF7 in RA-FLSs. Taken together, our results demonstrated that KLF7 which targeted by miR-9a-5p might participate in the pathogenesis of RA by promoting cell proliferation, pro-inflammatory cytokine release and MMP expression through the activation of NF-κB and JNK pathways in RA-FLSs. Hence, KLF7 could be a novel target for RA therapy.     

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3′ untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.     

Dok5 regulates proliferation and differentiation of osteoblast via canonical Wnt/β-catenin signaling

Objective:In bone tissue engineering, the use of osteoblastic seed cells has been widely adopted to mediate the osteogenic differentiation so as to prompt bone regeneration and repair. It is hypothesized that Dok5 can regulate the proliferation and differentiation of osteoblasts. In this study, the role of Dok5 in osteoblast proliferation and differentiation was investigated.Methods:A lentiviral vector to silence Dok5 was transferred to C3H10, 293T and C2C12 cells. CCK-8 assay was used to detect the cell proliferation. Cells were stained by ALP and AR-S staining. Western blot and RT-PCR were used to detect the expression levels of related factors.Results:Dok5 expression level was gradually up-regulated during the osteoblast differentiation. Dok5 silencing down-regulated the expression levels of osteogenic biosignatures OPN, OCN, and Runx2 and suppressed the osteogenesis. Additionally, the osteoblast proliferation and canonical Wnt/β-catenin signaling were suppressed upon Dok5 knockdown, β-catenin expression level was significantly down-regulated in the knockdown group, while the expression levels of GSK3-β and Axin, negative regulators in the Wnt signaling pathway, were up-regulated. Furthermore, overexpression of Dok5 promoted the proliferation and osteogenesis and activated the canonical Wnt/β-catenin signaling pathway.Conclusion:Dok5 may regulate the osteogenic proliferation and differentiation via the canonical Wnt/β-catenin signaling pathway.     

Butyrate inhibits IL-1β-induced inflammatory gene expression by suppression of NF-κB activity in pancreatic beta cells

Cytokine-induced beta cell dysfunction is a hallmark of type 2 diabetes (T2D). Chronic exposure of beta cells to inflammatory cytokines affects gene expression and impairs insulin secretion. Thus, identification of anti-inflammatory factors that preserve beta cell function represents an opportunity to prevent or treat T2D. Butyrate is a gut microbial metabolite with anti-inflammatory properties for which we recently showed a role in preventing interleukin-1β (IL-1β)-induced beta cell dysfunction, but how prevention is accomplished is unclear. Here, we investigated the mechanisms by which butyrate exerts anti-inflammatory activity in beta cells. We exposed mouse islets and INS-1E cells to a low dose of IL-1β and/or butyrate and measured expression of inflammatory genes and nitric oxide (NO) production. Additionally, we explored the molecular mechanisms underlying butyrate activity by dissecting the activation of the nuclear factor-κB (NF-κB) pathway. We found that butyrate suppressed IL-1β-induced expression of inflammatory genes, such as Nos2, Cxcl1, and Ptgs2, and reduced NO production. Butyrate did not inhibit IκBα degradation nor NF-κB p65 nuclear translocation. Furthermore, butyrate did not affect binding of NF-κB p65 to target sequences in synthetic DNA but inhibited NF-κB p65 binding and RNA polymerase II recruitment to inflammatory gene promoters in the context of native DNA. We found this was concurrent with increased acetylation of NF-κB p65 and histone H4, suggesting butyrate affects NF-κB activity via inhibition of histone deacetylases. Together, our results show butyrate inhibits IL-1β-induced inflammatory gene expression and NO production through suppression of NF-κB activation and thereby possibly preserves beta cell function.     

SNHG1 represses the anti-cancer roles of baicalein in cervical cancer through regulating miR-3127-5p/FZD4/Wnt/β-catenin signaling

As a flavonoid, baicalein exhibits remarkable anti-cancer roles in several cancers. However, the factors regulating the antitumorigenic roles of baicalein in cervical cancer remain undefined. Here, we revealed that long noncoding RNA SNHG1 is implicated in the tumor-suppressive roles of baicalein. Functional assays demonstrated that ectopic expression of SNHG1 attenuates the roles of baicalein in repressing cervical cancer cell viability, inducing apoptosis, and repressing migration. SNHG1 silencing promotes the tumor-suppressive roles of baicalein in cervical cancer cell viability, apoptosis, and migration. Xenograft assays showed that SNHG1 reverses the tumor-suppressive roles of baicalein in repressing cervical cancer growth in vivo. Mechanistic investigations revealed that SNHG1 directly binds miR-3127-5p and up-regulates FZD4, a target of miR-3127-5p. Via regulating miR-3127-5p/FZD4, SNHG1 activates Wnt/β-catenin signaling. Moreover, SNHG1 reverses the repressive role of baicalein on Wnt/β-catenin signaling. The effect of SNHG1 on the antitumorigenic process of baicalein was abolished by Wnt/β-catenin signaling inhibitor ICG-001. Together, our observations demonstrated that SNHG1 represses the tumor-suppressive roles of baicalein in cervical cancer through regulating miR-3127-5p/FZD4/Wnt/β-catenin axis, and suggested that targeting SNHG1 represents a potential strategy to enhance the tumor-suppressive roles of baicalein in cervical cancer.Impact statementBaicalein exhibits anti-cancer roles in several cancers. However, the factors influencing the antitumorigenic efficiencies of baicalein in CC remain largely unclear. Here, we provide convincing evidences that lncRNA SNHG1 attenuates the tumor-suppressive roles of baicalein in CC cell viability, apoptosis, migration, and CC tumor growth. This study further demonstrates that the influences of SNHG1 in the antitumorigenic process of baicalein are achieved through modulating the miR-3127-5p/FZD4Wnt/β-catenin axis. SNHG1 attenuates the repressive role of baicalein on Wnt/β-catenin. Therefore, SNHG1 is a novel modulator of the tumor-suppressive roles of baicalein and SNHG1 represents a therapeutic intervention target to reinforce the tumor-suppressive roles of baicalein in CC.     

Genistein-3′-sodium sulfonate Attenuates Neuroinflammation in Stroke Rats by Down-Regulating Microglial M1 Polarization through α7nAChR-NF-κB Signaling Pathway

Microglial M1 depolarization mediated prolonged inflammation contributing to brain injury in ischemic stroke. Our previous study revealed that Genistein-3′-sodium sulfonate (GSS) exerted neuroprotective effects in ischemic stroke. This study aimed to explore whether GSS protected against brain injury in ischemic stroke by regulating microglial M1 depolarization and its underlying mechanisms. We established transient middle cerebral artery occlusion and reperfusion (tMCAO) model in rats and used lipopolysaccharide (LPS)-stimulated BV2 microglial cells as in vitro model. Our results showed that GSS treatment significantly reduced the brain infarcted volume and improved the neurological function in tMCAO rats. Meanwhile, GSS treatment also dramatically reduced microglia M1 depolarization and IL-1β level, reversed α7nAChR expression, and inhibited the activation of NF-κB signaling in the ischemic penumbra brain regions. These effects of GSS were further verified in LPS-induced M1 depolarization of BV2 cells. Furthermore, pretreatment of α7nAChR inhibitor (α-BTX) significantly restrained the neuroprotective effect of GSS treatment in tMCAO rats. α-BTX also blunted the regulating effects of GSS on neuroinflammation, M1 depolarization and NF-κB signaling activation. This study demonstrates that GSS protects against brain injury in ischemic stroke by reducing microglia M1 depolarization to suppress neuroinflammation in peri-infarcted brain regions through upregulating α7nAChR and thereby inhibition of NF-κB signaling. Our findings uncover a potential molecular mechanism for GSS treatment in ischemic stroke.     

MicroRNA-384 inhibits nasopharyngeal carcinoma growth and metastasis via binding to Smad5 and suppressing the Wnt/β-catenin axis

Nasopharyngeal carcinoma (NPC) is a major otorhinolaryngological disease with limited effective therapeutic options. This work focused on the function of microRNA-384 (miR-384) on the NPC pathogenesis and the molecules involved. miR-384 expression in cancer tissues and cells was detected. Gain- and loss-of-functions of miR-384 were performed to identify its role in NPC progression. The target mRNA of miR-384 was predicted on an online system and validated through a luciferase reporter assay. The activity of Wnt/β-catenin signaling was detected. Consequently, miR-384 was found to be poorly expressed in NPC tissues and cell lines and was linked to unfavorable survival rates in patients. Overexpression of miR-384 in 6-10B cells suppressed growth, migration, invasion and resistance to apoptosis of cells, but inverse trends were presented in C6661 cells where miR-384 was downregulated. miR-384 targeted Smad5 mRNA. Upregulation of Smad5 counteracted the roles of miR-384 mimic in cells. The NPC-inhibiting effects of miR-384 mimic were also blocked by Wnt/β-catenin activation. To conclude, miR-384 targets Smad5 and inactivates the Wnt/β-catenin pathway, which exerts a suppressing role in NPC cell behaviors as well as tumor growth in vivo. The findings may offer novel thoughts into NPC therapy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10616-021-00458-3.     

Anisomycin protects against sepsis by attenuating IκB kinase-dependent NF-κB activation and inflammatory gene expression

Anisomycin is known to inhibit eukaryotic protein synthesis and has been established as an antibiotic and anticancer drug. However, the molecular targets of anisomycin and its mechanism of action have not been explained in macrophages. Here, we demonstrated the anti-inflammatory effects of anisomycin both in vivo and in vitro. We found that anisomycin decreased the mortality rate of macrophages in cecal ligation and puncture (CLP)- and lipopolysaccharide (LPS)-induced acute sepsis. It also declined the gene expression of proinflammatory mediators such as inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β as well as the nitric oxide and proinflammatory cytokines production in macrophages subjected to LPS-induced acute sepsis. Furthermore, anisomycin attenuated nuclear factor (NF)-κB activation in LPS-induced macrophages, which correlated with the inhibition of phosphorylation of NF-κB-inducing kinase and IκB kinase, phosphorylation and IκBα proteolytic degradation, and NF-κB p65 subunit nuclear translocation. These results suggest that anisomycin prevented acute inflammation by inhibiting NF-κB-related inflammatory gene expression and could be a potential therapeutic candidate for sepsis.