Figure 4.

MiR-30b-5p inhibits ferroptosis in the Ang II-induced CMEC

(A) MiR-30b-5p targeting site in TIMP1 3ʹUTR. (B) Luciferase assay. Cells were transfected with miR-30b-5p and then with luciferase constructs of WT TIMP1 3ʹUTR (TIMP1-wt) or mutated TIMP1 3ʹUTR (TIMP1-mut). Luciferase activity was analyzed. ^##p < 0.01 versus TIMP1-wt group. (C) The mRNA expression of miR-30b-5p in cardiac tissue of rats induced by AAC for 4 weeks. Data are represented by means ± SEM (n = 6). ^##p < 0.01 versus sham group. (D) The mRNA expression of miR-30b-5p in CMECs exposed to Ang II for 24 h. (E) The mRNA expression of miR-30b-5p in CMECs. (F and G) Cell viability of CMECs was detected. (H and I) Western blot analysis of TIMP1, MMP9, and TFR-1 in CEMCs. (J) Statistical analysis chart of Fe²⁺ content. (K) Immunofluorescence results of ROS in CMECs. (L) Changes of MDA content in CMECs. (M) The mRNA expression of ptgs2 in different groups. (N) Statistical analysis of CMEC mortality. AMO: AMO-miR-30b-5p. NC: negative control. Ang II group: CMECs were exposed to Ang II for 24 h. Ang II + miR-30b-5p group: CMECs were exposed to Ang II for 24 h and then treated with miR-30b-5p mimics for 24 h. Ang II + miR-30b-5p + AMO group: CMECs were exposed to Ang II for 24 h and then treated with miR-30b-5p mimics and AMO-miR-30b-5p for 24 h. Ang II + NC-miR-30b-5p group: CMECs were exposed to Ang II for 24 h and then treated with negative control of miR-30b-5p. Ang II + NC-AMO group: CMECs were exposed to Ang II for 24 h and then treated with negative control of AMO-miR-30b-5p. Data are represented by means ± SEM (n = 6). ^##p < 0.01 versus control group; ^∗∗p < 0.01 versus Ang II group. (O) qRT-PCR analysis of lncRNAs in the hearts of cardiac hypertrophy rats induced by AAC. AABR07017145.1: lncRNA AAB. Data are represented by means ± SEM (n = 6). ^#P < 0.05 and ^##p < 0.01 versus sham group. (P) The expression of the three lncRNAs in CMECs exposed to Ang II for 24 h. Data are represented by means ± SEM (n = 6). ^##p < 0.01 versus control group. (Q) lncRNA AAB contained a site complementary to miR-30b-5p. (R) Luciferase activity analyses in cells co-transfected with TIMP1-wt and luciferase reporters containing miR-30b-5p mimic or lncRNA AAB. Data are represented by means ± SEM (n = 6). ^##p < 0.01 versus TIMP1-wt group; ^∗∗p < 0.01 versus TIMP1-wt and miR-30b-5p mimic group. Scale bars, (G) 10 μm; (K) 50 μm.