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. Author manuscript; available in PMC: 2009 Sep 18.

Published in final edited form as: Circulation. 2009 Apr 20;119(17):2357–2366. doi: 10.1161/CIRCULATIONAHA.108.814145

Overexpression of miR-320 enhanced cell death and apoptosis in cultured adult rat cardiomyocytes upon simulated ischemia/reperfusion, while knock-down was cytoprotective. (A) Diagram of recombinant adenoviral vectors. The primary miR-320 (470 bp) was PCR-amplified and inserted in the sense or antisense direction to generate AdmiR-320 or AdasmiR-320. (B) Nearly 100% cardiomyocytes were infected after 48-h adenoviral infection, as indicated by GFP fluorescence, and there were no morphological changes among GFP-, miR-320-, and asmiR-320-infected cells. (C) The levels of miR-320 were determined after 60-h infection by Taq^man RT-PCR, as described in Figure 1, which confirmed the expression of mature miR-320 in AdmiR-320-infected cells, and knockdown of endogenous miR-320 in AdasmiR-320-cells. (D-G) miR-320-overexpressing cardiomyocytes were sensitive, while knockdown cells resistant to simulated ischemia/reperfusion (I/R)-induced cell death (D and E), and apoptosis (F and G), as determined by MTS incorporation (E), nuclear fragmentation by Hoechst staining (F), and cell-death-detection ELISA kit (G) (*P < 0.05 versus control AdGFP). Similar results were observed in three additional, independent experiments.

Overexpression of miR-320 enhanced cell death and apoptosis in cultured adult rat cardiomyocytes upon simulated ischemia/reperfusion, while knock-down was cytoprotective. (A) Diagram of recombinant adenoviral vectors. The primary miR-320 (470 bp) was PCR-amplified and inserted in the sense or antisense direction to generate AdmiR-320 or AdasmiR-320. (B) Nearly 100% cardiomyocytes were infected after 48-h adenoviral infection, as indicated by GFP fluorescence, and there were no morphological changes among GFP-, miR-320-, and asmiR-320-infected cells. (C) The levels of miR-320 were determined after 60-h infection by Taq^man RT-PCR, as described in Figure 1, which confirmed the expression of mature miR-320 in AdmiR-320-infected cells, and knockdown of endogenous miR-320 in AdasmiR-320-cells. (D-G) miR-320-overexpressing cardiomyocytes were sensitive, while knockdown cells resistant to simulated ischemia/reperfusion (I/R)-induced cell death (D and E), and apoptosis (F and G), as determined by MTS incorporation (E), nuclear fragmentation by Hoechst staining (F), and cell-death-detection ELISA kit (G) (*P < 0.05 versus control AdGFP). Similar results were observed in three additional, independent experiments.