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microRNA-34a-Mediated Down-Regulation of the Microglial-Enriched Triggering Receptor and Phagocytosis-Sensor TREM2 in Age-Related Macular Degeneration

Fig 4

Functional validation of a miRNA-34a-TREM2–3’UTR interaction.

(A) ribonucleotide sequence of the 299 nt TREM2-mRNA-3’-UTR is shown in the 5’-3’ direction; the 22 nt miRNA-34a-TREM2 3’UTR complementarity-interaction region is indicated by a black underline and the 8 nt TREM2-mRNA-3’-UTR seed sequence 5’-ACACUGCU-3’ is overlaid in yellow; a single arrowhead indicates the 5’ end of a poly A+ tail in the TREM2 mRNA (22 ‘A’ nt shown; the length of this poly A+ tail is variable); TREM2 mRNA sequence derived from NM_018965; TREM2 transcript is the major X1 variant (see also (Fig 3); (B) TREM2-mRNA-3’UTR expression vector luciferase reporter assay (pLightSwitch-3’UTR; Cat#S801178; Switchgear Genomics, Palo Alto CA); in this vector, the entire 299 nucleotide TREM2 3’UTR was ligated into the unique Nhe1-Xho1 site; (C) control C8B4 murine microglial cells, 1 week in culture; phase contrast bright field microscopy 20x; C8B4 cells transfected with the TREM2-mRNA-3’UTR expression vector luciferase reporter were treated exogenously with miRNA-34a, a scrambled control miRNA-34a (miRNA-sc) or control miRNA-183; see references and text for further details [18,19]; (D) compared to control, C8B4 cells transfected with a scrambled (sc) control pLightSwitch-3’UTR vector, the TREM2-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 MG cells) a control luciferase signal was generated and included separate appropriate 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); dashed horizontal line set to 1.0 for ease of comparison; N = 5; *p<0.001 (ANOVA). The results suggest a physiologically relevant miRNA-34a- TREM2-mRNA-3‘UTR interaction and a miRNA-34a-mediated down-regulation of TREM2 expression in stressed MG cells. This pathogenic ineraction may be related to the down-regulation of other immune system genes by up-regulated pro-inflammatory miRNAs in the CNS [12,19,22] and/or an impairment in cellular phagocytosis or related phagocytic signaling [57,20,21].

Fig 4