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. 2021 Apr 13;101(8):1060–1070. doi: 10.1038/s41374-021-00595-5

Fig. 4. The prediction and validation of miR-96-5p targets.

Fig. 4

a Online programs predict putative osteoarthritis-related target genes of miR-96-5p. TargetScanHuman identified 573 target genes (Context++ score < −0.15), and MicroRNA.org identified 358 target genes (mirSVR score < −0.8). One hundred and twenty-two candidate’s genes were designated putative target genes of miR-96-5p by both programs. b The expression of the 122 putative target genes was determined by real-time PCR in AMs transfected with miR-96-5p mimics or anti-miR-96-5p. c The expression of 12 potential target genes (HIF-2α, CHMP2B, BASP1, CTNND1 EPHA3, MPP1, MSN, PLOD2, RPS6KA6, RUNDC3B, ST7, and XKR4) was determined by real-time PCR in murine chondrocytes transfected with miR-96-5p mimics or anti-miR-96-5p. d Predicted miR-96-5p binding sites in the 3ʹ-UTR of HIF-2α. e Luciferase reporter assay with cotransfection of wild-type or mutant HIF-2α and miR-96-5p mimics, anti-miR-96-5p, mimics-control, or anti-miRNA-control in murine chondrocytes. f, g Western blot analysis of HIF-2α expression in chondrocytes transfected with miR-96-5p mimics, anti-miR-96-5p, mimic-control, or anti-miRNA-control. h, i Immunohistochemistry of HIF-2α expression in cartilage tissues transfected with miR-96-5p mimics, anti-miR-96-5p, mimic-control, or anti-miRNA-control. Bar = 50 μm. All the treatments in this figure were carried out in triplicate, and the results are displayed as the mean ± SD. *P < 0.05.