Rno_circ_0005139 regulates apoptosis by targeting Wnt5a in rat anorectal malformations

Figure 4 Rno_circ_0005139 directly binds to miR-324-3p, a direct upstream target of Wnt5a. A: Relative expression of microRNA (miR)-324-3p in anorectal malformations and normal control anorectal tissues was detected by quantitative reverse transcription polymerase chain reaction (qRT–PCR); B: Luciferase reporter assays were conducted to explore the correlation between rno_circ_0005139 and miR-324-3p; C: Expression of miR-324-3p was detected after transfection with si-rno_circ_0005139 (si-circ) and a negative control (si-NC) in IECs by qRT–PCR; D: Luciferase reporter assays were conducted to explore the correlation between miR-324-3p and Wnt5a; E: Expression of miR-324-3p was detected after transfection with miR-324-3p mimic, miR-324-3p inhibitor, or a negative control (mimic-NC/inhibitor-NC) by qRT–PCR. Expression of Wnt5a was detected after transfection with miR-324-3p mimic, miR-324-3p inhibitor, or negative control (mimic-NC/inhibitor-NC) by qRT–PCR; F and G: mRNA and protein expression levels of Wnt5a were detected after transfection with miR-324-3p mimic, miR-324-3p inhibitor, or negative control (mimic-NC/inhibitor-NC) by qRT–PCR and western blotting. ^aP < 0.05 vs normal, ^bP < 0.01 vs r-rno_circ_0005139-WT+NC, ^cP < 0.05 vs r-rno_circ_0005139-MUT1+NC, ^dP < 0.05 vs si-circ_0005139-NC; ^eP < 0.05 vs r-Wnt5a-WT+NC, ^fP < 0.05 vs r-Wnt5a-MUT+NC, ^gP < 0.05 vs Normal control, ^hP < 0.05 vs miR-324-3p mimic NC and ⁱP < 0.05 vs miR-324-3p inhibitor NC. ARM: Anorectal malformations; miR: microRNA.