Fig. 2

HBEGF serves as a direct target of miR-760 and mediates the function of miR-760 in chondrocytes. A, B After 24 h of transfection, the expression of 6 different putative miR-760 target genes in chondrocytes was assessed via qPCR following miR-760 overexpression or inhibition. n = 3 (1 technical replicate on 3 different donors). Student's t test (t test) was used for comparisons between two groups. *p < 0.05. C After 48 h, CAM2KG and HBEGF protein levels were analysed via western immunoblotting following miR-760 inhibition or overexpression. n = 3 (1 technical replicate on 3 different donors). D, E A dual-luciferase reporter assay was used to analyse interactions between miR-760 and HBEGF (or CAM2KG) mRNA. HEK-293 T cells were cotransfected with miR-760 mimic or NC and a luciferase reporter construct containing WT or MUT HBEGF (or WT or MUT CAM2KG). n = 3 with 2 technical replicates. Data were analysed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for comparison between the control and treatment groups. *p < 0.05. F Analyses of HBEGF expression in OA-associated degenerative and nondegenerative cartilage tissue samples. n = 20. Student's t test (t test) was used for comparisons between two groups. *p < 0.05. G, H HBEGF expression levels in TNF-α (10 ng/mL)- and IL-1β (10 ng/mL)-treated chondrocytes. n = 3 (1 technical replicate on 3 different donors). Data were analysed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for comparison between the control and treatment groups. *, p < 0.05 compared with 0 h. miRNAs, microRNAs; qPCR, real-time quantitative PCR; WT, wild type; MUT, mutant; OA, osteoarthritis; TNF, tumour necrosis factor; IL, interleukin; NC, negative control