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Roles of microRNA-34a targeting SIRT1 in mesenchymal stem cells.

Zhang F, Cui J, Liu X, Lv B, Liu X, Xie Z, Yu B - Stem Cell Res Ther (2015)

Bottom Line: The results of the current study showed that miR-34a was significantly up-regulated under H/SD conditions in MSCs, while overexpression of miR-34a was significantly associated with increased apoptosis, impaired cell vitality and aggravated senescence.Moreover, we found that the mechanism underlying the proapoptotic function of miR-34a involves activation of the SIRT1/FOXO3a pathway, mitochondrial dysfunction and finally, activation of the intrinsic apoptosis pathway.Further study showed that miR-34a can also aggravate MSC senescence, an effect which was partly abolished by the reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC).

View Article: PubMed Central - PubMed

Affiliation: Key Laboratories of Education Ministry for Myocardial Ischemia Mechanism, The Second Affiliated Hospital of Harbin Medical University, 148 Baojian Road, Harbin, 150086, P.R. China. 805137949@qq.com.

ABSTRACT

Introduction: Mesenchymal stem cell (MSC)-based therapies have had positive outcomes both in animal models of cardiovascular diseases and in clinical patients. However, the number and function of MSCs decline during hypoxia and serum deprivation (H/SD), reducing their ability to contribute to endogenous injury repair. MicroRNA-34a (miR-34a) is originally identified as a TP53-targeted miRNA that modulates cell functions, including apoptosis, proliferation, and senescence via several signaling pathways, and hence is an appealing target for MSC-based therapy for myocardial infarction.

Methods: Bone marrow-derived MSCs were isolated from 60-80 g male donor rats. Expression levels of miR-34a were determined by qRT-PCR. The roles of miR-34a in regulating cell vitality, apoptosis and senescence were investigated using the cell counting kit (CCK-8) assay, flow cytometric analysis of Annexin V-FITC/PI staining and senescence-associated β-galactosidase (SA-β-gal) staining, respectively. The expression of silent information regulator 1 (SIRT1) and forkhead box class O 3a (FOXO3a) and of apoptosis- and senescence-associated proteins in MSCs were analyzed by western blotting.

Results: The results of the current study showed that miR-34a was significantly up-regulated under H/SD conditions in MSCs, while overexpression of miR-34a was significantly associated with increased apoptosis, impaired cell vitality and aggravated senescence. Moreover, we found that the mechanism underlying the proapoptotic function of miR-34a involves activation of the SIRT1/FOXO3a pathway, mitochondrial dysfunction and finally, activation of the intrinsic apoptosis pathway. Further study showed that miR-34a can also aggravate MSC senescence, an effect which was partly abolished by the reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC).

Conclusions: Our study demonstrates for the first time that miR-34a plays pro-apoptotic and pro-senescence roles in MSCs by targeting SIRT1. Thus, inhibition of miR-34a might have important therapeutic implications in MSC-based therapy for myocardial infarction.

No MeSH data available.


Related in: MedlinePlus

Overexpression of miR-34a induces senescence in MSCs. Cells were left untreated or pretreated with miR-34a mimic, siRNA-SIRT1, ROS scavenger NAC (10 mM), and miR-34a inhibitor separately or in combination for 72 hours, and then cellular senescence was analyzed by SA-β-gal staining (a, b). Cellular ROS production was assessed by measuring the fluorescent intensity of DCFH-DA determined using flow cytometry (c, d). Cellular DNA damage and senescence-related proteins including γ-H2A.X, p53, p21, and p16 were determined with western blot (e, f). Each column represents mean ± SD from three independent experiments. *P <0.05 vs. scramble, △P <0.05 vs. miR-34a mimic, ▲P <0.05 vs. siRNA-SIRT1.cp. DCFH 2′,7′-dichlorodihydrofluorescein, MFI mean fluorescence intensity, miRNA microRNA, NAC N-acetylcysteine, SA-β-gal senescence-associated galactosidase, SIRT1 silent information regulator 1, siRNA small interfering RNA
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Fig5: Overexpression of miR-34a induces senescence in MSCs. Cells were left untreated or pretreated with miR-34a mimic, siRNA-SIRT1, ROS scavenger NAC (10 mM), and miR-34a inhibitor separately or in combination for 72 hours, and then cellular senescence was analyzed by SA-β-gal staining (a, b). Cellular ROS production was assessed by measuring the fluorescent intensity of DCFH-DA determined using flow cytometry (c, d). Cellular DNA damage and senescence-related proteins including γ-H2A.X, p53, p21, and p16 were determined with western blot (e, f). Each column represents mean ± SD from three independent experiments. *P <0.05 vs. scramble, △P <0.05 vs. miR-34a mimic, ▲P <0.05 vs. siRNA-SIRT1.cp. DCFH 2′,7′-dichlorodihydrofluorescein, MFI mean fluorescence intensity, miRNA microRNA, NAC N-acetylcysteine, SA-β-gal senescence-associated galactosidase, SIRT1 silent information regulator 1, siRNA small interfering RNA

Mentions: Considering that the regenerative capacity of MSCs contributed greatly to their function, we further examined cellular senescence in miR-34a mimic-transfected MSCs. SA-β-gal activity, which is a characteristic feature of senescence-related growth arrest [31], was assayed. Results revealed that overexpression of miR-34a significantly increased the percentage of SA-β-gal-positive cells compared with that of scramble (Fig. 5a, b). SIRT1 inhibition has been reported to be associated with premature senescence and impaired proliferative activity in EPCs [17]. Consistently, the percentage of SA-β-gal-positive senescent cells was remarkably increased following SIRT1 knockdown (Fig. 5a, b).Fig. 5


Roles of microRNA-34a targeting SIRT1 in mesenchymal stem cells.

Zhang F, Cui J, Liu X, Lv B, Liu X, Xie Z, Yu B - Stem Cell Res Ther (2015)

Overexpression of miR-34a induces senescence in MSCs. Cells were left untreated or pretreated with miR-34a mimic, siRNA-SIRT1, ROS scavenger NAC (10 mM), and miR-34a inhibitor separately or in combination for 72 hours, and then cellular senescence was analyzed by SA-β-gal staining (a, b). Cellular ROS production was assessed by measuring the fluorescent intensity of DCFH-DA determined using flow cytometry (c, d). Cellular DNA damage and senescence-related proteins including γ-H2A.X, p53, p21, and p16 were determined with western blot (e, f). Each column represents mean ± SD from three independent experiments. *P <0.05 vs. scramble, △P <0.05 vs. miR-34a mimic, ▲P <0.05 vs. siRNA-SIRT1.cp. DCFH 2′,7′-dichlorodihydrofluorescein, MFI mean fluorescence intensity, miRNA microRNA, NAC N-acetylcysteine, SA-β-gal senescence-associated galactosidase, SIRT1 silent information regulator 1, siRNA small interfering RNA
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4597437&req=5

Fig5: Overexpression of miR-34a induces senescence in MSCs. Cells were left untreated or pretreated with miR-34a mimic, siRNA-SIRT1, ROS scavenger NAC (10 mM), and miR-34a inhibitor separately or in combination for 72 hours, and then cellular senescence was analyzed by SA-β-gal staining (a, b). Cellular ROS production was assessed by measuring the fluorescent intensity of DCFH-DA determined using flow cytometry (c, d). Cellular DNA damage and senescence-related proteins including γ-H2A.X, p53, p21, and p16 were determined with western blot (e, f). Each column represents mean ± SD from three independent experiments. *P <0.05 vs. scramble, △P <0.05 vs. miR-34a mimic, ▲P <0.05 vs. siRNA-SIRT1.cp. DCFH 2′,7′-dichlorodihydrofluorescein, MFI mean fluorescence intensity, miRNA microRNA, NAC N-acetylcysteine, SA-β-gal senescence-associated galactosidase, SIRT1 silent information regulator 1, siRNA small interfering RNA
Mentions: Considering that the regenerative capacity of MSCs contributed greatly to their function, we further examined cellular senescence in miR-34a mimic-transfected MSCs. SA-β-gal activity, which is a characteristic feature of senescence-related growth arrest [31], was assayed. Results revealed that overexpression of miR-34a significantly increased the percentage of SA-β-gal-positive cells compared with that of scramble (Fig. 5a, b). SIRT1 inhibition has been reported to be associated with premature senescence and impaired proliferative activity in EPCs [17]. Consistently, the percentage of SA-β-gal-positive senescent cells was remarkably increased following SIRT1 knockdown (Fig. 5a, b).Fig. 5

Bottom Line: The results of the current study showed that miR-34a was significantly up-regulated under H/SD conditions in MSCs, while overexpression of miR-34a was significantly associated with increased apoptosis, impaired cell vitality and aggravated senescence.Moreover, we found that the mechanism underlying the proapoptotic function of miR-34a involves activation of the SIRT1/FOXO3a pathway, mitochondrial dysfunction and finally, activation of the intrinsic apoptosis pathway.Further study showed that miR-34a can also aggravate MSC senescence, an effect which was partly abolished by the reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC).

View Article: PubMed Central - PubMed

Affiliation: Key Laboratories of Education Ministry for Myocardial Ischemia Mechanism, The Second Affiliated Hospital of Harbin Medical University, 148 Baojian Road, Harbin, 150086, P.R. China. 805137949@qq.com.

ABSTRACT

Introduction: Mesenchymal stem cell (MSC)-based therapies have had positive outcomes both in animal models of cardiovascular diseases and in clinical patients. However, the number and function of MSCs decline during hypoxia and serum deprivation (H/SD), reducing their ability to contribute to endogenous injury repair. MicroRNA-34a (miR-34a) is originally identified as a TP53-targeted miRNA that modulates cell functions, including apoptosis, proliferation, and senescence via several signaling pathways, and hence is an appealing target for MSC-based therapy for myocardial infarction.

Methods: Bone marrow-derived MSCs were isolated from 60-80 g male donor rats. Expression levels of miR-34a were determined by qRT-PCR. The roles of miR-34a in regulating cell vitality, apoptosis and senescence were investigated using the cell counting kit (CCK-8) assay, flow cytometric analysis of Annexin V-FITC/PI staining and senescence-associated β-galactosidase (SA-β-gal) staining, respectively. The expression of silent information regulator 1 (SIRT1) and forkhead box class O 3a (FOXO3a) and of apoptosis- and senescence-associated proteins in MSCs were analyzed by western blotting.

Results: The results of the current study showed that miR-34a was significantly up-regulated under H/SD conditions in MSCs, while overexpression of miR-34a was significantly associated with increased apoptosis, impaired cell vitality and aggravated senescence. Moreover, we found that the mechanism underlying the proapoptotic function of miR-34a involves activation of the SIRT1/FOXO3a pathway, mitochondrial dysfunction and finally, activation of the intrinsic apoptosis pathway. Further study showed that miR-34a can also aggravate MSC senescence, an effect which was partly abolished by the reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC).

Conclusions: Our study demonstrates for the first time that miR-34a plays pro-apoptotic and pro-senescence roles in MSCs by targeting SIRT1. Thus, inhibition of miR-34a might have important therapeutic implications in MSC-based therapy for myocardial infarction.

No MeSH data available.


Related in: MedlinePlus