Tardif Ginette, Pelletier Jean-Pierre, Fahmi Hassan, Hum David, Zhang Yue, Kapoor Mohit, Martel-Pelletier Johanne
Arthritis Res Ther. 2013;15(6):R197. doi: 10.1186/ar4387.
MicroRNAs (miRNAs) down-regulate their target genes. The intronic miR-140, present in the WW domain containing E3 ubiquitin protein ligase 2 (WWP2) gene, decreases the expression of genes that play detrimental roles in osteoarthritis (OA). As the expression level of miR-140 is significantly decreased in human OA chondrocytes, we investigated its regulation in those cells.
Gene expression in human chondrocytes was determined by quantitative polymerase chain reaction (qPCR) and gene silencing was done in OA chondrocytes by transient transfection with specific small interfering RNAs (siRNAs). Binding sites of the miR-140 regulatory sequence (rsmiR-140) were identified by mutagenesis and chromatin immunoprecipitation (ChIP) in OA chondrocytes. The effects of translocation on OA chondrocytes were determined by immunocytochemistry and qPCR.
In contrast to miR-140, the expression of WWP2 was similar in both normal and OA cells, suggesting that miR-140 has an additional level of regulation. rsmiR-140 showed activity and predicted binding sites for nuclear matrix transcription factor 4 (NMP4), myc-associated zinc (MAZ), nuclear factor of activated T-cells (NFAT), and mothers against decapentaplegic homolog 3 (SMAD3). Silencing NFAT3 (P ≤0.01) and SMAD3 (P ≤0.05) differentially regulated miR-140 independently of WWP2. Silencing NFAT5 decreased both miR-140 and WWP2 (P ≤0.003 and P ≤0.05, respectively). NFAT3 activation increased and transforming growth factor-β (TGF-β) decreased rsmiR-140 activity. Mutagenesis of rsmiR-140 and ChIP assays identified binding sites at which NFAT3 (activator) and SMAD3 (repressor) directly regulated miR-140. TGF-β interfered with NFAT3 translocation, and subsequently with miR-140 expression.
This is the first study to provide evidence of a regulatory mechanism of miR-140 independent of WWP2, and new and differential roles for NFAT3 and SMAD3 in the OA process in the regulation of miR-140 transcription. Such knowledge could advance therapeutic strategies targeting OA.
微小RNA（miRNA）可下调其靶基因。内含子miR - 140存在于含WW结构域的E3泛素蛋白连接酶2（WWP2）基因中，可降低在骨关节炎（OA）中起有害作用的基因的表达。由于miR - 140在人OA软骨细胞中的表达水平显著降低，我们研究了其在这些细胞中的调控机制。
通过定量聚合酶链反应（qPCR）测定人软骨细胞中的基因表达，并通过用特异性小干扰RNA（siRNA）瞬时转染在OA软骨细胞中进行基因沉默。通过诱变和染色质免疫沉淀（ChIP）在OA软骨细胞中鉴定miR - 140调控序列（rsmiR - 140）的结合位点。通过免疫细胞化学和qPCR确定转位对OA软骨细胞的影响。
与miR - 140相反，WWP2在正常细胞和OA细胞中的表达相似，这表明miR - 140具有额外的调控水平。rsmiR - 140显示出活性，并预测了核基质转录因子4（NMP4）、与 myc 相关的锌指蛋白（MAZ）、活化T细胞核因子（NFAT）和抗五肢瘫蛋白同源物3（SMAD3）的结合位点。沉默NFAT3（P≤0.01）和SMAD3（P≤0.05）可独立于WWP2差异性地调控miR - 140。沉默NFAT5可降低miR - 140和WWP2的表达（分别为P≤0.003和P≤0.05）。NFAT3激活增加，而转化生长因子-β（TGF-β）降低rsmiR - 140活性。rsmiR - 140的诱变和ChIP分析确定了NFAT3（激活剂）和SMAD3（抑制剂）直接调控miR - 140的结合位点。TGF-β干扰NFAT3转位，随后干扰miR - 140表达。
这是第一项提供miR - 140独立于WWP2的调控机制证据的研究，以及NFAT3和SMAD3在OA过程中调控miR - 140转录的新的和差异性作用。这些知识可能会推动针对OA的治疗策略的发展。
