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.     

Regulation of COX-2 expression by miR-146a in lung cancer cells

Prostaglandins are a class of molecules that mediate cellular inflammatory responses and control cell growth. The oxidative conversion of arachidonic acid to prostaglandin H₂ is carried out by two isozymes of cyclooxygenase, COX-1 and COX-2. COX-1 is constitutively expressed, while COX-2 can be transiently induced by external stimuli, such as pro-inflammatory cytokines. Interestingly, COX-2 is overexpressed in numerous cancers, including lung cancer. MicroRNAs (miRNAs) are small RNA molecules that function to regulate gene expression. Previous studies have implicated an important role for miRNAs in human cancer. We demonstrate here that miR-146a expression levels are significantly lower in lung cancer cells as compared with normal lung cells. Conversely, lung cancer cells have higher levels of COX-2 protein and mRNA expression. Introduction of miR-146a can specifically ablate COX-2 protein and the biological activity of COX-2 as measured by prostaglandin production. The regulation of COX-2 by miR-146a is mediated through a single miRNA-binding site present in the 3′ UTR. Therefore, we propose that decreased miR-146a expression contributes to the up-regulation and overexpression of COX-2 in lung cancer cells. Since potential miRNA-mediated regulation is a functional consequence of alternative polyadenylation site choice, understanding the molecular mechanisms that regulate COX-2 mRNA alternative polyadenylation and miRNA targeting will give us key insights into how COX-2 expression is involved in the development of a metastatic condition.     

Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients

Introduction

MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis.

Methods

Total RNA was isolated from peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis, and healthy and disease control individuals, and the expression of miR-146a, miR-155, miR-132, miR-16, and microRNA let-7a was analyzed using quantitative real-time PCR.

Results

Rheumatoid arthritis peripheral blood mononuclear cells exhibited between 1.8-fold and 2.6-fold increases in miR-146a, miR-155, miR-132, and miR-16 expression, whereas let-7a expression was not significantly different compared with healthy control individuals. In addition, two targets of miR-146a, namely tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were similarly expressed between rheumatoid arthritis patients and control individuals, despite increased expression of miR-146a in patients with rheumatoid arthritis. Repression of TRAF6 and/or IRAK-1 in THP-1 cells resulted in up to an 86% reduction in tumor necrosis factor-α production, implicating that normal miR-146a function is critical for the regulation of tumor necrosis factor-α production.

Conclusions

Recent studies have shown that synovial tissue and synovial fibroblasts from patients with rheumatoid arthritis exhibit increased expression of certain microRNAs. Our data thus demonstrate that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts. The increased microRNA expression in rheumatoid arthritis patients is potentially useful as a marker for disease diagnosis, progression, or treatment efficacy, but this will require confirmation using a large and well defined cohort. Our data also suggest a possible mechanism contributing to rheumatoid arthritis pathogenesis, whereby miR-146a expression is increased but unable to properly function, leading to prolonged tumor necrosis factor-α production in patients with rheumatoid arthritis.     

Altered microRNAs in STHdh/Hdh cells: miR-146a targets TBP

We studied expression of 90 miRNAs in STHdh^Q111/Hdh^Q111 cells, a model for Huntington’s disease and compared with that obtained in STHdh^Q7/Hdh^Q7 cells. Fifteen miRNAs were down regulated and 12 miRNAs were up regulated more than 2-fold. Such changes were statistically significant. One hundred and forty-two genes are experimentally known targets of these altered miRNAs. It has been predicted that miR-146a may target Tata Binding Protein (TBP). Using luciferase reporter assays with 3′-UTRs of TBP, over-expression and inhibition of miR-146a, we showed that miR-146a targets TBP. Regulation of TBP by miR-146a may contribute to HD pathogenesis.     

miR-146a and miR-150 promote the differentiation of CD133+ cells into T-lymphoid lineage

MicroRNAs control the genes involved in hematopoietic stem cell (HSCs) survival, proliferation and differentiation. The over-expression of miR-146 and miR-150 has been reported during differentiation of HSCs into T-lymphoid lineage. Therefore, in this study we evaluated the effect of their over-expression on CD133⁺ cells differentiation to T cells. miR-146a and miR-150 were separately and jointly transduced to human cord blood derived CD133⁺ cells (>97 % purity). We used qRT-PCR to assess the expression of CD2, CD3ε, CD4, CD8, CD25, T cell receptor alpha (TCR-α) and Ikaros genes in differentiated cells 4 and 8 days after transduction of the miRNAs. Following the over-expression of miR-146a, significant up-regulation of CD2, CD4, CD25 and Ikaros genes were observed (P < 0.01). On the other hand, over-expression of miR-150 caused an increase in the expression of Ikaros, CD4, CD25 and TCR-α. To evaluate the combinatorial effect of miR-146a and miR-150, transduction of both miRNAs was concurrently performed which led to increase in the expression of Ikaros, CD4 and CD3 genes. In conclusion, it seems that the effect of miR-150 and miR-146a on the promotion of T cell differentiation is time-dependant. Moreover, miRNAs could be used either as substitutes or complements of the conventional differentiation protocols for higher efficiency.     

Impaired miR-146a expression links subclinical inflammation and insulin resistance in Type 2 diabetes

Molecular and Cellular Biochemistry - Type 2 diabetes patients exhibit subclinical inflammation but the regulatory mechanisms are poorly understood. We sought to evaluate the role of miR-146a...     

miR-146b Reverses epithelial-mesenchymal transition via targeting PTP1B in cisplatin-resistance human lung adenocarcinoma cells

Epithelial-mesenchymal transformation (EMT) is associated with drug resistance in human lung adenocarcinoma cells, but its specific mechanism has not been clarified. In this study, we investigated the effect of miRNA-146b on EMT in cisplatin (DDP) resistant human lung adenocarcinoma cells and the corresponding mechanism. Cisplatin resistant (CR) human lung adenocarcinoma cells (A549/DDP and H1299/DDP) were established, and the EMT characteristics and invasion and metastasis ability of CR cells were determined by tumor cell-related biological behavior experiments. The role of miR-146b in EMT of CR cells was determined by in vitro functional test. The targeted binding of miR-146b to protein tyrosine phosphatase 1B (PTP1B) was verified by biological information and double luciferin gene reporting experiments. The effect of miR-146b on tumor growth and EMT phenotype in vivo was investigated by establishing the xenotransplantation mouse model. Compared with the control group, H1299/DDP and A549/DDP cells showed the enhanced EMT phenotypes, invasion and migration ability. Besides, miR-146b was lowly expressed in H1299/DDP and A549/DDP cells. More importantly, overexpressed miR-146b could specifically bind to PTP1B, thus inhibiting the EMT process and ultimately reducing CR in H1299/DDP and A549/DDP cells. Finally, overexpressed miR-146b observably inhibited tumor growth in xenograft model mice and inhibited the EMT phenotype of A549/DDP cells in vivo by regulating the expressions of EMT-related proteins. Overexpressed miR-146b could reverse the EMT phenotype of CR lung adenocarcinoma cells by targeting PTP1B, providing new therapeutic directions for CR of lung adenocarcinoma cells.     

Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells

The embryonic stem cell-specific cell cycle-regulating (ESCC) family of microRNAs (miRNAs) enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. Here we show that the human ESCC miRNA orthologs hsa-miR-302b and hsa-miR-372 promote human somatic cell reprogramming. Furthermore, these miRNAs repress multiple target genes, with downregulation of individual targets only partially recapitulating the total miRNA effects. These targets regulate various cellular processes, including cell cycle, epithelial-mesenchymal transition (EMT), epigenetic regulation and vesicular transport. ESCC miRNAs have a known role in regulating the unique embryonic stem cell cycle. We show that they also increase the kinetics of mesenchymal-epithelial transition during reprogramming and block TGFβ-induced EMT of human epithelial cells. These results demonstrate that the ESCC miRNAs promote dedifferentiation by acting on multiple downstream pathways. We propose that individual miRNAs generally act through numerous pathways that synergize to regulate and enforce cell fate decisions.     

miR-146a参与不同疾病的研究进展*

miR-146a是近年来miRNA研究的热点,其在不同物种中高度保守,并参与了各种类型疾病的发生与发展,如炎症、自身免疫性疾病、癌症与肥胖症等,其机制主要通过TLR4、MyD88、NF-κB和Akt等信号通路来发挥作用。就miR-146a在不同疾病过程中的作用及其机制进行综述,为深入研究miR-146a在各类疾病中的调节作用提供资料。     

The expression and role of miR-301a in human umbilical cord-derived mesenchymal stromal cells

Background aimsToll-like receptors (TLRs) are expressed in human umbilical cord-derived mesenchymal stromal cells (UC-MSCs), and activation of TLRs plays an important role in proliferation, differentiation and immunoregulatory activity of UC-MSCs. We investigated whether TLRs regulated the expression of microRNAs (miRNAs) in UC-MSCs and the role of miRNAs.Methods and ResultsWith miRNA microarray analysis, we demonstrated that the expression of many miRNAs varied when UC-MSCs were stimulated with the ligand of toll-like receptor 4 (TLR4), lipopolysaccharide (LPS). The expression of some miRNAs was verified by polymerase chain reaction. It was found that microRNA-301a (miR-301a) was up-regulated by the ligands of TLR3 and TLR4, LPS and polyinosinic acid:polycytidylic acid poly(I:C). However, the inhibitors of nuclear factor κB NF-κB and interferon regulatory factor 3 IRF3 signal attenuated the effect of LPS and poly(I:C) on miR-301a expression. Over-expression or lower expression of miR-301a affected the cytokine secretion of UC-MSCs.ConclusionsThe expression of miR-301a in UC-MSCs was regulated by TLRs, and miR-301a affected the cytokine secretion of UC-MSCs.     

Serum miR-146a and miR-223 as potential new biomarkers for sepsis

Objective

Current biomarkers cannot completely distinguish sepsis from systemic inflammatory response syndrome (SIRS) caused by other non-infectious diseases. Circulating microRNAs (miRNAs) are promising biomarkers for several diseases, but their correlation with sepsis is not totally clarified.

Methods

Seven miRNAs related to inflammation or infection were included in the present study. Serum miRNA expression was investigated in 50 patients diagnosed with sepsis, 30 patients with SIRS and 20 healthy controls to evaluate the diagnostic and prognostic value. Expression levels of serum miRNAs were determined by quantitative PCR using the Qiagen miScript system. Serum CRP and IL-6 levels were determined by enzyme linked immunosorbent assay.

Results

Serum miR-146a and miR-223 were significantly reduced in septic patients compared with SIRS patients and healthy controls. The areas under the receiver operating characteristic curve of miR-146a, miR-223 and IL-6 were 0.858, 0.804 and 0.785, respectively.

Conclusion

Serum miR-146a and miR-223 might serve as new biomarkers for sepsis with high specificity and sensitivity. (ClinicalTrials.gov number, NCT00862290.)     

miR-146a Polymorphism Influences Levels of miR-146a,IRAK-1, and TRAF-6 in Young Patients with Coronary Artery Disease

Modulation of nuclear factor KappaB (NF-κB) activation may play a role in regulating inflammatory conditions associated with coronary artery disease (CAD). MicroRNA-146a (miR-146a) primarily targets interleukin-1 receptor-associated kinase 1 (IRAK-1) and tumour necrosis factor receptor associated factor 6 (TRAF-6), which results in inhibition of NF-κB via the TLR pathway. This study investigated the influence of the miR-146a GC rs2910164 on miR-146a expression in young South African Indians with CAD. CAD patients and controls were genotyped by PCR–RFLP and miRNA-146a levels were measured by qPCR. IRAK-1, TRAF-6 and NF-κB expression was determined by Western blot. No differences in genotypic frequency was found (GG: 45 vs. 47 %, GC: 46 vs. 41 %, CC: 9 vs. 12 %) in controls and patients respectively (odds ratio = 1.025; 95 % confidence interval 0.6782–1.550; p = 0.9164). Significantly higher levels of miR-146a was associated with CAD patients with the CC genotype (6.25-fold increase relative to controls and patients with the wildtype variant, p < 0.0001). Significantly lower levels of IRAK-1 (0.38 ± 0.02; p = 0.0072) and TRAF-6 (0.44 ± 0.02; p = 0.0146) was found in CAD patients with the CC genotype. The lowest levels of NF-κB and C-reactive protein were found in patients with the homozygous C allele compared to the heterozygous GC and wildtype variants. We propose a role for miR-146a in TLR signalling through a negative feedback mechanism involving the attenuation of NF-κB by down-regulation of IRAK-1 and TRAF-6. Our observations implicate miR-146a as a target for lowering inflammation in CAD patients.     

Differentially expressed miR-20, miR-21, miR-100, miR-125a and miR-146a as a potential biomarker for prostate cancer

Prostate cancer is the leading cause of death among men worldwide. Deregulation of microRNAs has been reported in many cancers. Expression of microRNAs miR-20a-5p, miR-21-5p, miR-100-5p, miR-125a-5p and miR-146a-5p in tissue blocks of histologically confirmed prostate cancer patients compared with BPH patients, to identify potential microRNA biomarker for prostate cancer. MicroRNA was isolated and expression was quantified by qRT-PCR using Taqman Advanced microRNA assay kits. The interactions between the microRNA:target mRNA were predicted by using bioinformatics tools such as miRwalk and miRTargetlink. The experimentally validated targets were analysed using gprofiler to identify their molecular function, biological process and related pathways. The expression analysis revealed that miR-21 and miR-100 were significantly down-regulated whereas miR-125a was up-regulated in prostate cancer patients. Comparative analysis of the expression levels with tumor grading reveal that miR-100 was significantly down-regulated (p?<?0.05) in high grade tumor, indicating that miR-100 associated with prostate cancer. ROC analysis revealed that combined analysis of down-regulated miRNAs (miR-21 and miR-100) shown AUC of 0.72 (95% CI 0.65–0.79). The combined analysis of all five miRNAs showed AUC of 0.87 (95% CI 0.81–0.92). The targets prediction analysis revealed several validated targets including BCL2, ROCK1, EGFR, PTEN, MTOR, NAIF1 and VEGFA. Our results provide evidence that combined analysis of all the five miRNAs as a panel can significantly improve the prediction level of the presence of prostate cancer and may be used as a potential diagnostic biomarker.