Abstract
SummaryObjectiveMicroRNA 140 (miR-140) is a chondrocyte-specific endogenous gene regulator implicated in osteoarthritis (OA). As mechanical injury is a primary aetiological factor in OA, we investigated miR-140-dependent mechanosensitive gene regulation using a novel CRISPR-Cas9 methodology in primary human chondrocytes.MethodPrimary (passage 1/2) human OA chondrocytes were isolated from arthroplasty samples (six donors) and transfected with ribonuclear protein complexes or plasmids using single guide RNAs (sgRNAs) targeting miR-140, in combination with Cas9 endonuclease. Combinations of sgRNAs and single/double transfections were tested. Gene editing was measured by T7 endonuclease 1 (T7E1) assay. miRNA levels were confirmed by qPCR in chondrocytes and in wild type murine femoral head cartilage after acute injury. Predicted close match off-targets were examined. Mechanosensitive miR-140 target validation was assessed in 42 injury-associated genes using TaqMan Microfluidic cards in targeted and donor-matched control chondrocytes. Identified targets were examined in RNAseq data from costal chondrocytes from miR-140−/− mice.ResultsHigh efficiency gene editing of miR-140 (90–98%) was obtained when two sgRNAs were combined with double RNP-mediated CRISPR-Cas9 transfection. miR-140 levels fell rapidly after femoral cartilage injury. Of the top eight miR-140 gene targets identified (P < 0.01), we validated three previously identified ones (septin 2, bone morphogenetic protein 2 and fibroblast growth factor 2). Novel targets included Agrin, a newly recognised pro-regenerative cartilage agent, and proteins associated with retinoic acid signalling and the primary cilium.ConclusionWe describe a highly efficient CRISPR-Cas9-mediated strategy for gene editing in primary human chondrocytes and identify several novel mechanosensitive miR-140 targets of disease relevance.
Highlights
Osteoarthritis (OA) is generally accepted as a biologically driven disease where mechanical stresses, combined with other factors, lead to imbalance between catabolic and repair activities within the joint[1]
We examine the regulation of MicroRNA 140 (miR-140) upon ex vivo murine cartilage injury and explore the regulation of a number of previously described and novel targets that are relevant to chondrocyte mechanobiology and OA pathogenesis
We first assessed the impact of two different transfection methods on isolated human osteoarthritic chondrocytes (OA hACs)
Summary
Osteoarthritis (OA) is generally accepted as a biologically driven disease where mechanical stresses, combined with other factors, lead to imbalance between catabolic and repair activities within the joint[1]. The chondrocyte is regarded as a critical disease player; being highly mechanosensitive, able to synthesise its own degradative enzymes and having limited renewal, leading to poor tissue repair (reviewed in[3]). Multiple pathways have been associated with disease modification and many of these have been validated in murine models where the molecule of interest is deleted or suppressed pharmacologically. MicroRNAs (miRs) are important regulatory molecules in all animal and plant cells. They are small endogenous RNAs, typically 20e25 nucleotides in length, that suppress specific mRNAs, by binding and targeting the mRNA for degradation or by suppressing protein translation[5]. The mature miR is loaded onto the Argonaute protein to form the active RNA-induced silencing complex[8]
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