Figure 3.

MIAT regulates human carotid artery smooth muscle cell proliferation through the ERK/ELK1/EGR1 pathway. A and B, Proliferation (A) or apoptosis (B) of human carotid smooth muscle cells (hCASMCs) on knockdown of MIAT (KD) monitored through live-cell imaging over time (0–72 hours). Data were analyzed by 2-way ANOVA. C and D, Proliferation (C) or apoptosis (D) of hCASMCs on KD of MIAT determined by Ki-67 or Caspase 3 immunofluorescent staining (white arrows indicate Ki-67/Caspase 3–positive cells). Bar=50 µm. E, In silico analysis of transcription factors associated with MIAT (predicted binding free energy of <–30 Kcal/mol) using the regulatory RNA elements/RegRNA tool. F, MIAT fragment containing 2 predicted ELK1 binding sites (MIAT-ELK1_BS1_BS2) was transiently transfected and coimmunoprecipitated with endogenous ELK1 in hCASMCs. IgGs were used as control of immunoprecipitation (IP) specificity. MIAT or GAPDH (as unrelated target) enrichment in ELK1 IP fraction was quantified with quantitative real-time polymerase chain reaction and expressed as (2^ΔCt)×100 ELK1 IP÷(2^ΔCt)×100 IgG. ΔCt was calculated based on input. RNA content in IP or IgG was normalized on RPLPO mRNA. ELK1 IP efficiency was monitored by Western blot using an anti-ELK1 antibody. G, Transfected cells were lysed after 24 hours, and total protein was extracted. P-ERK normalized to total ERK was monitored using Western Blot. Quantification of the Western Blot was done with Fiji Image J software. H, MIAT-ELK1_BS1_BS2 was transiently transfected, and proliferation was monitored using the IncuCyte Live Cell Imaging System. Bar=50 µm. Data were analyzed by 2-way repeated-measures ANOVA (A), area under the curve (B), and Student t test (C, D, F, G). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Casp indicates caspase; Ctrl, control; DAPI, 4′,6-diamidino-2-phenylindole; EV, empty vector (control); and IgG, immunoglobulin G.