Fig. 3: AC008 interacts with miR-328-3p.

a The expression level of AC008 in the nucleus and cytoplasm of primary chondrocytes was analyzed by qRT-PCR. β-actin and U6 were used as internal controls for the cytoplasmic and nuclear fractions, respectively. b FISH was performed to determine the subcellular localization of AC008 in chondrocytes. 18S rRNA and U6 were labeled with cyanine 3 (Cy3, red), and nuclei were stained with DAPI (blue). 18S rRNA and U6 were used as cytoplasmic and nuclear markers, respectively. Scale bar: 10 μm. c RIP experiments were performed in primary chondrocytes, and the coprecipitated RNAs were subjected to qRT-PCR to analyze AC008 expression. The fold enrichment of AC008 in the anti-Ago2 precipitate is shown relative to that in the corresponding IgG control precipitate. d The potential miR-328-3p binding sites in AC008 were predicted with the RNAhybrid database. e The expression level of miR-328-3p in normal and osteoarthritic cartilage was evaluated by qRT-PCR (n = 39). f The association between AC008 and miR-328-3p expression in osteoarthritic cartilage was assessed by Spearman correlation analysis. g Top: the designed wild-type AC008 sequence and mutant AC008 sequence (in which only the putative miR-328-3p binding site was mutated). Bottom: luciferase reporter assay in HEK293T cells transfected with psiCHECK2-AC008 (WT or Mut) and the miR-328-3p mimic or mimic control. h qRT-PCR was performed to determine the expression level of miR-328-3p in chondrocytes after transfection with pcDNA-AC008 or AC008 ASO. OA osteoarthritis, WT wild-type, MUT mutant, ns no significant difference. The data are presented as the means ± SDs. Statistical differences were determined using unpaired two-tailed Student’s t test (c, e, g, h) or Spearman correlation analysis (f). ^**P < 0.01; ^***P < 0.001.