Figure 4.
The interaction between DLEU2 and miR-212-5p in the anti-tumor effects of Huaier. (A) DLEU2 wild-type (wt) sequence containing the putative miR-212-5p binding sites and DLEU2 sequence with the mutant (mut) miR-212-5p binding sites. (B) The miR-212-5p level in A549 cells and the noncancerous lung epithelial cell line BEAS-2B. (C) The miR-212-5p level in Huaier-treated and untreated A549 cells. (D and E) Luciferase reporter assay in HEK-293T. HEK-293T was cotransfected with pRL-TK carrying wild-type or mutant DLEU2 sequence and the miR-212-5p mimic or miR-212-5p inhibitor, and the luciferase activity was detected 48 h after transfection. (F) The miR-212-5p level in NSCLC cells transfected with DLEU2 siRNA (siDLEU2) or control (siNC). (G) The expression of DLEU2 in NSCLC cells transfected with miR-212-5p mimic or mimic control. (H) Colony formation assay in DLEU2-silenced A549 cells transfected with miR-212-5p inhibitor (siDLEU2 + miR-212-5p inhibitor) or inhibitor control (siDLEU2 + miRNA NC). Colony formation assay in Huaier-treated A549 cells transfected with miR-212-5p inhibitor (Huaier + miR-212-5p inhibitor) or inhibitor control (Huaier + miRNA NC). (I, J and K) A549 cells were treated as in (H). wound healing assay (I), transwell assay (J) and western blot assay (K) were performed.