Pgl3 vector

Three online prediction programs, TargetScan, miRanda, and PicTar, were used to identify candidate targets for miR-144. The gene for ADAM10, predicted by all three databases and associated with cognitive function, was further studied as a potential target. Sequence of segments with WT or mutant 3’-UTR region of ADAM10 were synthesized and cloned into the pGL3 vector (GeneChem, Shanghai, China). All constructs were verified by sequencing. First, the N2A or HEK293 cells were seeded at 0.5×10⁵ cell per well in 24-well plates 24 h prior to transfection. The following day, the pGL3 vector containing WT 3’-UTR of ADAM10 mRNA or mutant forms was co-transfected with LV-miR-144 into N2A or HEK293 cells. After 48 h, all cells were harvested according to manufacturer's protocol (Promega, Madison, WI), and the Firefly and Renilla luciferase activity were determined using dul-luciferase reporter assay system (Promega, Madison, WI) with a Victor X machine (PerkinElmer, Boston, MA). Firefly luciferase activity was normalized to Renilla luciferase activity. Three independent experiments were performed in triplicate.

Wild-type (WT) or mutant of 3′ untranslated region (Mut) sequences of ColA1 were inserted into the Fse I and Xba I sites of the pGL3 vector (GeneChem). Cells were co-transfected with pRL-TK and collected according to the manufacturer's protocol (Promega)

Lentiviruses carrying miR-346 or a miRNA negative control (miR-NC) were produced by RiboBio (Guangzhou, China). Small interfering (si) RNA targeting NFIB and control si-noncoding (siNC) oligonucleotides were purchased from GenePharma (Shanghai, China). Human NFIB cDNA was inserted into the pGL3 vector (GeneChem, Shanghai, China) to generate pGL3-NFIB. All oligonucleotides and plasmids were transfected using Lipofectamine 2000 Transfection Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions.

Dual-luciferase 3′ UTR reporter assay was carried out to validate SND1 as a direct target of miR-361-5p. Wild-type (WT) or mutant of 3′ untranslated region (UTR) sequences of SND1 were inserted into the Fse I and Xba I sites of the pGL3 vector (GeneChem). HEK293 cells infected with anti-miR-361-5p lentivirus or negative control (NC) lentivirus were plated into 96-well plates. pGL3 vector (40 ng) with the above sequence was cotransfected with 10 ng of pRL-TK vector into cells by Lipofectamine LTX (Invitrogen). Twenty-four hours later, cells were collected according to the manufacturer's protocol (Promega) and firefly and Renilla luciferase activity was detected using Dual-luciferase Reporter Assay System Kits (Promega) with a Victor X machine (PerkinElmer). Additionally, a mutant SND1 3′ UTR reporter construct was made by site-directed mutagenesis in the putative target site of miR-361-5p using Quickchange XL site-directed mutagenesis kit (Agilent Technologies). All PCR products were verified by DNA sequencing. The normalized luciferase activity was expressed as a ratio of firefly luciferase to Renilla luciferase units.

To validate that IR/p110α gene is indeed the target of miR-378b, we purchased a plasmid harboring luciferase with the 3' UTR sequence of IR/p110α mRNA. Then, the mutant or wild-type of 3' UTR sequences of IR/p110α were inserted into the XbaI restriction sites of the pGL3 vector (GeneChem, Shanghai, China). 293T cells (Cell Bank of Chinese Academy of Sciences, Shanghai, China) infected with miR-378 mimics or ctrl RNA were seeded into 24-well plates. 0.6 μg pGL3 vector with above sequence was cotransfected with 0.05 μg pRL-TK vector into the cells by Lipofectamine 2000 (11668-019, Invitrogen, Carlsbad, CA, USA). At 48 h after transfection, 293T cells were harvested. The Dual-Luciferase Reporter Assay System (E2920, Promega, Madison, WI, USA) was used to measure luciferase activity.