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let-7b suppresses apoptosis and autophagy of human mesenchymal stem cells transplanted into ischemia/reperfusion injured heart 7by targeting caspase-3.

Ham O, Lee SY, Lee CY, Park JH, Lee J, Seo HH, Cha MJ, Choi E, Kim S, Hwang KC - Stem Cell Res Ther (2015)

Bottom Line: Let-7b-transfected MSCs (let-7b-MSCs) showed high expression of survival-related proteins, including p-MEK, p-ERK and Bcl-2, leading to a decrease in Annexin V/PI- and TUNEL-positive cells under ROS-rich conditions.Moreover, autophagy-related genes, including Atg5, Atg7, Atg12 and beclin-1, were significantly downregulated in let-7b-MSCs.Using a rat model of acute myocardial infarction, we found that intramyocardial injection of let-7b-MSCs markedly enhanced left ventricular (LV) function and microvessel density, in accordance with a reduced infarct size and the expression of caspase-3.

View Article: PubMed Central - PubMed

Affiliation: Catholic Kwandong University International St. Mary's Hospital, Incheon Metropolitan City, 404-834, Republic of Korea. onju1336@gmail.com.

ABSTRACT

Introduction: Mesenchymal stem cells (MSCs) have therapeutic potential for the repair of myocardial injury. The efficacy of MSC therapy for myocardial regeneration mainly depends on the survival of cells after transplantation into the infarcted heart. In the transplanted regions, reactive oxygen species (ROS) can cause cell death, and this process depends on caspase activation and autophagosome formation.

Methods: A Software TargetScan was utilized to search for microRNAs (miRNAs) that target caspase-3 mRNA. Six candidate miRNAs including let-7b were selected and transfected into human MSCs in vitro. Expression of MEK-EKR signal pathways and autophagy-related genes were detected. Using ischemia/reperfusion model (I/R), the effect of MSCs enriched with let-7b was determined after transplantation into infarcted heart area. Miller catheter was used to evaluate cardiac function.

Results: Here, we report that let-7b targets caspase-3 to regulate apoptosis and autophagy in MSCs exposed to ROS. Let-7b-transfected MSCs (let-7b-MSCs) showed high expression of survival-related proteins, including p-MEK, p-ERK and Bcl-2, leading to a decrease in Annexin V/PI- and TUNEL-positive cells under ROS-rich conditions. Moreover, autophagy-related genes, including Atg5, Atg7, Atg12 and beclin-1, were significantly downregulated in let-7b-MSCs. Using a rat model of acute myocardial infarction, we found that intramyocardial injection of let-7b-MSCs markedly enhanced left ventricular (LV) function and microvessel density, in accordance with a reduced infarct size and the expression of caspase-3.

Conclusions: Taken together, these data indicate that let-7b may protect MSCs implanted into infarcted myocardium from apoptosis and autophagy by directly targeting caspase-3 signaling.

No MeSH data available.


Related in: MedlinePlus

H2O2-induced apoptosis. a Cell survival was evaluated after treating cells with increasing concentrations of H2O2 for 6 hours (***p <0.0001, **p <0.001). b Expression of cleaved caspase-3 and PARP was measured by western blotting in cells treated with increasing concentrations of H2O2 for 6 hours (*p <0.05). c Schematic presentation of miRNA binding site in the 3′ UTR of human caspase-3. Candidate miRNAs predicted to target caspase-3 were selected based on the TargetScan miRNA-target prediction database [19]. miRNAs with an aggregation Pct value ≥0.2 were selected. d Cell survival was measured in candidate miRNA-transfected hMSCs. All samples were treated with 500 μM H2O2 after miRNA transfection. e Effect of H2O2 on endogenous let-7b expression was evaluated by real-time PCR. Quantitative data expressed as mean ± standard deviation of at least three independent experiments. (*p <0.05, and #p <0.05)
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Fig1: H2O2-induced apoptosis. a Cell survival was evaluated after treating cells with increasing concentrations of H2O2 for 6 hours (***p <0.0001, **p <0.001). b Expression of cleaved caspase-3 and PARP was measured by western blotting in cells treated with increasing concentrations of H2O2 for 6 hours (*p <0.05). c Schematic presentation of miRNA binding site in the 3′ UTR of human caspase-3. Candidate miRNAs predicted to target caspase-3 were selected based on the TargetScan miRNA-target prediction database [19]. miRNAs with an aggregation Pct value ≥0.2 were selected. d Cell survival was measured in candidate miRNA-transfected hMSCs. All samples were treated with 500 μM H2O2 after miRNA transfection. e Effect of H2O2 on endogenous let-7b expression was evaluated by real-time PCR. Quantitative data expressed as mean ± standard deviation of at least three independent experiments. (*p <0.05, and #p <0.05)

Mentions: We used H2O2 to simulate ROS-mediated cell death in our experiments. To induce apoptosis, hMSCs were treated with varying concentrations of H2O2. After 6 hours of treatment, cell survival significantly decreased when the concentration of H2O2 was higher than 500 μM (Fig. 1a). Especially with 500 μM H2O2 treatment, the amount of both cleaved caspase-3 and PARP also increased (Fig. 1b), suggesting that H2O2 at this concentration effectively induced apoptosis hMSCs.Fig. 1


let-7b suppresses apoptosis and autophagy of human mesenchymal stem cells transplanted into ischemia/reperfusion injured heart 7by targeting caspase-3.

Ham O, Lee SY, Lee CY, Park JH, Lee J, Seo HH, Cha MJ, Choi E, Kim S, Hwang KC - Stem Cell Res Ther (2015)

H2O2-induced apoptosis. a Cell survival was evaluated after treating cells with increasing concentrations of H2O2 for 6 hours (***p <0.0001, **p <0.001). b Expression of cleaved caspase-3 and PARP was measured by western blotting in cells treated with increasing concentrations of H2O2 for 6 hours (*p <0.05). c Schematic presentation of miRNA binding site in the 3′ UTR of human caspase-3. Candidate miRNAs predicted to target caspase-3 were selected based on the TargetScan miRNA-target prediction database [19]. miRNAs with an aggregation Pct value ≥0.2 were selected. d Cell survival was measured in candidate miRNA-transfected hMSCs. All samples were treated with 500 μM H2O2 after miRNA transfection. e Effect of H2O2 on endogenous let-7b expression was evaluated by real-time PCR. Quantitative data expressed as mean ± standard deviation of at least three independent experiments. (*p <0.05, and #p <0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4546263&req=5

Fig1: H2O2-induced apoptosis. a Cell survival was evaluated after treating cells with increasing concentrations of H2O2 for 6 hours (***p <0.0001, **p <0.001). b Expression of cleaved caspase-3 and PARP was measured by western blotting in cells treated with increasing concentrations of H2O2 for 6 hours (*p <0.05). c Schematic presentation of miRNA binding site in the 3′ UTR of human caspase-3. Candidate miRNAs predicted to target caspase-3 were selected based on the TargetScan miRNA-target prediction database [19]. miRNAs with an aggregation Pct value ≥0.2 were selected. d Cell survival was measured in candidate miRNA-transfected hMSCs. All samples were treated with 500 μM H2O2 after miRNA transfection. e Effect of H2O2 on endogenous let-7b expression was evaluated by real-time PCR. Quantitative data expressed as mean ± standard deviation of at least three independent experiments. (*p <0.05, and #p <0.05)
Mentions: We used H2O2 to simulate ROS-mediated cell death in our experiments. To induce apoptosis, hMSCs were treated with varying concentrations of H2O2. After 6 hours of treatment, cell survival significantly decreased when the concentration of H2O2 was higher than 500 μM (Fig. 1a). Especially with 500 μM H2O2 treatment, the amount of both cleaved caspase-3 and PARP also increased (Fig. 1b), suggesting that H2O2 at this concentration effectively induced apoptosis hMSCs.Fig. 1

Bottom Line: Let-7b-transfected MSCs (let-7b-MSCs) showed high expression of survival-related proteins, including p-MEK, p-ERK and Bcl-2, leading to a decrease in Annexin V/PI- and TUNEL-positive cells under ROS-rich conditions.Moreover, autophagy-related genes, including Atg5, Atg7, Atg12 and beclin-1, were significantly downregulated in let-7b-MSCs.Using a rat model of acute myocardial infarction, we found that intramyocardial injection of let-7b-MSCs markedly enhanced left ventricular (LV) function and microvessel density, in accordance with a reduced infarct size and the expression of caspase-3.

View Article: PubMed Central - PubMed

Affiliation: Catholic Kwandong University International St. Mary's Hospital, Incheon Metropolitan City, 404-834, Republic of Korea. onju1336@gmail.com.

ABSTRACT

Introduction: Mesenchymal stem cells (MSCs) have therapeutic potential for the repair of myocardial injury. The efficacy of MSC therapy for myocardial regeneration mainly depends on the survival of cells after transplantation into the infarcted heart. In the transplanted regions, reactive oxygen species (ROS) can cause cell death, and this process depends on caspase activation and autophagosome formation.

Methods: A Software TargetScan was utilized to search for microRNAs (miRNAs) that target caspase-3 mRNA. Six candidate miRNAs including let-7b were selected and transfected into human MSCs in vitro. Expression of MEK-EKR signal pathways and autophagy-related genes were detected. Using ischemia/reperfusion model (I/R), the effect of MSCs enriched with let-7b was determined after transplantation into infarcted heart area. Miller catheter was used to evaluate cardiac function.

Results: Here, we report that let-7b targets caspase-3 to regulate apoptosis and autophagy in MSCs exposed to ROS. Let-7b-transfected MSCs (let-7b-MSCs) showed high expression of survival-related proteins, including p-MEK, p-ERK and Bcl-2, leading to a decrease in Annexin V/PI- and TUNEL-positive cells under ROS-rich conditions. Moreover, autophagy-related genes, including Atg5, Atg7, Atg12 and beclin-1, were significantly downregulated in let-7b-MSCs. Using a rat model of acute myocardial infarction, we found that intramyocardial injection of let-7b-MSCs markedly enhanced left ventricular (LV) function and microvessel density, in accordance with a reduced infarct size and the expression of caspase-3.

Conclusions: Taken together, these data indicate that let-7b may protect MSCs implanted into infarcted myocardium from apoptosis and autophagy by directly targeting caspase-3 signaling.

No MeSH data available.


Related in: MedlinePlus