The Functional Analysis Of Micrornas In Chondrogenesis

Osteoarthritis is one of the prevailing skeletal diseases worldwide. Articular cartilage and embryonic cartilage share common ancestors which are differentiated from the condensed mesenchymal progenitor cells. As the function of microRNAs in the vertebrate developmental process is reported, the role of the microRNAs in chondrogenesis comes to be more attractable to explore therapeutic target for arthritis diseases. Recently, several investigations of microRNAs in chondrogenesis were performed, however it still remains unclear for the function of the microRNAs that are specially involved in chondrogenesis and etiology of arthritis. Here the exclusive functional microRNAs in chondrogenesis are identified through comparative microRNA profiling, followed by intensively studies on their transcriptional regulation and chondrogenic function.In this study, the comparative microRNA profiling between chondrogenesis and Wnt/?-catenin induced non-chondrogenic process was conducted in micromass culture of E12.5 mouse limb bud cells. First, we found that miR-140, miR-140* and miR-455 were specifically expressed in chondrogenesis, and the myoblast-related microRNAs of miR-1, miR-133a, miR-133b and miR-206 were significantly induced by Wnt/?-catenin signaling. Secondly, the primary miR-140/140* was an intron-retained transcript and was specifically co-expressed with its host gene Wwp2-C in chondrogenesis, revealed through the approaches of RT-PCR, RACE and Whole mount in situ hybridization. The expression pattern and the protein coding capability of the host gene Wwp2-C were also presented here for the first time. Third, to investigate the transcriptional regulation of miR-140/140*, the promoter of primary miR-140/140* was evaluated by luciferase assay and ChIP experiment with chondrogenic transcription factor Sox9. These findings suggest that the Sox9 directly bind to the -200 motif upstream of the promoter to drive the exclusive expression of miR-140 in chondrogenesis. Meanwhile, the RCAS::miR-140-Sponge was applied to knock down the mature miR-140 in the chondrogenic process, implicating that miR-140 was critical to maintain the chondrocyte proliferation. In addition, Sp1, the cell cycle regulator was verified as a target of miR-140 in chondrogenesis.Taken together, miR-140 was identified as an exclusive microRNA involved in chondrogenesis through the microRNA comparative profiling. It was co-expressed with host gene Wwp2-C and directly induced by Sox9. Furthermore, miR-140 mantained the proliferation of chondrocytes through target cell cycle regulator Sp1 protein. Those findings provide a better understanding of the role of microRNA in chondrogenesis, which could be regarded as a therapeutic target for osteoarthritis.