Fig. 4

HBEGF reverses the effects of miR-760 on OA progression. A, B After 24 and 48 h of transfection, OA-associated cartilage metabolism gene expression at the mRNA (A, n = 3 with 3 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) and protein (B, n = 3; three different donors) levels were assessed in chondrocytes following miR-760 mimic transfection plus HBEGF overexpression. C, D OA-associated cartilage metabolism gene expression at the mRNA (C, n = 3 with 3 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) and protein (D, n = 3; three different donors) levels were assessed in chondrocytes following miR-760 inhibitor transfection plus HBEGF knockdown. The miR-760/HBEFG axis regulates OA progression in vivo. Sham: incision of the right knee of mice that did not undergo ACLT surgery. ACLT: right knee articular cartilage that underwent anterior cruciate ligament transection. E, F Changes in cartilage metabolism-related gene expression at the mRNA level (E, n = 3 with 3 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) and protein level (F, n = 3; three different animals) in the control group (Sham + PBS) and two OA experimental groups (ACLT + AVV-miR-760 mimic or ACLT + AAV-miR-760 mimic + AAV-OE HBEGF). G Safranin-O/fast green staining and COL2A1, SOX9 IHC staining in the control mice (Sham + PBS), and ACLT-induced OA articular cartilage (medial tibia) of mice injected with miR-760 mimic or miR-760 mimic + HBEGF overexpression. n = 6; six different animals. Scale bars = 50 μm. H OARSI scoring was performed according to staining results in the control group (Sham + PBS) and two OA experimental groups (ACLT + AVV-miR-760 mimic or ACLT + AAV-miR-760 mimic + AAV-OE HBEGF); n = 6; six different animals. 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 < 0.01. I, J Positive chondrocyte percentages of COL2A1 and SOX9 in the control group (Sham + PBS) and two OA experimental groups (ACLT + AVV-miR-760 mimic or ACLT + AAV-miR-760 mimic + AAV-OE HBEGF). Six mice were evaluated for each group, and eight sections at different sites were measured for each mouse. 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, ***p < 0.001. OA, osteoarthritis; IHC, immunohistochemistry; ACLT, anterior cruciate ligament transection; miRNAs, microRNAs; ECM, extracellular matrix. OE, overexpression; Sh, short hairpin RNA; NC, negative control