ABSTRACT
Osteoarthritis (OA) is the most common joint disease, characterized by articular cartilage degradation and changes in all other joint tissues. MicroRNAs (miRNAs) play an important role in mediating the main risk factors for OA. This study aimed to investigate the effect of miR-26a/26b on the proliferation and apoptosis of human chondrocytes by targeting fucosyltransferase 4 (FUT4) through NF-κB signaling pathway. We revealed the differential expression profiles of FUT4 and miR-26a/26b in the articular cartilage tissues of OA patients and normal people. The ability of miR-26a/26b to specifically interact with the 3'UTR of FUT4 was demonstrated via a luciferase reporter assay in chondrocytes. Further results showed altered levels of miR-26a/26b and FUT4 could regulate the process of IL-1ß-induced extracellular matrix degradation in chondrocytes. Forced miR-26a/26b expression was able to affect chondrocytes proliferation and apoptosis, while altered expression of FUT4 in chondrocytes modulated progression upon transfection with miR-26a/26b mimic or inhibitor. In OA mice, the overexpression of miR-26a/26b by intra-articular injection significantly attenuated OA progression. In addition, regulating FUT4 expression markedly modulated the activity of NF-κB signaling pathway, and this effect could be reversed by miR-26a/26b. In short, miR-26a/-26b/FUT4/NF-κB axis may serve as a predictive biomarker and a potential therapeutic target in OA treatment.
Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/metabolism , Fucosyltransferases/antagonists & inhibitors , Lewis X Antigen/antagonists & inhibitors , MicroRNAs/metabolism , NF-kappa B/metabolism , Osteoarthritis/metabolism , Signal Transduction , 3' Untranslated Regions , Animals , Apoptosis , Cartilage, Articular/immunology , Cartilage, Articular/pathology , Cell Proliferation , Cells, Cultured , Chondrocytes/immunology , Chondrocytes/pathology , Disease Progression , Enzyme Repression , Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Genes, Reporter , Humans , Injections, Intra-Articular , Interleukin-1beta/metabolism , Lewis X Antigen/genetics , Lewis X Antigen/metabolism , Male , MicroRNAs/administration & dosage , MicroRNAs/antagonists & inhibitors , MicroRNAs/therapeutic use , Osteoarthritis/pathology , Osteoarthritis/physiopathology , Osteoarthritis/therapy , RNA/administration & dosage , RNA/therapeutic use , RNA Interference , RNA Isoforms/administration & dosage , RNA Isoforms/antagonists & inhibitors , RNA Isoforms/metabolism , RNA Isoforms/therapeutic use , Rats, Sprague-DawleyABSTRACT
MicroRNAs are short noncoding, ~22-nucleotide RNAs that regulate protein abundance. The growth in our understanding of this class of RNAs has been rapid since their discovery just over a decade ago. We now appreciate that miRNAs are deeply embedded within the genetic networks that control basic features of metazoan cells including their identity, metabolism, and reproduction. The Drosophila melanogaster model system has made and will continue to make important contributions to this analysis. Intended as an introductory overview, here we review the current methods and resources available for functional analysis of fly miRNAs for those interested in performing this type of analysis.
Subject(s)
Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation , MicroRNAs/genetics , RNA-Binding Proteins/genetics , Ribonuclease III/genetics , Animals , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Gene Expression Profiling , Genes, Reporter , Genetic Techniques , MicroRNAs/antagonists & inhibitors , MicroRNAs/classification , MicroRNAs/metabolism , Nucleic Acid Conformation , Oligonucleotide Array Sequence Analysis , RNA Isoforms/antagonists & inhibitors , RNA Isoforms/genetics , RNA Isoforms/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/metabolism , Ribonuclease III/metabolism , Signal TransductionABSTRACT
Integrins are heterodimeric cell-surface adhesion receptors that play a critical role in tissue development. Characterization of the full-length mRNA encoding the ß1 subunit (Itgb1) revealed an alternative functional cleavage and polyadenylation site that yields a new Itgb1 mRNA isoform 578 bp shorter than that previously reported. Using a variety of experimental and bioinformatic approaches, we found that the two Itgb1 isoforms are expressed at different levels in a variety of mouse tissues, including the mammary gland, where they are differentially regulated at successive developmental stages. The longer mRNA species is prevelant during lactation, whereas the shorter is induced after weaning. In 3D cultures, where expression of integrin ß1 protein is required for normal formation of acini, experimental blockade of the longer isoform induced enhanced expression of the shorter species which allowed normal morphological mammary differentiation. The short isoform lacks AU-rich motifs and miRNA target sequences that are potentially implicated in the regulation of mRNA stability and translation efficiency. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. In summary, the results of the present study identify a new regulatory instance involved in the fine-tuning of Itgb1 expression during mammary gland development and function.
Subject(s)
Integrin beta1/metabolism , Mammary Glands, Animal/growth & development , Mammary Glands, Animal/metabolism , RNA Isoforms/metabolism , RNA Processing, Post-Transcriptional , RNA Stability , RNA, Messenger/metabolism , Animals , Cell Culture Techniques , Cell Differentiation , Cell Line , Data Mining , Female , Gene Expression Regulation, Developmental , Integrin beta1/chemistry , Integrin beta1/genetics , Lactation/metabolism , Mammary Glands, Animal/cytology , Mice , Mice, Inbred BALB C , Polyadenylation , Pregnancy , RNA Isoforms/antagonists & inhibitors , RNA, Messenger/antagonists & inhibitors , RNA, Small Interfering , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Specific Pathogen-Free Organisms , WeaningABSTRACT
OBJECTIVE: Dopamine agonists normalize prolactin (PRL) levels and reduce tumour size in responsive prolactinoma. However, several cases have shown resistance to dopamine agonists upon initial treatment. Infrequently, prolactinoma initially responds, but then becomes refractory to prolonged treatment (secondary resistance). We investigated the possible mechanisms of resistance to dopamine agonists. SUBJECTS AND METHODS: Twelve cases of prolactinoma were surgically resected and classified according to the responsiveness of PRL levels and tumour size to dopamine agonists: good responders (n = 5), poor responders (n = 5), or secondary resistance (n = 2). We examined the expression of dopamine D(2) receptor (D(2)R) isoform (short: D(2)S and long: D(2)L) mRNA and protein. We investigated DNA methylation patterns in the promoter region of the DRD2 gene. RESULTS: The predominant D(2)R isoform expressed in prolactinoma was D(2)L. Levels of D(2)L mRNA were significantly lower in secondary resistance and poor responders than in good responders. Expression of D(2)R protein was variable among cases. Almost no CpG sites of the DRD2 gene promoter region were methylated. CONCLUSION: Resistance of prolactinoma to dopamine agonists is correlated with a reduction in D(2)L isoform mRNA levels. Silencing of the DRD2 gene by methylation in the promoter region is unlikely to play a role in dopamine agonist resistance in prolactinoma.
