MicroRNA-let-7a promotes E2F-mediated cell proliferation and NFκB activation in vitro.
Epigenetic factors, including altered microRNA (miRNA) expression, may contribute to aberrant immune cell function in systemic lupus erythematosus (SLE). MiRNA-let-7a (let-7a) has been shown to directly alter cell cycle progression and proinflammatory cytokine production. Due to the crucial role of let-7a in cell division and inflammation, we investigated let-7a-mediated proliferation and NFκB translocation in J774A.1 macrophages and MES 13 mesangial cells in vitro. In immune-stimulated cells transfected with let-7a, cell proliferation was significantly increased over time. There was a significant increase in the number of immune-stimulated cells in S and G2 phases. Immune-stimulated cells overexpressing let-7a had increased nuclear translocation of NFκB. Bioinformatical analysis revealed that the E2F family, critical regulators of the G1-S transition, has potential binding sites for let-7a in their mRNA transcripts. Let-7a overexpression significantly increased the expression of the cell cycle activator E2F2 and increased retinoblastoma protein (Rb) phosphorylation in immune-stimulated cells. The cell cycle inhibitor E2F5 was significantly decreased in let-7a-transfected cells that were immune-stimulated. Bioinformatical analysis revealed E2F2 and NFκB are transcription factors predicted to regulate the let-7a promoter. We analyzed transcriptional regulation of let-7a by real-time RT-PCR using chromatin immunoprecipitation with E2F2 and NFκB antibodies. There was an increase in E2F2 and NFκB binding in DNA enriched for the let-7a promoter in immune-stimulated cells. Silencing E2F2 or NFκB significantly decreased let-7a expression and IL-6 production in immune-stimulated cells. Taken together, our results suggest that overexpression of let-7a may contribute to hyperplasia and the proinflammatory response in SLE.
Department of Biomedical Sciences & Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA.
Cristen B
CB
Chafin
Department of Biomedical Sciences & Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA.
Nicole L
NL
Regna
Department of Pathology/Comparative Medicine, Wake Forest University Primate Center, Wake Forest School of Medicine, Winston-Salem, NC, USA.
David L
DL
Caudell
1] Department of Biomedical Sciences & Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA [2] Edward Via College of Osteopathic Medicine, Blacksburg, VA, USA.
Christopher M
CM
Reilly
D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle; D049109:Cell Proliferation; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D050703:E2F2 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D050527:Mesangial Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000093242:Interleukin-6 Inhibitors
D050703:E2F2 Transcription Factor; D015850:Interleukin-6; D035683:MicroRNAs; D016328:NF-kappa B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016160:Retinoblastoma Protein; D000093242:Interleukin-6 Inhibitors; C484940:E2f2 protein, mouse; C542687:mirnlet7 microRNA, mouse