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. 2019 Feb 6;10:28. doi: 10.3389/fneur.2019.00028

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Copyright © 2019 Zhao, Jaber, LeBeauf, Sharfman and Lukiw.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Luciferase reporter vector-based studies of miRNA-34a and SHANK3 expression - Functional validation of a miRNA-34a-SHANK3–3′UTR interaction. (A) partial ribonucleotide sequence of the 1986 nt SHANK3-mRNA-3′-UTR is shown in the 5′-3′ direction; the 22 nucleotide (nt) miRNA-34a-SHANK3-3′UTR complementarity-interaction region is indicated by a black underline and the 8 nt SHANK3-mRNA-3′-UTR seed sequence is overlaid in yellow; a single vertical red arrow indicates the 5′ end of a poly A+ tail in the SHANK3 mRNA; the SHANK3 mRNA sequence derived from NM_018965; (B) SHANK3-mRNA-3′UTR expression vector luciferase reporter assay (pLightSwitch-3′UTR; Cat#S801178; Switchgear Genomics, Palo Alto CA); in this vector, the entire 1986 nucleotide SHANK3 3′UTR was ligated into the unique Nhe1-Xho1site; not drawn to scale; (C) HNG cells, 2 weeks in primary culture; neurons (red stain; λ_max = 690 nm), DAPI (blue nuclear stain; λ_max = 470 nm) and glial fibrillary associated protein (GFAP; glial-specific green stain; λ_max = 520 nm); the HNG cell culture is about 60% confluent and at 2 weeks of culture contains 70% neurons and 30% astroglia; human neurons do not culture well in the absence of glia; neurons also show both extensive arborization and display electrical activity (unpublished; Lonza); 40X magnification; HNG cells were transfected with the SHANK3-mRNA-3′UTR expression vector luciferase reporter were treated exogenously with a stabilized miRNA-34a, a scrambled control miRNA-34a (miRNA-34a-sc) or control miRNA-183; see references and text for further details; (D) compared to control, HNG cells transfected with a scrambled (sc) control pLightSwitch-3'UTR vector, the SHANK3-mRNA-3′UTR vector exhibited decreased luciferase signal to a mean of 0.16-fold of controls in the presence of miRNA-34a; this same vector exhibited no change in the presence of the control miRNA-34a-sc or miRNA-183; for each experiment (using different batches of HNG cells) a control luciferase signal was generated and included separate controls with each analysis; in addition a control vector β-actin-3′UTR showed no significant effects on the relative luciferase signal yield after treatment with either miRNA-183 or miRNA-34a (data not shown); a dashed horizontal line set to 1.0 is included for ease of comparison; N = 6; *p < 0.001 (ANOVA). The results suggest a physiologically relevant miRNA-34a-SHANK3-mRNA-3′UTR interaction and a miRNA-34a-mediated down-regulation of SHANK3 expression in HNG cells. This pathogenic interaction may be related to the down-regulation of other immune, inflammatory, and synaptic system genes by up-regulated miRNAs in the CNS resulting in an impairment in trans-synaptic signaling and synaptic cytoarchitecture.