RESUMO
miR-140 is selectively expressed in cartilage. Deletion of the entire Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5' and 3' end, with >99% having one of two seed sequences (5' bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3'UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140-null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.
Assuntos
Cartilagem/química , MicroRNAs/genética , Análise de Sequência de RNA/métodos , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Condrogênese , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Camundongos , Anotação de Sequência Molecular , Especificidade de Órgãos , Regulação para CimaRESUMO
eIF4A is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of mRNA translation. The Arabidopsis genome encodes two isoforms, one of which (eIF4A-1) is required for the coordination between cell cycle progression and cell size. A T-DNA mutant eif4a1 line, with reduced eIF4A protein levels, displays slow growth, reduced lateral root formation, delayed flowering and abnormal ovule development. Loss of eIF4A-1 reduces the proportion of mitotic cells in the root meristem and perturbs the relationship between cell size and cell cycle progression. Several cell cycle reporter proteins, particularly those expressed at G2/M, have reduced expression in eif4a1 mutant meristems. Single eif4a1 mutants are semisterile and show aberrant ovule growth, whereas double eif4a1 eif4a2 homozygous mutants could not be recovered, indicating that eIF4A function is essential for plant growth and development.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Tamanho Celular , Fator de Iniciação 4A em Eucariotos/fisiologia , Óvulo Vegetal/crescimento & desenvolvimento , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ciclo Celular/genética , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Flores/citologia , Flores/genética , Flores/fisiologia , Genoma de Planta , Homeostase , Meristema/citologia , Meristema/genética , Meristema/fisiologia , Mitose/genética , Mutação , Óvulo Vegetal/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Raízes de Plantas/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologiaRESUMO
UNLABELLED: MicroRNAs have been shown to function in cartilage development and homeostasis, as well as in progression of osteoarthritis. The objective of the current study was to identify microRNAs involved in the onset or early progression of osteoarthritis and characterise their function in chondrocytes. MicroRNA expression in mouse knee joints post-DMM surgery was measured over 7 days. Expression of miR-29b-3p was increased at day 1 and regulated in the opposite direction to its potential targets. In a mouse model of cartilage injury and in end-stage human OA cartilage, the miR-29 family was also regulated. SOX9 repressed expression of miR-29a-3p and miR-29b-3p via the 29a/b1 promoter. TGFß1 decreased expression of miR-29a, b, and c (3p) in primary chondrocytes, whilst IL-1ß increased (but LPS decreased) their expression. The miR-29 family negatively regulated Smad, NFκB, and canonical WNT signalling pathways. Expression profiles revealed regulation of new WNT-related genes. Amongst these, FZD3, FZD5, DVL3, FRAT2, and CK2A2 were validated as direct targets of the miR-29 family. These data identify the miR-29 family as microRNAs acting across development and progression of OA. They are regulated by factors which are important in OA and impact on relevant signalling pathways. KEY MESSAGES: Expression of the miR-29 family is regulated in cartilage during osteoarthritis. SOX9 represses expression of the miR-29 family in chondrocytes. The miR-29 family is regulated by TGF-ß1 and IL-1 in chondrocytes. The miR-29 family negatively regulates Smad, NFκB, and canonical Wnt signalling. Several Wnt-related genes are direct targets of the miR-29 family.