Figure 5.

lncRNA-MEG3 Binds to and Decreases the Expression of miR-328-3p

(A) Diagram showing the genomic structure of the relative oligos, miR-328-3p binding sequences, mutation sequence used for luciferase experiments, PCR product for MST, and overexpression segment (left panel). miR-328-3p expression detected by real-time PCR in mPASMCs transiently transfected with 30 nM NC siRNA or siRNA against MEG3 (siMEG3), followed by hypoxia for 24 h (right panel). (B) Detection of miR-328-3p expression in PAs, lung, and right ventricular from hypoxia PH mouse. (C) Detection of lncRNA-MEG3 (left panel) and miR-328-3p (right panel) expression in PAs from SUGEN-hypoxia mouse models. (D) Expression of miR-328-3p was detected in iPAH-PASMCs (n = 3). (E) mPASMCs were transiently transfected with the indicated agents, followed by transient transfection with 25 ng of psiCHECK plasmid harboring the lncRNA-MEG3 binding region (nt 2071–2094) (left panel) or mutated sequence (right panel). Luciferase activity was assessed 36 h after transfection and normalized to the activity of Renilla luciferase. (F) MST analysis indicated that miR-328-3p mimics interaction with lncRNA-MEG3 (2030–2193, 163-nt PCR product), IGF1R, and NC nucleotide. (G) RNA immunoprecipitation by biotin-labeled MEG3 segment (nt 2030–2193) was subjected to pulldown, following real time-PCR to detect miR-328-3p expression. Data represent means ± SEM from six (except where indicated) independent experiments. Student’s t test (for two means) or one-way ANOVA followed by Dunnett’s test (for >2 means). *p < 0.05, **p < 0.01.