Research Paper Volume 13, Issue 5 pp 7676—7690

Downregulation of microRNA-29b by DNMT3B decelerates chondrocyte apoptosis and the progression of osteoarthritis via PTHLH/CDK4/RUNX2 axis

DNMT3B suppresses the expression of miR-29b by enhancing DNA methylation in the miR-29b promoter region. (A, B) The methylation level of miR-29b in cartilage tissues from OA patients (n = 46) and normal cartilage tissues (n = 46) detected by MSP (Nor, normal cartilage tissue; OA, cartilage tissues in patients with OA; U; unmethylated status; M, methylated status). (C) Immunofluorescence staining of Col2A1 and Aggrecan in chondrocytes (× 200, scale bar = 50 μm). (D) The methylation level of miR-29b in chondrocytes under IL-1β and 5-Aza treatment by MSP (U; unmethylated status; M, methylated status). (E) The expression of miR-29b in chondrocytes measured by RT-qPCR. (F) The mRNA expression of DNMT3B in chondrocytes measured by RT-qPCR. (G) The protein expression of DNMT3B in chondrocytes normalized to β-actin measured by Western blot analysis. (H) The methylation level of miR-29b in response to overexpression of DNMT3B determined by MSP. (I) The expression of miR-29b in response to overexpression of DNMT3B determined by RT-qPCR. *p t-test was conducted for comparison between the two groups. The one-way ANOVA was used for comparison among multiple groups followed by Tukey’s post hoc test. The experiment was repeated 3 times independently.

Figure 2. DNMT3B suppresses the expression of miR-29b by enhancing DNA methylation in the miR-29b promoter region. (A, B) The methylation level of miR-29b in cartilage tissues from OA patients (n = 46) and normal cartilage tissues (n = 46) detected by MSP (Nor, normal cartilage tissue; OA, cartilage tissues in patients with OA; U; unmethylated status; M, methylated status). (C) Immunofluorescence staining of Col2A1 and Aggrecan in chondrocytes (× 200, scale bar = 50 μm). (D) The methylation level of miR-29b in chondrocytes under IL-1β and 5-Aza treatment by MSP (U; unmethylated status; M, methylated status). (E) The expression of miR-29b in chondrocytes measured by RT-qPCR. (F) The mRNA expression of DNMT3B in chondrocytes measured by RT-qPCR. (G) The protein expression of DNMT3B in chondrocytes normalized to β-actin measured by Western blot analysis. (H) The methylation level of miR-29b in response to overexpression of DNMT3B determined by MSP. (I) The expression of miR-29b in response to overexpression of DNMT3B determined by RT-qPCR. *p < 0.05. Statistical data were measurement data and described as the mean ± standard deviation. The independent sample t-test was conducted for comparison between the two groups. The one-way ANOVA was used for comparison among multiple groups followed by Tukey’s post hoc test. The experiment was repeated 3 times independently.