Figure 5.

FXR1 promotes the maturation of pre-miR-503 in keloid-derived fibroblasts. (A) Bioinformatics-based (starBase) prediction of FXR1 binding sites on pre-miR-503. (B) Pre-miR-503 levels in keloid and adjacent normal skin tissue were measured by RT-qPCR. (C) Association between pre-miR-503 levels in keloid and adjacent normal skin tissue was determined. (D and E) Pre-miR-503 levels in normal human fibroblasts and keloid-derived fibroblasts in the presence or absence of FXR1 knockdown was evaluated by RT-qPCR. (F and G) miR-503-3p and miR-503-5p levels in normal human fibroblasts and keloid-derived fibroblasts. (H and I) miR-503-3p and miR-503-5p levels in keloid-derived fibroblasts in the presence or absence of FXR1 knockdown was detected by RT-qPCR. (J) Keloid-derived fibroblast lysate was prepared for RNA pulldown with biotinylated pre-miR-503 or antisense transcripts and western blotting analysis was performed using anti-FXR1. GAPDH was used as a control. (K) Interaction between pre-miR-503 and FXR1 was investigated by immunoprecipitation of pre-miR-503 using whole-cell lysate of 293T cells, followed by immunoblotting for FXR1. (L) RNA immunoprecipitation assay was performed using anti-FXR1 or anti-IgG antibodies and pre-miR-503 levels were measured via RT-qPCR; GAPDH and TERC mRNA were used as the negative and positive controls, respectively. (M) Co-immunoprecipitation was performed to investigate the association between Dicer and FXR1 in 293T cells. Northern blot assay was performed to assess the levels of mature miR-503 processed from transfected pre-miR-503 in 293T cells (N) following FXR1 overexpression or (O) in the absence of FXR1. ^**P<0.01 vs. Ctrl or human normal fibroblasts. FXR1, Fbxo4-mediated FMR1 autosomal homolog 1; miR, microRNA; RT-q, reverse transcription-quantitative; TERC, telomerase RNA component; Ctrl, control; si, small interfering.