Research Paper Volume 13, Issue 2 pp 2864—2884

Figure 3. The underlying mechanism between LHPP, miR-217 and GAS5. (A) A schematic diagram of the miR-217 sequence with LHPP and with LHPP mutated at the putative binding site. (B) Luciferase reporter activity in A549/DDP and H1299/DDP cells was measured after co-transfection with pPG-miR-217 (or the empty vector as a control) and the luciferase empty vector (pmiR-GLo), or the vector containing the wild-type LHPP (pmiR-GLo-LHPP-wt) or mutant transcripts (pmiR-GLo-LHPP-mut). (C) Luciferase reporter activity in A549/DDP and H1299/DDP cells was measured after co-transfection with pPG-miR-217 (or the empty vector as a control) and the luciferase empty vector (pmiR-GLo), or the vector containing the wild-type GAS5 (pmiR-GLo-GAS5-wt) or mutant transcripts (pmiR-GLo-GAS5-mut). (D) The amount of GAS5 bound to SNRNP70 (positive control), Ago 2 or lgG (negative control) was determined by qRT-PCR after RIP in H1299 cells. MiR-217 exert their miRNA-mediated gene silencing function by binding to Ago2, a core component of the RNA-induced silencing complex (RISC). When miR-217 forms a RISC complex, it is wrapped by Ago protein, mainly Ago2. Pulling down the Ago2 protein will pull down the miR-217 bound to it. LncRNA GAS5 bound by RISC will also be pulled down. GAS5 expression levels were measured by qPCR. (E) A549/DDP cells were transfected with biotinylated NC (Bio-NC), biotinylated wild-type miR-217 (BiomiR-217) or biotinylated mutant miR-217 (Bio-miR-217-mut), and biotin-based miRNA pull-down assays were conducted after 48 h of transfection. biotinylated wild-type miR-217 can combine lncRNA GAS5. Bead combines biotinylated and lncRNA GAS5 will be pulled down. GAS5 expression levels were measured by qPCR. *p < 0.05, **p < 0.01, ***p < 0.001. The same experiments were performed at least three times.