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 upregulates PTHLH through the downregulation of miR-29b in IL-1β-treated chondrocytes. (A) Prediction of the binding site between miR-29b and PTHLH available at the Targetscan database. (B) The mRNA expression of PTHLH in OA cartilage tissues (n = 46) and normal cartilage tissues (n = 46) by RT-qPCR. (C) Pearson’s analysis of the correlation between PTHLH and miR-29b expression. (D) Pearson’s analysis of the correlation between PTHLH and DNMT3B expression. (E) Luciferase activity of cells co-transfected with PTHLH-WT or PTHLH-MUT and miR-29b mimic or mimic-NC. The firefly luciferase activity was normalized to Renilla luciferase activity. (F) The protein expression of PTHLH in response to transfection with miR-29b mimic or inhibitor normalized to β-actin determined by Western blot analysis. (G) The expression of DNMT3B after transfection with oe-DNMT3B or si-DNMT3B measured by RT-qPCR or Western blot analysis. (H) The protein expression of PTHLH in response to transfection with oe-DNMT3B or si-DNMT3B normalized to β-actin determined by Western blot analysis. (I) The protein expression of PTHLH in response to co-transfection with oe-DNMT3B and miR-29b mimic or si-DNMT3B and miR-29b inhibitor normalized to β-actin determined by Western blot analysis. *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 3. DNMT3B upregulates PTHLH through the downregulation of miR-29b in IL-1β-treated chondrocytes. (A) Prediction of the binding site between miR-29b and PTHLH available at the Targetscan database. (B) The mRNA expression of PTHLH in OA cartilage tissues (n = 46) and normal cartilage tissues (n = 46) by RT-qPCR. (C) Pearson’s analysis of the correlation between PTHLH and miR-29b expression. (D) Pearson’s analysis of the correlation between PTHLH and DNMT3B expression. (E) Luciferase activity of cells co-transfected with PTHLH-WT or PTHLH-MUT and miR-29b mimic or mimic-NC. The firefly luciferase activity was normalized to Renilla luciferase activity. (F) The protein expression of PTHLH in response to transfection with miR-29b mimic or inhibitor normalized to β-actin determined by Western blot analysis. (G) The expression of DNMT3B after transfection with oe-DNMT3B or si-DNMT3B measured by RT-qPCR or Western blot analysis. (H) The protein expression of PTHLH in response to transfection with oe-DNMT3B or si-DNMT3B normalized to β-actin determined by Western blot analysis. (I) The protein expression of PTHLH in response to co-transfection with oe-DNMT3B and miR-29b mimic or si-DNMT3B and miR-29b inhibitor normalized to β-actin determined by Western blot analysis. *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.