Fig. 2

CircZNF800 sponges miR-140-3p, miR-382-5p & miR-579-3p to up-regulate expression of ALK7, FZD3 & WNT5A. A, B Properties of in vitro transcribed and circularized circZNF800: RNase R resistance shown in a 2% formaldehyde-agarose gel (A) and co-localization of circZNF800-Cy3 RNA transfected in WiDr cells observed under a fluorescence microscope (B). C, D For CRISPR-Cas13d knockdown of circZNF800, the two CRISPR RNA (crRNA) constructs, crRNA1 and crRNA2 (C), and the knockdown efficiency (D) are shown. In (D), CrSC was a crRNA construct with scrambled sequence used as a reference control. Relative expression levels of circZNF800 and ZNF800 post-crRNA transfection were determined by qRT-PCR, normalizing to the levels of the control crSC-transfected cells. **p < 0.01 and ***p < 0.001 were values relative to the CrSC cells. E Mapping of the miRNA seed sequence-interacting sites on circZNF800 (top panel) and the 3’-UTR sequences of the targeted transcripts (bottom panel) (see Suppl. Figure S1 for sequence alignment details). The relative positions of the miRNAs, the GenBank accession numbers of the mRNA sequences and the 3’-UTR lengths are shown. CDS, coding sequence; An, polyA tail. F RNA pulldown assays using biotinylated circZNF800 in HCT-15 and WiDr cells. The pulled-down RNA products were quantified in stem loop qRT-PCR (see Suppl. Table S1 for primer details) using a scramble RNA as a control and normalized to 1.0. G, H CircZNF800 over-expression (OE) sponges while knockdown (KD) up-regulates expression of the targeted miRNAs (G) and mRNAs (H) in CRC cells relative to the controls without OE and KD transfection, normalized to 1.0 as shown by the horizontal control (Ctrl) dashed lines. OE and KD were achieved by transfection of the in vitro synthesized circZNF800 and the CRIPSR constructs, respectively, in CRC cells for 48 h before RNA quantification by stem loop qRT-PCR relative to the control values (horizontal Ctrl dashed lines). *P < 0.05, **P < 0.01 and ***P < 0.001 were values relative to the specified controls