Journal: Translational Psychiatry
Article Title: MicroRNA-132/212 negatively modulates opioid reward by targeting dopamine transporter in the ventral tegmental area
doi: 10.1038/s41398-026-03915-9
Figure Lengend Snippet: A, B The effect of miR-132 on the level of DAT mRNA expression in SK-N-SH cells. DAT mRNA levels were measured by qRT‒PCR (n = 3). DAT protein levels were analyzed by western blotting (n = 3). C, D The transfection of miR-212 results in increased expression of DAT in SK-N-SH cells. Meanwhile, miR-132/212 inhibitors decreased DAT expression (n = 3). The data are shown as the mean values ± SEM and were obtained from at least three independent experiments performed in triplicate. E The binding site of miR-132/212 in the SLC6A3 3’UTR and the miR-132/212 seed region mutation of the SLC6A3 3’UTR. F The sequences near the miR-132/212 seed region in the SLC6A3 3’UTR are highly conserved among the indicated mammalian species. G The effect of miR-132/212 on the activity of the luciferase reporter construct containing the wild-type 3’UTR of SLC6A3 (n = 3). H The effect of miR-132/212 on the activity of the luciferase reporter construct containing the SLC6A3 3’UTR with miR-132/212 seed region mutation (n = 3). The data are shown as the mean values ± SEM and were obtained from at least three independent experiments performed in triplicate. I Fluorescence microscopy of DAT overexpression. After transfection with ctrl-miR or the miRNAs for 72 h, DAT-overexpressing HepG2 cells were incubated at 37 °C with 10 ng/ml Hoechst 33342 for 15 min. HepG2 cells were then visualized and photographed using a fluorescence microscope with a 10× objective. miR-132/212 can significantly reduce the expression of DAT-GFP. ‘Blank’ indicates cells that were not transfected with the DAT overexpression plasmid. Scale bar, 25 μm. A representative result from 3 independent experiments is shown. (* P < 0.05, ** P < 0.01 vs ctrl-miR; NS, not significant. miR-132, hsa-miR-132-3p; miR-212, hsa-miR-212-3p; ctrl-miR, control miRNA; Hsa Homo sapiens, Ptr Pan troglodytes, Mml Macaca, mulatta, Oga Otolemur garnetti, Mmu, Mus musculus, Rno Rattus norvegicus, Dno Dasypus novemcinctus.).
Article Snippet: The dopaminergic SK-N-SH neuroblastoma cell line (ATCC HTB-11) was maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1% antibiotic penicillin/streptomycin (P/S) and 0.1 g/L sodium pyruvate in a humidified incubator under 5% CO 2 /95% air at 37 °C.
Techniques: Expressing, Western Blot, Transfection, Binding Assay, Mutagenesis, Activity Assay, Luciferase, Construct, Fluorescence, Microscopy, Over Expression, Incubation, Plasmid Preparation, Control
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![(A) Proportion of editing events detected in P gene mRNA amplified by either negative (-) sense or oligo-dT reverse transcription (RT) primers from isolated total RNA from CedV-infected SK-N-SH, HeLa, and PATGV cells. Frequencies of A and G insertions [1 =100%] are indicated by green and blue, respectively. (B) Western blot of Strep-tagged P variants in BSR-T7/5 cells expressed from the cloned and mutated P gene cDNA sequences at 20 h post transfection. (C) Position 3312 insertions (red letters) result in a frameshift and expression of a 69-aa alternative V-like CTD (C-terminal domain, blue letters) comprising the identified peptides 1, 2 and 3. (D) Intracellular localization of expressed Strep-tagged CedV and NiV P protein variants expressed from their cDNA sequences in BSR-T7/5 cells at 20 h post transfection. Ctrl: BSR-T7/5 cells transfected with the empty vector. The nuclei are marked by a white star.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_48/10__64898_slash_2026__02__27__708548/10__64898_slash_2026__02__27__708548___F2.large.jpg)
