MicrRNA-26a deficiency attenuates the severity of frozen shoulder in a mouse immobilization model

The main pathogenesis of the frozen shoulder is thought to be the inflammation of the intra-articular synovium and subsequent fibrosis of the shoulder joint capsule. However, the molecular pathogenesis of the frozen shoulder is still unknown. A class of non-coding RNAs, microRNAs (miRNAs) contribute to various diseases including musculoskeletal diseases. MicroRNA-26a (miR-26a) has been reported to be associated with fibrosis in several organs. This study aims to reveal the role of miR-26a on fibrosis in the shoulder capsule using a frozen shoulder model in miR-26a deficient (miR-26a KO) mice. MiR-26 KO and wild type (WT) mice were investigated using a frozen shoulder model. The range of motion of the shoulder, histopathological analysis such as synovitis, and fibrosis related-genes expression in the model mice were evaluated to determine the role of miR-26a. In WT mice, both inflammatory cell infiltration and thickening of the inferior shoulder joint capsule were observed after 1 week of immobilization, and this thickening further progressed over the subsequent 6 weeks. However, the immobilized shoulder in miR-26a KO mice consistently exhibited significantly better range of motion compared with WT mice at each point, and histological changes were notably less severe. The expression of inflammation- and fibrosis-related genes was decreased in the miR-26a KO mice compared with WT mice at 1 and 6 weeks. Together, miR-26a deficiency attenuated the severity of frozen shoulder in the immobilization model mouse. The present study suggests that miR-26a has the potential to be a target miRNA for therapeutic approach to frozen shoulder. Overall design: To understand its mechanisms including target genes of miR-26a, synovial fibroblasts (SFBs) derived from shoulder joint capsule tissue of WT and miR-26a KO mice one week after immobilization were isolated by outgrowth method (Ref) in Dulbecco's modified Eagle's medium (DMEM) (FUJIFILM Wako, Osaka, Japan). Isolated SFBs were cultured in DMEM with 10 % fetal bovine serum. Experiments were carried out at passage 2. Double-stranded RNA oligonucleotides representing mature sequences that 10 nM miR-26a mimic (sense: 5'-UUCAAGUAAUCCAGGAUAGGCU-3' and anti-sense: 5'-CCUAUUCUUGGUUCCUUGCACG-3') or Silencer Negative Control siRNA#1 (Themo Fisher Scientific) were transfected into miR-26a KO SFBs using Lipofectamine RNAiMax Reagent (Themo Fisher Scientific) for 24 hours according to the manufacturers' instructions. Total RNA of wild-type and, miR-26a KO SFBs were extracted and underwent RNA-sequencing (RNA-seq) analysis (BGI Genomics, China).