Fig. 3

CircNTRK2 acts as a sponge for miR-140-3p of in ESCC cells. (a) The subcellular distribution of circNTRK2 in Eca-109 and KYSE-150 cells was determined by nuclear mass separation experiment. (b) Venn diagram showed the overlapping candidate targets of circNTRK2 predicted by CircInteractome, CircBank, and StarBase 3.0. (c) Schematic representation of miR-140-3p binding sites on circNTRK2. The red part is the mutated sequences of circNTRK2. (d) The level of miR-140-3p in Eca-109 and KYSE-150 cells transfected with si-circNTRK2 #2 or circNTRK2 was measured via qRT-PCR. (e) qRT-PCR was performed to evaluate the the expression of circNTRK2 in Eca-109 and KYSE-150 cells with miR-140-3p overexpression or depletion. (f) Dual-luciferase reporter assay was performed in Eca-109 and KYSE-150 cells after transfection with miR-NC or miR-140-3p and circNTRK2-wt or circNTRK2-mut reporter. (g) RIP assays were carried out in miR-NC- or miR-140-3p-transfected Eca-109 and KYSE-150 cells by using anti-Ago2 or anti-IgG, then qRT-PCR was used to examine the enrichment of circNTRK2 in immunoprecipitated RNA. (h) RNA pull-down assay was performed to confirm the direct binding between circNTRK2 and miR-140-3p. (i) The level of miR-140-3p in ESCC tissues was tested by qRT-PCR. (j) Correlation between miR-140-3p and circNTRK2 was detected in ESCC tissues via Pearson’s test. (k) Differential expression of circNTRK2 in ESCC cell lines (Eca-109, EC-9706, KYSE-30, KYSE-150, TE-1) and normal esophageal epithelial cell line Het-1A. *P < 0.05, **P < 0.01