Fig 2: Increased miRNA-7 and deficits in ciRS-7 in AD. (A): up-regulated miRNA-7 in AD brain; lack of the miRNA-7 (ciRS-7) sponge by insufficient ciRS-7 suggests the miRNA-7 would be increased in the same brain regions where ciRS-7 is down-regulated as is observed; data in (A) quantified in bar graph format in (B); down-regulation of a family of miRNA-7-sensitive mRNA targets in the sporadic AD brain includes UBE2A; other mRNA targets may be involved; (C): detection of circRNA for miRNA-7 (ciRS-7) in sporadic AD and age-matched control hippocampal CA1 (control (CON) N = 4; AD (ALZ) N = 6]; in (C) the single upper ciRS-7 (~1400 nt) band contains ~70 selectively conserved miRNA-7 binding sites as previously described [8,10,11]; a lower 28S RNA served as an internal loading and reference control; all samples depleted of rRNA were treated with 50 units of RNaseR prior to electrophoresis see [8,10,23]); (D): AD ciRS-7 is significantly reduced to ~0.18-fold of control (CON) in AD (ALZ) hippocampal CA1 (HIPP CA1), to ~0.22 of control in the superior temporal lobe (Brodmann A22; NCTX A22) but not the brain stem (BRAINSTEM); this implicates loss of miRNA-7 sponge effects, and ambient up-regulation of miRNA-7 in at least two anatomical areas targeted by the AD process; there were no significant differences between age for control or AD tissues; (mean +/- one standard deviation (SD) = 71.6 ± 6.3 years (N = 6; control); 73.5 ± 6.1 years (N = 12; AD)); all AD cases were from moderate-to-advanced stages of AD; all postmortem intervals were 2.3 h or less; there were no significant differences in age, ApoE allele status, RNA quality (all RIN values were 8.1–9.0) or yield between the control or AD groups; Figure 1C has been redrawn in part from [10]; values in (B) and (D) are normalized to control levels; a dashed horizontal line at 1.0 or 10.0 is included for ease of comparison; (* p < 0.01, ANOVA); ** p < 0.001 (ANOVA).

Fig 3: A strong miRNA-7-UBE2A mRNA 3’-UTR interaction. (A) Complimentarity map of human miRNA-7 and the UBE2A mRNA 3'-UTR—an “|” represents a hydrogen bond between the miRNA-7 and the UBE2A mRNA 3’-UTR and “:” represents a partial hydrogen bond; “seed” sequences are highlighted in yellow for miRNA-7 and in red for the UBE2A mRNA 3'-UTR; an energy of association (EA) between the miRNA-7 and the UBE2A mRNA 3'-UTR of -22.9 kcal/mol is very favorable thermodynamically; due to a deficit in ciRS-7 there are insufficient miRNA-7 sponging effects and an increase ambient miRNA-7 levels (Figure 2), resulting in a down-regulation in the expression of miRNA-7 targeted mRNA-3’-UTRs (such as UBE2A mRNA 3’-UTR) which is observed; other circRNAs may be involved; (B) levels of UBE2A protein in Brodmann area A22 and Alzheimer (ALZ) hippocampal CA1 compared to control (CON) brain postmortem samples; as in Figure 2, mean and one standard deviation are shown; UBE2A is observed to be reduced to 3.6- and 2.7-fold of controls, respectively, in Brodmann A22 (the superior temporal neocortex) and the hippocampal CA1 of AD brains and the results are highly significant; insufficient UBE2A might be expected to contribute to deficits in the ubiquitination cycle that helps to clear amyloid peptides via autophagy, thus contributing to amyloidogenesis in the AD brain; in (B) there were no significant differences in age, ApoE allele status, RNA quality (all RIN values were 8.1–9.0) or yield between the control (CON) or Alzheimer (ALZ) groups; control UBE2A levels in the Brodmann A22 (the superior temporal neocortex) and the hippocampal CA1 of AD were arbitrarily set to 10.0; a dashed horizontal line at 10.0 is included for ease of comparison; (* p < 0.001 (ANOVA).