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. 2021 Jan 14;41(1):BSR20203130. doi: 10.1042/BSR20203130

Figure 3. Circ_0061012 interacts with miR-194-5p in HaCaT cells.

Figure 3

(A) The possible miRNA targets of circ_0061012 were predicted by StarBase software, and the putative binding sites between miR-194-5p and circ_0061012 were shown. The mutant miR-194-5p-binding sites in circ_0061012 were also shown. (B) Dual-luciferase reporter assay was implemented to test the interaction between miR-194-5p and circ_0061012. Luciferase plasmid (WT-circ_0061012 or MUT-circ_0061012) and miR-194-5p or miR-NC were co-transfected into HaCaT cells, and the luciferase intensities in four groups were determined after transfection for 48 h. (C) RIP assay was used to confirm the interaction between miR-194-5p and circ_0061012. Ago2 antibody was used to pull down miRNA-contained RNA-induced silencing complex (RISC) to test if there was spatial interaction between miR-194-5p and circ_0061012 in RISC. (D) The expression of miR-194-5p in healthy skin tissues (n=27) and psoriasis skin tissues (n=27) was analyzed by qRT-PCR. (E) Linear correlation between the expression of miR-194-5p and circ_0061012 was analyzed by Spearman’s correlation coefficient. (F) The level of miR-194-5p was determined in HaCaT cells treated with different doses of IL-22 (50, 100 or 200 ng/ml) by 24 h via qRT-PCR. (G) HaCaT cells transfected with circ_0061012 or Vector were subsequently exposed to 100 ng/ml IL-22 for 24 h, and the expression of circ_0061012 was examined by qRT-PCR. (H) qRT-PCR was implemented to measure the expression of miR-194-5p in Control group, IL-22 group, IL-22 + si-NC group, IL-22 + si-circ_0061012 group, IL-22 + Vector group and IL-22 + circ_0061012 group. The experiments were independently repeated for three times with at least three technical repetitions. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.