Martinez-Sanchez Aida, Lazzarano Stefano, Sharma Eshita, Lockstone Helen, Murphy Christopher L
Kennedy Institute of Rheumatology, University of Oxford, Oxford OX3 7FY, UK.
Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.
Life (Basel). 2020 May 11;10(5):58. doi: 10.3390/life10050058.
MicroRNAs (miRNAs) play key roles in cartilage development and homeostasis and are dysregulated in osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of . Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs, critical if miR-145 is to be considered a target for cartilage repair. We performed RIP-seq (RNA-immunoprecipitation and high-throughput sequencing) of miRISC (miRNA-induced silencing complex) in HACs overexpressing miR-145 to identify miR-145 direct targets and used cWords to assess enrichment of miR-145 seed matches in the identified targets. Further validations were performed by RT-qPCR, Western immunoblot, and luciferase assays. MiR-145 affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3'UTR or, more commonly, the coding region. MiR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion leads to MMP13 upregulation, suggesting a contribution towards the effect of miR-145 on MMP13 expression. In conclusion, miR-145 directly targets several genes involved in the expression of the extracellular matrix and inflammation in primary chondrocytes. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.
微小RNA（miRNA）在软骨发育和稳态中起关键作用，且在骨关节炎中表达失调。MiR-145的调节可诱导人关节软骨细胞（HAC）表型发生深刻变化，部分是通过直接抑制……实现的。由于miRNA可同时沉默多个靶标，我们旨在确定HAC中miR-145的完整靶标组，这对于将miR-145视为软骨修复靶点至关重要。我们对过表达miR-145的HAC中的miRISC（miRNA诱导沉默复合体）进行了RIP-seq（RNA免疫沉淀和高通量测序），以鉴定miR-145的直接靶标，并使用cWords评估已鉴定靶标中miR-145种子匹配序列的富集情况。通过RT-qPCR、Western免疫印迹和荧光素酶测定进行了进一步验证。MiR-145影响350多个基因的表达，并通过3'UTR或更常见的编码区直接靶向50多个mRNA。MiR-145在翻译水平靶向参与软骨组织和发育的DUSP6。DUSP6的缺失导致MMP13上调，表明其对miR-145对MMP13表达的影响有作用。总之，miR-145直接靶向原代软骨细胞中参与细胞外基质表达和炎症的多个基因。因此，我们提出miR-145是软骨细胞功能的重要调节因子和软骨修复的新靶点。
