Regulation of Growth Plate Chondrocyte Hypertrophic Differentiation By Mir-374-5p, Mir-379-5p, and Mir-503-5p

Presentation Number: OR17-2
Date of Presentation: April 2nd, 2017

Youn Hee Jee*1, Jinhee Wang1, Melissa Jennings1, Ola Nilsson2, Julian Lui1 and Jeffrey Baron1
1NIH, Bethesda, MD, 2Karolinska Institutet


Background: Growth plate chondrocytes undergo sequential differentiation to form the resting (RZ), proliferative (PZ), and hypertrophic zones (HZ). The important role of miRNAs in the growth plate was revealed by cartilage-specific ablation of dicer, an enzyme essential for biogenesis of microRNAs. Here, we sought to identify specific microRNAs that regulate differentiation of PZ chondrocytes to HZ chondrocytes.

Experimental Design: Individual growth plate zones were collected by microdissection from 4-day-old rat proximal tibias. Using solution hybridridization to color-coded probes (Nanostring) (n=4) and also RNAseq (n=3), we identified microRNAs that were downregulated in HZ vs PZ (> 2 fold and FDR < 0.05 by Nanostring). To prioritize these microRNAs for subsequent study, we identified microRNAs that were 1) highly expressed in growth plate chondrocytes compared to osteoblasts, 2) predicted to regulate multiple genes that are differentially expressed in PZ vs HZ, and 3) predicted to target critical regulatory genes in the growth plate, such as Ihh, Pth1r, Gdf10, Col10a1, Bmp2/6, Mmp13, Vegfa, Nppc, and Npr2. For the four highest priority microRNAs, inhibitors were transfected into primary rat growth plate chondrocytes for evaluation of cell proliferation (thymidine labeling) and changes in expression (RT-PCR) of genes that are upregulated in the PZ to HZ transition.

Results: Four microRNAs (mir-369-3p, mir-374-5p, mir-379-5p, mir-503-5p) that were downregulated in HZ vs PZ were selected based on the prioritization analysis. When inhibitors for these microRNAs were transfected into primary chondrocytes, proliferation decreased (thymidine incorporation 38, 26, 46, 49%, respectively, P<0.001, n=5) vs control (transfected with scrambled microRNA). The inhibitors for three of the microRNAs (mir-374-5p, mir-379-5p, mir- 503-5p) also increased expression of genes physiologically upregulated in HZ (n=7): Ihh (7.6, 6.9, 8.0 fold changes, respectively, P<0.01), Bmp2 (7.6, 5.4, 5.6 fold, P<0.01), Bmp6 (3.5, 2.9, 3.2 fold, P=0.008, 0.06, 0.047), and Col10a1 (5.3, 4.5, 4.4 fold, P=0.015, 0.051, 0.04).

Conclusion: Our findings suggest that mir-374-5p, mir-379-5p, and mir- 503-5p are downregulated in the PZ to HZ transition, thereby contributing to the inhibition of proliferation and stimulation of hypertrophic differentiation, which are important steps in endochondral bone formation at the growth plate.


Nothing to Disclose: YHJ, JW, MJ, ON, JL, JB