http://www.mircode.org/) were used to identify the potential binding sequences of miR-140-5p and LINC00504. SKBR3 and MDA-MB-231 cells were seeded in 24-well plates. The cells were cotransfected with miR-140-5p or microRNA NC and luciferase reporter constructs containing WT-LIC00504 or Mut-LINC00504, as previously described.
13 The luciferase activity was detected using a Dual Luciferase Reporter Gene Assay Kit (RG027, Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer’s protocol. Mutations in the putative binding sites of LINC00504 were made using a Quick-change site-directed mutagenesis kit (200518, Agilent Technologies, Santa Clara, USA). The primers were as follows: LINC00504, Forward primer: 5ʹ-GTGACTCGAGCTTGCCTCTGCCATGT-3ʹ, Reverse primer: 5ʹ-GTGAGCGGCCGCTTTCAGAGTGAAACAATACTT-3ʹ; Mut-LINC00504, Forward primer: 5`-GGATTACAGGCGTGAGGGTGAC-3`; and Reverse primer: 5`-GTCACCCTCACGCCTGTAATCC-3`.