MiR-20a-5p facilitates cartilage repair in osteoarthritis via suppressing mitogen-activated protein kinase kinase kinase 2

Figures & data

Table 1. Primer sequences used for RT-qPCR

Gene	Sequence (5’$\to$3’)
mmu-miR-20a-5p forward	AGGGCTAAAGTGCTTATAGTGC
mmu-miR-20a-5p reverse	TCCTCCTCTCCTCTCCTCTC
Znfx1 forward	GCCGGGTGTCAGTTACAGG
Znfx1 reverse	CGGAAAGGAGCAGGATGGTT
Il25 forward	GGCAACTCCGTCCCACTTTA
Il25 reverse	GCAGAGTTGGCATGCACAAA
Ezh1 forward	TCAGGGTAGCTGGGCCTAAT
Ezh1 reverse	AGGGAATGGTTAGCAAGGGC
Map3k2 forward	GGAAGCTGTCCGTCACTTGA
Map3k2 reverse	GCCAGCTCTCTTCCGGTATC
Epha4 forward	AGATCCCAGCTCCCCTGAAT
Epha4 reverse	CACCAGCGATGGACAGAGTT
GAPDH forward	AGGTCGGTGTGAACGGATTTG
GAPDH reverse	GTAGACCATGTAGTTGAGGTCA
U6 forward	GTGCTCGCTTCGGCAGCACA
U6 reverse	TGGCAGGGTCCGAGGT

Figure 1. MiR-20a-5p is overexpressed during chondrogenic differentiation. a. Flow cytometry analysis for surface antigen expression of BMSCs. b. Morphological characteristics of BMSCs under a microscope. c. Alcian blue staining for detecting chondrogenic differentiation of BMSCs. d. Western blotting for evaluating protein levels of cartilage genes at 0, 7 and 14 days. e. RT-qPCR analysis of miR-20a-5p level. *p < 0.05, **p < 0.01.

Figure 2. Depletion of miR-20a-5p inhibits chondrogenic differentiation of BMSCs. a. RT-qPCR analysis for detection of transfection efficiency of miR-20a-5p inhibitor. b-c. Alcian blue staining for assessing chondrogenic differentiation of miR-20a-5p inhibitor-transfected BMSCs. d-e. Western blotting for assessing protein levels of cartilage genes (Aggrecan and Col2), catabolic markers (ADAMTS5, MMP13) and hypertrophic chondrocyte marker (Col10) in BMSCs with above transfection. **p < 0.01, ***p < 0.001.

Figure 3. Upregulation of miR-20a-5p ameliorates OA. a. HE staining for histopathological observation of knee articular cartilage. b. Modified Mankin scores assigned to cartilage tissues. c-e. Micro-CT images of the mice in four groups for assessments of BV/TV, Tb. Th, and Tb. Sp. f-g. Western blotting for assessing the protein levels of cartilage markers (Aggrecan and Col2), catabolic markers (ADAMTS5, MMP13) and hypertrophic chondrocyte marker (Col10). **p < 0.01, ***p < 0.001 versus Sham + AAV-NC group; ^##p < 0.01 versus Sham + AAV-miR-20a-5p group. n = 10 mice/group.

Figure 4. MiR-20a-5p targets Map3k2. a. miRDB used for screening miR-20a-5p target genes. b. RT-qPCR analysis of mRNA levels of the predicted genes in miR-20a-5p-depleted BMSCs. c. Western blotting for assessing Map3k2 protein level in above cells. d. TargetScan for predicting the binding site of miR-20a-5p on Map3k2. e. Luciferase reporter assay for identifying the binding relation between Map3k2 and miR-20a-5p in BMSCs. **p < 0.01, ***p < 0.001.

Figure 5. The expression of Map3k2 and miR-20a-5p in murine cartilage tissues was negatively correlated. a-b. RT-qPCR of miR-20a-5p and Map3k2 levels in murine cartilage tissues. c. The expression correlation between Map3k2 and miR-20a-5p analyzed by Pearson analysis. ^&&p < 0.01 versus Sham + AAV-NC group; **p < 0.01 versus Sham + AAV-NC group; ^##p < 0.01 versus OA + AAV-NC group.

Figure 6. Map3k2 knockdown reverses the suppressive influences of miR-20a-5p inhibition on chondrogenic differentiation. a. Western blotting of Map3k2 protein level in BMSCs transfected with Sh-Map3k2. b-c. Alcian blue staining for examining chondrogenic differentiation of BMSCs transfected with miR-NC, miR-20a-5p inhibitor or miR-20a-5p inhibitor + Sh-Map3k2. d-e. Western blotting of Aggrecan, Col2, ADAMTS5, MMP13 and Col10 protein levels in BMSCs with above transfection. **p < 0.01, ***p < 0.001 versus sh-NC/miR-NC group; ^#p < 0.05, ^##p < 0.01 versus miR-20a-5p inhibitor group.

Data Availability Statement

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.