Fig 1: Non-competitive binding of hsa-let-7a-2 with AGTR2. (A) The kinetics of pre-miRNA binding to AGTR2 assayed by microscale thermophoresis (MST), with purified AGTR2-GFP protein as the receptor and hsa-let-7a-1, 2, and 3 as the ligands. (B) The binding kinetics of AGTR2 with its inhibitor, PD123319, as a positive control. (C, D) Competitive inhibition assay performed with [125-I]-sar1-Ile8-angiotensin II as a radioligand (C) or fluorescent ligand (D) to confirm the binding kinetics. Data are presented as means ± SD; n = 3 experiments.
Fig 2: Hsa-let-7a-2 negatively regulated Ang II-AGTR2 induced RhoA phosphorylation. (A) Under AGTR1 inhibition, the effect of hsa-let-7a-2 or hsa-let-7a-1/3 overexpression on RhoA serine phosphorylation in Dicer-/- cells was examined. ** P < 0.01 vs. p-CON. (B) RhoA serine phosphorylation was examined in Dicer-/-AGTR1-/- cells overexpressing hsa-let-7a-2 or hsa-let-7a-1/3. ** P < 0.01 vs. p-CON. (C) The changes in Ang II-induced RhoA serine phosphorylation were examined in AGTR1-/- hsa-let-7a-2+/- cells. *P < 0.01 vs. AGTR1-/-. n = 4-8 experiments.
Fig 3: Hsa-let-7a-2 negatively regulated Ang II-AGTR2 related cAMP and SHP-1 signals. (A) Under AGTR1 inhibition with losartan and adenylate cyclase stimulation with forskolin, the intracellular cAMP level was examined after Ang II treatment in cells overexpressing hsa-let-7a-2 (p-hsa-let-7a-2) or control plasmid (p-CON). ** P < 0.01 vs. p-CON. (B) Under the same conditions, Ang II-induced cAMP alterations were examined in Dicer-/- cells overexpressing hsa-let-7a-2 or hsa-let-7a-1/3. ** P < 0.01 vs p-CON. (C) Dicer-/-AGTR1-/- double-knockout cells overexpressing hsa-let-7a-2 or hsa-let-7a-1/3 were pretreated with forskolin and the Ang II-induced cAMP level was measured. * P < 0.05 vs. p-CON. (D) AGTR1-/-hsa-let-7a-2+/- cells and AGTR1-/- cells were pretreated with forskolin and the Ang II-induced cAMP level was compared. ** P < 0.01 vs. AGTR1-/-. (E) Under AGTR1 inhibition with losartan, the Ang II-induced SHP-1 activity was examined in Dicer-/- cells overexpressing hsa-let-7a-2 or hsa-let-7a-1/3. ** P < 0.01 vs. p-CON. (F) Effect of hsa-let-7a-2 or hsa-let-7a-1/3 on Ang II-induced SHP-1 activity in Dicer-/-AGTR1-/- cells. ** P < 0.01 vs. p-CON. (G) Knockdown of hsa-let-7a-2+/- enhanced SHP-1 activity in AGTR1-/- cells. ** P < 0.01 vs. AGTR1-/-. n = 4-8 experiments.
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