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. 2019 Mar 28;78(6):826–836. doi: 10.1136/annrheumdis-2018-214786

Figure 3.

Figure 3

CircSERPINE2 serves as a sponge for miR-1271 in HC cells. (A) Number of RBP sites between RBPs and CircSERPINE2 is shown. (B) AGO2 RIP assay was performed to detect CircSERPINE2 levels in SW1353 cells stably expressing AGO2. n=3. *p<0.05. (C) miRNA-mediated differential dysregulation of miRNAs was detected by miRNA high-throughput sequencing in OA cartilage samples and control cartilage samples (n=3). (D) Schematic illustration to show the overlapping of the target miRNAs of CircSERPINE2, as predicted by Miranda, TargetScan and miRNAs upregulated in OA identified by sequencing analysis. (E) Lysates prepared from SW1353 cells were subjected to an RNA pull-down assay and tested with RT-qPCR. Relative levels of CircSERPINE2 were normalised to the levels of input. n = 3. *p<0.05 versus control (lac Z) probe. (F–G) miR-1271 expression was higher in human OA cartilage than in control cartilage tissue, as determined by FISH (F, n=3; three different donors; *p<0.05; mean with 95% CI) and RT-qPCR (G, n=10, *p<0.05). Representative images are shown (scale bar: 50–200 µm). *p<0.05. (H) HEK-293T cells were co-transfected with miR-1271 mimic or NC and a luciferase reporter construct containing WT or MUT CircSERPINE2. n = 6. *p<0.05. Mean with 95% CI. (I) Fish images showing the co-localisation of CircSERPINE2 and miR-1271 in HCs. miR-1271 probes were labelled with Alexa fluor 488, whereas CircSERPINE2 probes were tagged with Cy3. Nuclei were stained with DAPI. n = 3 (three different donors). Scale bar, 50 µm. (J–L) HCs were transfected with miR-1271 mimic, inhibitor or NC at a final concentration of 20 nM. After 48 hours of transfection (J, n = 3; three different donors), protein levels of MMP3, MMP13, ADAMTS4, SOX9, COL2A1 and aggrecan in chondrocytes were detected; IF assay for MMP13, aggrecan, COL2A1 and ADAMTS4 expression detection in chondrocytes was performed (K, n = 3; three different donors; scale bar: 50 µm). mRNA levels of MMP3, MMP13, ADAMTS4, SOX9, COL2A1 and aggrecan in chondrocytes were evaluated (l, n=9; three different donors for three different experiments, *p<0.05). AGO2, Argonaute 2; DAPI, 4′,6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridisation; HC, human chondrocyte; IF, immunofluorescence; miRNAs, microRNAs; MUT, mutant; NC, negative control; OA, osteoarthritis; RBP, RNA-binding protein; RIP, RNA immunoprecipitation; RT-qPCR; real-time quantitative PCR; WT, wild type.