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. 2020 Nov 26;23(12):101863. doi: 10.1016/j.isci.2020.101863

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© 2020.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

miR-302a Inhibition in hiPSC-CMs Blocks Rapamycin (RAPA)-Induced Protection Against Simulated Ischemia and Reoxygenation (SI/RO) Injury Under Normal (NG) and High Glucose (HG) Conditions

(A) Representative picture (upper panel) showing transfection efficiency of miR-302a inhibitor (LNA)-labeled with FAM (Green) dye of hiPSC-CMs and necrosis of following SI/RO.n = 6; ∗p < 0.0001 versus control, SI/RO + RAPA, RAPA, miR-302a-i (NG/HG); ^αp<0.05 versus SI/RO (HG) and miR-302a-i + SI/RO (NG).

(B) Representative immunoblots showing the effect of miR-302a inhibitor on expression of Bcl-2 and Bax following SI/RO with or without RAPA (100 nM; during RO). Right panel shows the densitometry analysis of Bcl-2 to GAPDH (n = 3; ∗p < 0.01 versus control and SI/RO + RAPA; ^αp<0.001 versus SI/RO + RAPA); Bax to GAPDH (n = 3; ∗p < 0.01 versus control, SI/RO & 302a-i + SI/RO) and Bcl-2 to Bax ratio (n = 3; ∗p < 0.01 versus 302i + SI/RO; ^αp<0.001 versus SI/RO (HG)).

(C) Phosphorylation of S6 and AKT under NG and HG condition following SI/RO with or without RAPA (100 nM; during RO). Right panel: densitometry analysis of the ratios of p-S6/S6 (n = 3; ∗p < 0.0001 versus control, miR-302a-i, SI/RO + RAPA, and miR-302a-i + SI/RO + RAPA; ^αp<0.01 versus SI/RO (NG); ^βp<0.001 versus SI/RO (HG)); S6/GAPDH (n = 3; ∗p < 0.001 versus control (NG) and SI/RO + RAPA (NG); ^αp<0.01 versus control (NG); ^βp<0.05 versus control (HG) and miR-302a-i (HG)); p-AKT/AKT (n = 3; ∗p < 0.001 versus others; ^αp<0.05 versus control, miR-302a-i and SI/RO + RAPA (HG); ^βp<0.0001 versus SI/RO (NG)); AKT/GAPDH (n = 3; ∗p < 0.01 versus others).

(D) PTEN expression under NG and HG conditions following SI/RO with or without RAPA treatment (100 nM; during RO). Lower panel shows densitometry analysis of PTEN with GAPDH as loading control. n = 3; ∗p < 0.01 versus control and SI/RO + RAPA; ^αp<0.05 versus miR-302a-i + SI/RO (NG). Statistics: one-way ANOVA. Data are represented as mean ± SEM.