MicroRNA-319 Regulates Chondrogenic Differentiation of Human Mesenchymal Stem Cells by Targeting SOX9

Abstract

Introduction Cartilage is an avascular tissue without innervations. Thus, it has really limited regeneration and repair capacity. Tissue-engineered cartilage is highly demanded for repairing cartilage defects in cartilage-related diseases. Mesenchymal stem cells (MSCs) are self-renewal and multipotent stem cells, which can differentiate into osteocytes, chondrocytes, and adipocytes. Several studies have shown that epigenetic regulation plays an important role in the chondrogenesis of MSCs. MicroRNAs (miRNAs) are a group of small noncoding single-strand RNAs which can bind to target mRNAs to modulate various biological processes. We are intended to find roles of miR-319 during chondrogenic differentiation of mesenchymal stem cells. Materials and Methods To investigate the roles of miR-319 in chondrogenic differentiation of human MSCs, we induced chondrogenic differentiation of MSCs. Chondrogenic differentiation of MSCs was identified by Alcian staining and RT-qPCR assays. Next, we used lentiviruses as vehicles to upregulate and downregulate miR-319. In addition, dual-luciferase reporter gene assay was done to identify target gene of miR-319. Western blot was done to identify changes of target protein. Results RT-qPCR assays were done on day 0, 7, 14, and 21. Results showed that level of miR-319 was decreased during chondrogenic differentiation of MSCs. Overexpression of miR-319 greatly inhibited Alcian staining and downregulated mRNA levels of chondrogenic marker genes, such as COL2A1, COMP, COL9a2, COL11A1, and ACAN. Similarly, underexpression of miR-319 promoted Alcian staining and upregulated mRNA levels of chondrogenic marker genes. Next, dual-luciferase reporter gene assay identified that SOX9 was the target gene of miR-319. Importantly, changes of miR-319 did not change mRNA level of SOX9, but changed protein level of SOX9. The results showed that miR-319 modulated SOX9 at posttranscriptional level. Conclusion In summary, our studies demonstrated that miR-319 inhibited chondrogenic differentiation of human MSCs by targeting SOX9. Disclosure of Interest None declared