Alu Elements in ANRIL Non-Coding RNA at Chromosome 9p21 Modulate Atherogenic Cell Functions through Trans-Regulation of Gene Networks
(A) Cell lines over-expressing variants of ANRIL2 (ANRIL2a, 2b, 2c) and ANRIL4 (ANRIL4a, 4b) devoid of Alu motif sequences highlighted by boxes. Validation of over-expression using qRT-PCR assays. (B) Reversal of up-regulation (TSC22D3) and (C) down-regulation (COL3A1) of ANRIL target-gene mRNA expression in ANRIL2a-2c and ANRIL4a,4b compared to ANRIL2 and ANRIL4. Reversal of (D) cell adhesion, (E) apoptosis, and (F) proliferation in cell lines over-expressing mutant ANRIL isoforms. (G) Stable cell lines containing mutated forms of the Alu motif in ANRIL2. Positions of nucleotide exchanges (0, 25%, 33%, and 100%) in the 48 base-pair Alu motif are indicated in red. (H,I) Reversal of trans-regulation in mutant cell lines compared to ANRIL2. (J) Cell adhesion, (K) apoptosis, and (L) proliferation in mutant cell lines. (B,C,H,I,F,L) 3–4 biological replicates/isoform.(D,E,J,K) quadruplicate measurements per pool of 2–3 biological replicates. Error bars indicate s.e.m.