Pmir rb report luciferase vector

HEK-293 T cells were seeded into a 96-well plate and allowed to reach 50–70% confluence prior to transfection. The pmiR-RB-Report™ luciferase vector (RiboBio, China) was modified by the introduction of either wild-type or mutant TET2 fragments (524 bp) into its restriction sites and designated as LUC-WT-TET2 or LUC-MUT-TET2, respectively. The transfection was performed using lipofectamine 3000 (Invitrogen, USA) with 150 ng of plasmids of LUC-WT-TET2 and LUC-MUT-TET2, and then exposed to 100 nM of m-NC and miR-20b-5p mimic, 300 nM of i-NC and miR-20b-5p inhibitor. The culture medium was replaced 6 h post-transfection. After 48-h incubation, the Dual-Luciferase system (Promega, USA) was utilized to determine the firefly and Renilla luciferase activities. The medium was aspirated, and the cells were treated with 50 μL of PBS and luciferase reagent. The 96-well plate was then incubated on a shaker at room temperature for 10 min before the determination of firefly luciferase activity. The Renilla luciferase activity was determined by adding 30 μL of stop reagent per well and shaking for 10 min. The luciferase activities were then detected using Agilent BioTek Synergy LX. Finally, the differences between firefly and Renilla luciferase activities were calculated to determine the relative luciferase activity.

Cloning of 3′UTR of ZHX1 into pmiR-RB-REPORT™ luciferase vector, transfection and validation were conducted by Ribo Bio (China).

The region of the HDAC4 3′-UTR containing the predicted seed sequences for miR-381 was PCR amplified using the following primers: Forward, 5′-CGGGCGATCGCTGGAGGTTGCATGGACTGT-3′; Reverse, 5′-AATGCGGCCGCAACACGCTCAGCTTCGTTA-3′. The 3′-UTR fragment was then inserted into the pmiR-RB-REPORT™ luciferase vector (RiboBio) using the SgfI/NotI restriction sites, generating Luc-HDAC4-3′-UTR. Meanwhile, the following primers were used for mutation of two predicted seed sequences within the HDAC4 3′-UTR, generating Luc-HDAC4-3′-UTR-mut: Site 1 forward, 5′-CGGGCGATCGCTGGAGGTTGCATGGACTGTACGACCGGCATGACTTTATAAACATAACAGATTTTGCACGCCAA-3′ and Site 1 reverse 5′-AATGCGGCCGCAACACGCTCAGCTTCGTTA-3′; Site 2 forward, 5′-AGAGTTTAAACATATTATGTGGAAACAGTGTT-3′ and Site 2 reverse, 5′-CCACATAATATGTTTAAACTCTATTATTGGTA-3′.
For luciferase assay analyses, SW1353 cells were seeded into 96-well plates and transfected with 50 nM miR-381 mimic or NC mimic, and 100 ng Luc-HDAC4-3′-UTR or Luc-HDAC4-3′-UTR-mut using Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer’s instructions. The transfected cells were cultured in fresh culture medium for an additional 48 h. Luciferase reporter assays were then performed using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA), according to the manufacturer’s instructions.