MiR-27b Inhibits Chondrocyte Hypertrophy By Targeting PPAR-γ

The formation and development of skeletons of vertebrates are mainly controlled by two mechanisms, intramembranous and endochondral ossification. Longitudinal bone growth exclusively happens in the cartilage growth plate and experienced a series of regulated processes of endochondral ossification. This process begins when mesenchymal cells form condensations and differentiate into chondrocytes, subsequently, the chondrocytes undergo processes of proliferation, maturation, hypertrophy, exiting the cell cycle, getting mature and differentiating into hypertrophic chondrocytes. Chondrocyte hypertrophy is a key step during bone development where chondrocytes sharply alter their program and turn to synthesize type X collagen(COL X) and other mineralizing cartilage matrix, which is significant in osteoclasts genesis and recruiting blood vessels to the ossification center. Along with the invading vascular tissue, osteoblasts arise and synthesize bone matrix laying on the degraded cartilage scaffold replacing the cartilage matrix. When the process of chondrocyte maturation and hypertrophy are disturbed, abnormal endochondral ossification occurs, as a consequence, skeleton dysplasia and diseases such as osteoarthritis may rise. Therefore, further study of the molecular mechanisms of chondrocyte hypertrophy is significant for the prevention and treatment of chondrocyte hypertrophy related diseases.Accumulating evidence shows that chondrogenic induction and differentiation are regulated by post-transcriptional mechanisms, most significantly by temporally expressed micro RNAs. This article aims to explore the role of mi R-27 b in the process of chondrocyte hypertrophy and the regulatory mechanism of PPAR-γ, which is one of the target genes of mi R-27 b.In this study, we isolated and harvested chondrocytes from the knee articular cartilage of newborn Wistar rats(1-3 days). Using immunohistochemistry and RT-PCR detected the expression of normal chondrocyte marker COL II and the hypertrophy markers COL X and MMP-13. We analyzed the distribution of mi R-27 b in situ hybridization experiments and RT-q PCR on the resting zone, proliferative zone and hypertrophic zone of cartilage. Using bioinformatics website, dual luciferase reporter assay system and Western blot we identified that PPARγ was a target gene of mi R-27 b. Afterwards, through mimics-mediated overexpression of mir-27 b in chondrocytes we confirmed the inhibition roles of mi R-27 b on the chondrocyte hypertrophy.Results: 1. Histological staining shown that in articular cartilage the chondrocytes have the columnar arrangements, and from the surface to the depth of the cartilage, chondrocytes volume as well as the expression of COL X and MMP-13 gradually increased.2. Mi R-27 b mainly expressed in the nucleus, and decreased when differentiated into hypertrophic chondrocytes. RT-q PCR results shown that compared with resting hypertrophic chondrocytes, in hypertrophic chondrocytes, expression of mi R-27 b was 6.2-fold higher(P<0.01).3. Bioinformatics website analysis confirmed that there is binding site of mi R-27 b in the PPAR-γ 3’-UTR sequence; Dual luciferase reporter gene experiments confirmed that the fluorescence expression of psi CHECK was significantly inhibited by rno-mir-27b-3p(p<0.01). Western blot shown that in the mi R-27 b mimic transfection group, the expression of PPAR-γ2 was significantly reduced.4. In the mimics-mediated overexpression of mir-27 b experiments, 7 days later, the experiment group and control group displayed lipid droplets, which were positive to oil red O staining. Comparing the experiment group with the transfection reagent control group and negative control group, the number of lipid droplets decreased significantly(P<0.01).5. RT-q PCR results shown that after the overexpression of mi R-27 b, the expression of COL II and SOX-9 increased(P<0.01), and the expression of MMP-13 and PPAR-γ decreased(P<0.01); Western blot results shown, after mi R-27b’s over-expression, the protein expression of PPAR-γ2 and COL X were significantly inhibited.Conclusions: 1. Mi R-27 b was a negative regulatory factor of chondrocyte hypertrophy. 2. Ectopically enhancing of mi R-27 b can delay the hypertrophic process of chondrocytes. 3. PPAR-γ is a relevant target gene of mi R-27 b and it may play a positive role in the regulation of COL X through some undefined mechanisms.