Fig. 3

Linc-ROR shares miRNA binding sites with SOX9. a qRT-PCR analysis of the expression of linc-ROR and SOX9 in EC9706 cells transfected with 12 different miRNA mimics versus scramble control. b qRT-PCR analysis of linc-ROR and SOX9 expression after treatment with miR-145 inhibitor; a mixture of miR-15b, miR-33a, and miR-129 inhibitors (3 miR-inh mix); and inhibitor cocktail of miR-15b, miR-33a, miR-129, miR-145, and miR-206 (5 miR-inh mix). c qRT-PCR analysis of stemness-associated genes CD44, KLF4, NANOG, OCT4, and SOX2 expression following treatment with miR-145 mimics in EC9706 or 5 miR-inh mix in Eca109 cells. Transcription levels were normalized to GAPDH expression. d Prediction for five candidate miRNA-binding elements on linc-ROR transcript and SOX9 3′-UTR. e Sequence alignment of miR-15b, miR-33a, miR-129, miR-145, and miR-206 seed sequence in linc-ROR and SOX9 3′-UTR. f Amount of linc-ROR and SOX9 bound to Ago2 was determined by qRT-PCR in the presence of inhibitor cocktail of all candidate miRNAs or negative control. IgG was used as a negative control. g SOX9 protein level in EC9706 cells following treatment with linc-ROR siRNA or miR-145 mimics was measured by Western blot (top). Luciferase assay of 293 T cells co-transfected with pmiR-REPORT-SOX9 3′-UTR and indicated RNA oligos. siRNA targeting SOX9 3′-UTR was used as a positive control (bottom)