The role of miR-140 AND miR-455 in murine skeletal development and osteoarthritis pathogenesis

Abstract

Introduction: Osteoarthritis (OA) is the most common musculoskeletal disorder, which is complex and characterised by degradation of articular cartilage of joint tissue. Epigenetic changes alter transcriptional regulation and disrupt signalling pathways involved in cartilage homeostasis leading to OA, and dysregulation of and by microRNA (miRNA) (e.g. miR-140 and miR-455) correlate with bone and OA development. Bioinformatic analysis of RNAsequencing (RNA-seq) could reveal the mechanism of the function of miR-140 and miR-455 in murine skeletal development and OA pathogenesis. Aim: Generation of Mir140-null and Mir455-null mouse model to understand their role in relation to murine skeletal and OA development. Material and Methods: By CRISPR/Cas9 technology we generated two mouse lines, for Mir140 (miR-140-/- ) and Mir455 (miR-455-/- ). These mice were interbred to derive mice with deletions of both Mir140 and Mir455 (DKO). On the mutant mice, we performed phenotyping of skeletal X-ray, weight growth curve, and tibial growth plate analysis, including histological staining and immunocytochemistry (BrdU labelling to monitor proliferation and TUNEL labelling to monitor apoptosis). Destabilisation of the medial meniscus (DMM) surgery was performed on mutant mice to assess knee articular cartilage integrity following joint trauma. Differential expression of genes within the growth plate of the mutant mice were identified by RNA-seq and potential upregulated miRNA predicted targets validated by 3’UTR luciferase analysis. The expression of two growth plate targets (Creb3l1 and Nrf2) were assessed by western blot analysis in isolated chondrocytes. Transcriptome analysis was also performed on micro-dissected knee articular cartilage (preand post-DMM surgery) and on laser micro-dissected growth plates. Results: Genotyping analysis confirmed the deletion of miRNAs in our mutant mice. Phenotyping of our Mir140-null mice matched that of previously published work, including mild short stature, domed skull, and a short tail. Our Mir455-null mice were dwarf when compared to control mice, however, Mir455-null mice housed at our collaborator were undisguisable from age-matched control mice. Our DKO mice shared a similar phenotype III with Mir140-null mice. Histological staining revealed abnormal tibial growth plate morphology and delayed secondary ossification in 1-week and 3-week-old mice. A reduction in tibial growth plate chondrocyte proliferation and increased apoptosis was detected in mutant mouse lines. Mutant mice exhibited different transcriptome profiles of costal chondrocyte by RNA-seq, and three miR-140 growth plate predicted target genes (Creb3l1, Nrf2 and Zeb1) were confirmed to be targets and to attenuate chondrocyte proliferation by WST-1 assay analysis. Following DMM-surgery all mutant mice showed higher OA score with either Glasson scoring system or OARSI scoring system, and all exhibited significant proteoglycan staining loss compared to control mice. Comparison of transcriptome profiles before and after DMM surgery had led to understand the function of miR-140 during OA pathogenesis. Discussion and conclusion: Our mutant mice showed clear growth phenotypes compared to control mice, however the phenotyping of our Mir455-null mouse line was different to recent literature and to similar animals at our collaborator facility. Histological and immunohistochemistry analysis confirmed that mutant mice exhibited abnormal growth plate morphology, with reduced chondrocyte proliferation and increased apoptosis. Transcriptome profiling from costal chondrocyte and knee cartilage chondrocyte (pre and post-DMM surgery) represents an important tool to identify miRNA target genes and, herein, to gain insights about miR-140 and miR-455 in skeletal developments and OA pathogenesis.