Luciferase reporter assay
For transfections, host H9C2 cells were plated in triplicate into
24-well plates containing Lipofectamine 2000 (Invitrogen, Carlsbad, USA)
and 0.8 μg pEZX-miR-210 (or pEZX-miR-Sc) per well. The precursor miR-210
expression clone was established via a
feline
immunodeficiency virus using the lentiviral vector system
(pEZX-miR-210). Subsequently, we designed the luciferase reporter
targeting the 3-UTR of Caspase8ap2 to surround the
binding sites of miR-210. The
Dual-Luciferase Reporter Assay System kit (Promega, Madison, USA) was
used to measure the amount of luciferase and Renilla luciferase activity
with a fluorescence spectrophotometer. The relative transcriptional
activity was normalized to its
corresponding value.