Quick site directed mutation kit

The link between HDAC5 3ʹ-UTR and miR-331-3p was predicted by TargetScan (Version 7.1, http://www.targetscan.org/vert_71/). The sequence of the HDAC5 3ʹ-UTR containing the predicted miR-331-3p binding site was cloned into pGL2-Basic Vector (Promega, Madison, WI, USA), and named as HDAC5-WT, while mutated HDAC5 3ʹ-UTR which was obtained by introducing two site mutations into pGL2-HDAC5 plasmid with a Quick Site-directed mutation kit (Agilent Technologies, Inc.) was used as NC (HDAC5-MUT). Next, the vector (pGL2-HDAC5-WT or pGL2-HDAC5-MUT) was co-transfected with miR-331-3p mimic and miR-NC mimic into A549 and CALU‐1 cells. The A549 and CALU‐1 cells were harvested at 48 h after transfection, next, the luciferase activity was measured by the Dual Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions. Firefly luciferase was normalized to Renilla luciferase (Promega).

The target of miR-155 was predicted by the online database Targetscan 7.2 [http://www.targetscan.org/vert_72/ (28 (link))]. The 3'UTR of TGFBR2 containing the predicted miR-155 specific binding site was amplified by PCR from genomic DNA, and inserted into the pMIR-REPORT™ luciferase reporter vector (Thermo Fisher Scientific, Inc.) to obtain the wild-type luciferase reporter plasmid p-TGFBR2-wt. PCR was performed using the following thermocycling conditions: 94˚C for 2 min, followed by 35 cycles of 94˚C for 2 sec, 60˚C for 60 sec and 72˚C for 1 min. The PCR products were amplified using cDNA and fused to the firefly luciferase gene of the pGL3-control plasmid (Promega Corporation) with the restriction enzyme sites of KpnI and XhoI. Two site mutations were introduced to WT-TGFBR2-3'-UTR to construct the mutant (MUT) TGFBR2-3'-UTR using a Quick Site-directed mutation kit (Agilent Technologies, Inc.). The cells were co-transfected with pGL3 constructions including 200 ng pGL3-WT-TGFBR2 and 200 ng pGL3-Mut-TGFBR2, 10 nM miR-NC or 10 nM miR-155 mimics and 26 ng pRL-TK in 24-well plates using Lipofectamine® 2000 (Invitrogen, USA). At 24 h of transfection, luciferase activity (firefly and Renilla) was determined using the dual-luciferase reporter assay system (Promega, USA).

TargetScan release 7.1 (http://www.targetscan.org/vert_71/) was used in the present study for the prediction of binding site between miR-27b and VEGF. The 3′UTR of VEGF mRNA was amplified from the cDNA of HEB cells and inserted into a pGL3-basic plasmid (Promega Corporation). The pGL3-VEGF 3′UTR-mutant (Mut) was created by introducing VEGF 3′UTR site mutations using a Quick Site-Directed Mutation kit (Agilent Technologies, Inc.). U251 and U87 cells were co-transfected with miR-27b mimics (20 nM) or miR-NC (20 nM), pGL3-VEGF 3′UTR-wild type (WT; 0.4 mg) or pGL3-VEGF 3′UTR-Mut (0.4 mg) and Renilla luciferase vectors using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. A dual luciferase assay was performed at 48 h after transfection using a Dual Luciferase kit (Promega Corporation). Activities were normalized to that of Renilla luciferase.