Multiple reports have suggested that the increased thickness of the acetabular roof cartilage, in DDH children and animal models, is associated with delayed endochondral ossification [10, 13, 22, 23]. This study has successfully induced DDH in New Zealand white rabbits. We also demonstrated that the MRI manifestation of the rabbit DDH acetabular roof is similar to the MRI data from DDH children, especially in terms of producing a thicker acetabular roof cartilage (also confirmed using gross specimen inspection and HE staining).
Growing evidences suggest a crucial role of miRNA in cartilage physiology [24]. Miyaki et al.[16], for instance, discovered the presence of miR-140 in the growth plate of X, which modulates target gene Adamts-5, an important matrix protease that hydrolyzes proteoglycans and type II collagen, to maintain healthy cartilage structure and function. Alternately, miR-140-knockout mice developed severe osteoarthritis. Nakamura et al. [17], on the other hand, demonstrated, using miR-140-knockout mice, that the Dnpep-mediated bone morphogenetic protein (BMP) signaling pathway was affected, which stimulated articular chondrocytes differentiation into hypertrophic chondrocytes and, thus, developed chondrogenic disorder. Likewise, Kobayashi et al. [18], specifically knocked out the Dicer gene in a mouse model which dramatically reduced miRNAs expression in chondrocytes and strongly suppressed bone formation and chondrocyte proliferation in the growth plate. Similarly, Zhang et al. [19], confirmed the low expression of miR-150-5p in osteoarthritis, which negatively impacted AKT Serine/Threonine Kinase 3 (AKT3) pathway to promote chondrocyte proliferation and inhibit apoptosis and degradation of extracellular matrix in chondrocytes. In another example, Bluhm et al. [20], reported high miR-332 expression, which modulated the RAF/MEK/ERK pathway to upregulate chondrocyte differentiation and develop achondroplasia. Taken together, miRNA not only modulates chondrocyte proliferation, differentiation, apoptosis, endochondral ossification, and cartilage development, but also osteoarthritis and achondroplasia [16–20]. However, as a member of miRNA, the expression and function of mir-1-3p in chondrocytes have not been elucidated, and its mechanism of action in acetabulum abnormal endochondral ossification in DDH remained unknown.
Based on existing research, miR-1-3p is a tumor-related miRNA that is involved in the viability, proliferation, and apoptosis of multiple cancerous cells. In fact, miR-1-3p was shown to be severely down-regulated in gastric cancer and its mechanism of action was shown to include negative regulation of STC2 to suppress cell proliferation and invasion to form gastric cancer [25]. Similarly, Gao et al. [26], reported that miR-1-3p can suppress BDNF expression and phosphorylation of TrkB to halt proliferation and invasion of bladder cancer cells. Likewise, Zhang et al. [27], confirmed low levels of miR-1-3p in hepatocellular carcinoma cell lines, which improved Sox9 expression, cell proliferation, and suppressed apoptosis in HCCLM3 and Bel-7474 cells. In addition, myogenic factors like MyoD, Mef2, and SRF were reported to increase miR-1-3p expression, whereas skeletal muscle hypertrophy decreased miR-1-3p expression [28, 29]. Moreover, in a recent study involving Chinese osteoporotic patients, miR-1-3p was reported to be significantly downregulated and SFRP1 was upregulated with reduced bone formation and bone mass [30]. In this study, we demonstrated low miR-1-3p levels in the ARC of DDH rabbits,which is consistent with the high-throughput sequencing data we conducted previously[13]. To ascertain the function of miR-1-3p in chondrocytes, we first confirmed its expression in normal human and rabbit chondrocytes, using FISH. We also demonstrated that with low miR-1-3p levels, in these chondrocytes, there also existed mineralized nodules, as evidenced by ARS staining. Analysis of endochondral osteogenesis-related genes showed reduced expression of RUNX2 and collagen type X following miR-1-3p suppression. To identify downstream targets of miR-1-3p, we screened out bioinformatics data and found SOX9. SOX9 is normally expressed in all chondrogenic progenitors and chondrocytes of the articular cartilage throughout adulthood and is a master transcription factor regulating multiple events involving chondrogenesis [31–34]. Moreover, several studies suggested a role of SOX9 in endochondral ossification. In particular, SOX9 suppression in the normal growth plate is essential for endochondral ossification, whereas high expressing Sox9 in the growth plate retards this process [35, 36]. In accordance with other studies, we showed a significant upregulation of SOX9 in mir-1-3p-silenced chondrocytes in vitro. To confirm whether miR-1-3p downregulation reduces RUNX2 and collagen type X expression via SOX9, we performed rescue experiments. In brief, we demonstrated that SOX9 silencing restored RUNX2 and collagen type X expression in cells treated with miR-1-3p inhibitor verses control. Prior studies have reported that Sox9 negatively regulates Runx2 and type X collagen expression in order to modulate endochondral ossification-related disorders [36, 37]. To verify the miR-1-3p-mediated regulation of endochondral ossification genes, the levels of SOX9, RUNX2, and collagen type X were examined in the ARC of DDH and healthy rabbit acetabula, using immunohistochemistry, qPCR, and western blot analysis. As expected, our results showed that low miR-1-3p levels in the ARC resulted in high expression of SOX9 and low expression level of RUNX2 and collagen type X in the DDH rabbit acetabula verses controls. These results suggest a strong involvement of miR-1-3p in the modulation of abnormal endochondral ossification of the ARC in DDH.