Research Paper Volume 12, Issue 16 pp 16304—16325

Figure 5. HOXD-AS1 functioned as a sponge for miR-520c-3p in CCA cells. (A) Subcellular localization of HOXD-AS1 was tested by subcellular fractionation assays. GAPDH and U6 were used as endogenous controls for cytoplasm and nucleus, respectively. (B) The expression levels of predicted miRNAs were detected after knocking down or amplifying HOXD-AS1 in CCLP-1 and QBC939 cells, respectively. (C) The expression of miR-520c-3p in CCA tissues and paired adjacent nontumor bile duct tissues. (D) The correlation between relative HOXD-AS1 expression and relative miR-520c-3p expression in CCA tissues. (E) The miR-520c-3p expression in CCA cells (CCLP-1, QBC939, TFK-1, RBE) and normal HIBEC. (F) Luciferase reporter plasmids were constructed with miR-520c-3p-binding site region of HOXD-AS1 sequence, including wild type and mutant type. (G) Luciferase reporter assays showed that cotransfected miR-520c-3p mimics significantly inhibited luciferase activity of HOXD-AS1 wild type. (H) AGO2 RIP assays further suggested the binding of miR-520c-3p to HOXD-AS1. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.