Fig. 4

ADAMTS1 is a direct target of miR-548k in ESCC. a, Candidate targets of miR-548k. b, The predicted binding sequence of human hsa-miR-548k and its binding site in the 3′-untranslated region (3’-UTR) of ADAMTS1 were presented for alignment. ADAMTS1–3’-UTR-mut was the mutated sequences of 3’-UTR of ADAMTS1 without the binding sites of miR-548k. c, Quantitative real time PCR results show that ADAMTS1 could be downregulated KYSE30 cells (left) and KYSE510 cells (right) (p < 0.001, t-test). Data were expressed as mean ± S.E.M of three independent experiments. d, Luciferase assay was used to confirm the interaction of miR-548k with ADAMTS1. 3’-UTR of ADAMTS1 containing the target binding site (ADAMTS1–3’-UTR-wt) and the mutated sequences of 3’-UTR of ADAMTS1 (ADAMTS1–3’-UTR-mut) were cloned into downstream of a firefly luciferase gene. The plasmids were transfected into empty vector and miR-548k stably expressing cells (KYSE30-lenti-miR-548k). Renilla luciferase plasmid was co-transfected for normalisation. GV126-Control vector was co-transfected as positive control (*p < 0.05). e, Western blot analysis the expression level of ADAMTS1 in miR-548k overexpression cells and control cells. f, Ectopic expressed ADAMTS1 open reading frame plasmid without 3’-UTR (cannot be targeted by miR-548k) in miR-548k overexpression cells (KYSE30-Lenti-548 k and KYSE510-Lenti-548 k) and control cells. g and h, Representative images (left) and quantitative data (right) of HDLECs cultured with conditioned medium derived from miR-548k overexpressing cells and control cells with or without ectopic expression of ADAMTS1. g, Transwell migration assays, left representative images; right, quantitative data. h Matrigel tube formation assay, left representative images; right, quantitative data. CM, conditioned medium. Error bars represent the mean ± S.E.M from three independent experiments, ns, no significance