Validating miRNA-target interactions

The interactions between LDLRAD3 and miR-20a-5p or miR-20a-5p and SLC7A5 were indicated by ENCORI. The oligo sequence of LDLRAD3 and 3′-UTR of SLC7A5 carrying the wild type or mutant binding sites of miR-20a-5p and were amplified using Q960 PCR instrument (Dinai, China) and inserted into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, USA) to create the plasmids (LDLRAD3-WT and LDLRAD3-MT; SLC7A5-WT and SLC7A5-MT). The mimic-NC and miR-20a-5p mimic were cotransfected with the above vectors using Hieff Trans Liposomal Transfection Reagent kit (Yeasen, China), and the luciferase activity was measured with DLR Gene Assay Kit (Yeasen, China) and GloMax 20/20 Luminometer (Promega, USA). The luminescence activity of firefly luciferase substrate was normalized to Renilla luciferase substrate [25 (link)].