Psicheck2 vector

The 3′‐UTR of MCU was synthesized and inserted into the psiCHECK‐2 vector (Ambion). The possible miR-129-3p binding sites in the 3′-UTR (MCU: AAGGGCT) were replaced with TCACTGA and validated via sequencing. The two reporter plasmids named MCU-WT and MCU-MUT were co-transfected into rat primary hippocampal neuronal cells with miR-129-3p mimic or NC mimic, respectively. The Dual Luciferase Reporter Assay kit (Promega, USA) with renilla luciferase as an internal control was used to perform luciferase assays, and The dual-luciferase activity was detected by Promega’s Digital-Luciferase Reporter Assay System (Promega, USA).

The psiCHECK-2 dual luciferase vector (Promega Corporation, Madison, WI) was used to construct the plasmid containing the 3-untranslated region (3-UTR) of DEK. The fragments containing the predicted wild and mutant sites were directly synthesized (Genewiz, Inc., Suzhou, China) and then subcloned into the psiCHECK-2 vector following digestion with XhoI and NotI restriction enzymes (Thermo Fisher Scientific, Inc., Pittsburgh, PA) to generate the DEK-3′-UTR-wild and DEK-3-UTR-mutant vectors. Subsequently, PK-1 cells (1 × 10⁵/well) were seeded into a 24-well plate and co-transfected with 50 ng/well DEK-3-UTR-wild or DEK-3-3′-UTR-mutant vector and 50 nM/well miR-200a mimics or scrambled microRNA negative control. Following culture for 48 hours, the PK-1 cells were collected, and the luciferase activities were measured by the Dual-Luciferase Reporter Assay kit (Promega Corporation) on a TD20/20 Luminometer (Turner Designs, Westport, MA). Each experiment was performed in triplicate. The results were expressed as relative Renilla luciferase activities, which were obtained following normalization to firefly luciferase activities. All the transient transfections were performed using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA).

We designed the following oligonucleotides and plasmids according to the method of Koibuchi et al.^{29 (link)} F2 (chick lysozyme TRE at nucleotides −2358 to −2326; half-sites arranged as an inverted palindrome with nucleotide gap of 6 (ref. 35 (link))); palindromic (typical TREs, which designed not to contain a putative RORE); DR4-1 (DR4 sequence containing AT-rich sequences at the 5′ end of the upstream half-site to serve as a putative RORE); and DR4-2 (DR4 oligonucleotide lacking an AT-rich sequence).^{36 (link)} These oligonucleotides were cloned in the psi-CHECK2 vector (Thermo Fisher Scientific, USA) containing a viral thymidine kinase promoter coupled to the firefly luciferase-labelled gene which was used as the internal reference to remove the transfection efficiency differences between groups, and the Renilla luciferase reporter gene.^{37 (link)} These reporter plasmids were sequenced to ensure that only a single copy of the TRE had been incorporated (Fig. 2A).
Accelrys DS Gene software (Accelrys Software, Inc., U.S.A.) was adopted to analyze the enzyme-cutting site of the psiCHECK2 promoter between the Poly A and hsv-tk promoter. The Bbs I enzyme was selected and the cutting site is shown in Fig. 2B. The nucleotide sequences of the double-stranded oligonucleotides containing TRE or RORE are shown in Fig. 2C.