ABSTRACT
AIMS: Bacterial lipopolysaccharides (LPS) induce innate immune inflammatory responses in endothelial cells by activating toll-like receptor 4 (TLR4) signalling. Here, we investigate the effects of angiopoietin-1 (Ang-1) on LPS-induced TLR4 signalling and the role of the miR-146 family of micro RNAs in the effects of Ang-1 on TRL4 signalling. METHODS AND RESULTS: Leucocyte adhesion to human umbilical vein endothelial cells (HUVECs) was detected using fluorescence microscopy. Adhesion molecule, pro-inflammatory cytokine, miR-146a, and miR-146b-5p expressions in HUVECs were quantified using real-time PCR. TLR4 signalling protein levels were measured using immunoblotting. Exposure of HUVECs to LPS for 4-6 h induces robust inflammatory responses, including enhanced leucocyte adhesion, up-regulation of adhesion molecule expression (VCAM1, ICAM1, E-SELECTIN), enhanced cytokine production (TNFα, IL1ß, IL6, and IL8), and increased NFκB luciferase reporter activity. Addition of Ang-1 to the culture medium for 24 h prior to LPS exposure significantly attenuates these responses. Prolonged Ang-1 exposure significantly decreases IRAK1 and TRAF6 protein levels but has no effect on TLR4, MYD88, IRAK4, or TAK1 expressions. Ang-1 triggers significant up-regulation of miR-146b-5p levels but has no effect on miR-146a or miR-146b-3p expressions. Transfection of HUVECs with a miR-146b-5p mimic significantly attenuates LPS-induced inflammatory responses and IRAK1 and TRAF6 expressions. In HUVECs transfected with a miR-146b-5p inhibitor, Ang-1 has no effect on LPS-induced inflammatory responses or IRAK1 and TRAF6 expressions. CONCLUSION: Ang-1 disrupts TLR4 signalling, resulting in inhibition of LPS-induced inflammatory responses in endothelial cells. This inhibition occurs through selective targeting of IRAK1 and TRAF6 proteins by miR-146b-5p.
Subject(s)
Angiopoietin-1/pharmacology , Anti-Inflammatory Agents/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Inflammation/prevention & control , MicroRNAs/metabolism , Toll-Like Receptor 4/antagonists & inhibitors , Cell Adhesion/drug effects , Coculture Techniques , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells/immunology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Inflammation Mediators/metabolism , Interleukin-1 Receptor-Associated Kinases/genetics , Interleukin-1 Receptor-Associated Kinases/metabolism , Leukocytes/drug effects , Leukocytes/immunology , Leukocytes/metabolism , Lipopolysaccharides/pharmacology , MicroRNAs/genetics , Signal Transduction/drug effects , TNF Receptor-Associated Factor 6/genetics , TNF Receptor-Associated Factor 6/metabolism , Time Factors , Toll-Like Receptor 4/agonists , Toll-Like Receptor 4/metabolism , Transfection , U937 CellsABSTRACT
TGF-ß plays an important role in breast cancer progression as a prometastatic factor, notably through enhancement of cell migration. It is becoming clear that microRNAs, a new class of small regulatory molecules, also play crucial roles in mediating tumor formation and progression. We found TGF-ß to down-regulate the expression of the microRNA miR-584 in breast cancer cells. Furthermore, we identified PHACTR1, an actin-binding protein, to be positively regulated by TGF-ß in a miR-584-dependent manner. Moreover, we found TGF-ß-mediated down-regulation of miR-584 and increased expression of PHACTR1 to be required for TGF-ß-induced cell migration of breast cancer cells. Indeed, both overexpression of miR-584 and knockdown of PHACTR1 resulted in a drastic reorganization of the actin cytoskeleton and reduced TGF-ß-induced cell migration. Our data highlight a novel signaling route whereby TGF-ß silences the expression of miR-584, resulting in enhanced PHACTR1 expression, and further leading to actin rearrangement and breast cancer cell migration.
Subject(s)
Actins/metabolism , Breast Neoplasms/mortality , Cell Movement , MicroRNAs/biosynthesis , Microfilament Proteins/biosynthesis , Neoplasm Proteins/biosynthesis , RNA, Neoplasm/biosynthesis , Transforming Growth Factor beta/metabolism , Actins/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cytoskeleton/genetics , Cytoskeleton/metabolism , Cytoskeleton/pathology , Female , Gene Expression Regulation, Neoplastic/genetics , Gene Silencing , Humans , MicroRNAs/genetics , Microfilament Proteins/genetics , Neoplasm Proteins/genetics , RNA, Neoplasm/genetics , Signal Transduction/genetics , Transforming Growth Factor beta/geneticsABSTRACT
MicroRNA-181 (miR-181) is a multifaceted miRNA that has been implicated in many cellular processes such as cell fate determination and cellular invasion. While miR-181 is often overexpressed in human tumors, a direct role for this miRNA in breast cancer progression has not yet been characterized. In this study, we found this miRNA to be regulated by both activin and TGFß. While we found no effect of miR-181 modulation on activin/TGFß-mediated tumor suppression, our data clearly indicate that miR-181 plays a critical and prominent role downstream of two growth factors, in mediating their pro-migratory and pro-invasive effects in breast cancer cells miR-181 acts as a metastamir in breast cancer. Thus, our findings define a novel role for miR-181 downstream of activin/TGFß in regulating their tumor promoting functions. Having defined miR-181 as a critical regulator of tumor progression in vitro, our results thus, highlight miR-181 as an important potential therapeutic target in breast cancer.
