Generation of dual-luciferase miRNA target
expression constructs and their stable transfectants. (a)
Commercially available original vectors, pmirGLO and psiCHECK-1,
which were designed to quantitatively evaluate miRNA activity via
the insertion of miRNA target sites on the 3′ UTR of the
firefly gene (luc2) (pmirGLO) or 3′ UTR of
the Renilla gene (hRluc) (psiCHECK-1). (b)
Insertion of annealed oligonucleotide pairs, which contain the
miR-182 (or miR-183) target sequence with appropriate restriction
sites (PmeI and XbaI for pmirGLO, SgfI and PmeI for psiCHECK-1)
downstream of luc2 in the pmirGLO and downstream of
hRluc in the psiCHECK-1. These vectors were
digested with KpnI and BamHI and the fragments which contained the
reporter units were isolated/ligated. (c) The final construct
consists of a dual reporter system utilizing both firefly and
Renilla luciferase. (d) (1) Using SHSY5Y cells, stable transfectants
of the engineered reporter constructs were created (specific for
either miR-182 [represented] or miR-183). (2) In order to maintain
minimal basal levels of activity of both luciferases, the stable
host cells were transduced with lentiviral particles containing
miR-182 (or miR-183) shMIMIC microRNAs. (3) These stable
transfectants (miR-182 or miR-183 target sequence in
pmirGLO/psiCHECK1 plus lentiviral particles containing miR-182 or
miR-183 shMIMIC) were usable for high-throughput screens. The final
stable reporter cell line was designed to identify small compounds
(e.g. HDAC inhibitor Panobinostat) that inhibit miR-182 (and/or
miR-183) generation or function, thereby resulting in the activation
of the luciferases (both firefly and Renilla).