Research Paper Volume 13, Issue 3 pp 4299—4316

Overexpression of long noncoding RNA ANRIL inhibits phenotypic switching of vascular smooth muscle cells to prevent atherosclerotic plaque development in vivo

Knockdown of lncRNA-ANRIL ablates pharmacological activations of AMPK in cultured VSMCs. (A–C) Cultured VSMCs were infected with adenovirus expressing scramble or lncRNA-ANRIL shRNA for 48 hours and then treated with metformin (1 mM) or AICAR (0.5 mM) for 6 hours. (A) The expression of lncRNA-ANRIL was determined by real-time PCR. N is 5 in each group. *PB) Total cell lysates were subjected to perform western blot analysis of phosphorylated AMPK (pAMPK) and total AMPK protein levels. (C) AMPK activity was assayed by P32-ATP method. N is 5 in each group. *P#PD) Cultured VSMCs were transfected antisense oligonucleotide of lncRNA-ANRIL (AO) for 48 hours and then treated with metformin (1 mM) for 6 hours. The pAMPK level was assayed by western blot in total cell lysates. N is 5 in each group. *P#PE) Cultured VSMCs were infected with adenovirus expressing lncRNA-ANRIL for 24 hours and then treated with metformin (1 mM) for 6 hours. The pAMPK level was assayed by western blot in total cell lysates. N is 5 in each group. *P#PF–I) Cultured VSMCs were infected with adenovirus expressing lncRNA-ANRIL shRNA for 24 hours and then treated with PDGF (5 ng/ml) for 48 hours. Total cell lysates were subjected to perform western blot analysis of pAMPK in F. The mRNA levels of the phenotypic switching markers, including α-SMA in (G), calponin in (H), and osteopontin in I were assessed by real-time PCR. N is 5 in each group. *P#P

Figure 3. Knockdown of lncRNA-ANRIL ablates pharmacological activations of AMPK in cultured VSMCs. (AC) Cultured VSMCs were infected with adenovirus expressing scramble or lncRNA-ANRIL shRNA for 48 hours and then treated with metformin (1 mM) or AICAR (0.5 mM) for 6 hours. (A) The expression of lncRNA-ANRIL was determined by real-time PCR. N is 5 in each group. *P<0.05 vs. adenovirus. (B) Total cell lysates were subjected to perform western blot analysis of phosphorylated AMPK (pAMPK) and total AMPK protein levels. (C) AMPK activity was assayed by P32-ATP method. N is 5 in each group. *P<0.05 vs. scramble shRNA alone. #P<0.05 vs. scramble shRNA plus metformin or AICAR. (D) Cultured VSMCs were transfected antisense oligonucleotide of lncRNA-ANRIL (AO) for 48 hours and then treated with metformin (1 mM) for 6 hours. The pAMPK level was assayed by western blot in total cell lysates. N is 5 in each group. *P<0.05 vs. control. #P<0.05 vs. metformin alone. (E) Cultured VSMCs were infected with adenovirus expressing lncRNA-ANRIL for 24 hours and then treated with metformin (1 mM) for 6 hours. The pAMPK level was assayed by western blot in total cell lysates. N is 5 in each group. *P<0.05 vs. adenovirus alone. #P<0.05 vs. adenovirus plus metformin. (FI) Cultured VSMCs were infected with adenovirus expressing lncRNA-ANRIL shRNA for 24 hours and then treated with PDGF (5 ng/ml) for 48 hours. Total cell lysates were subjected to perform western blot analysis of pAMPK in F. The mRNA levels of the phenotypic switching markers, including α-SMA in (G), calponin in (H), and osteopontin in I were assessed by real-time PCR. N is 5 in each group. *P<0.05 vs. scramble shRNA alone. #P<0.05 vs. lncRNA-ANRIL shRNA alone.