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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

miR-34a expression increases under H/SD, and correlates with decreased cell survival and increased apoptosis. a Rat MSCs were cultured in normal condition or exposed to H/SD for 6 hours and were transiently transfected with miR-34a mimic, NC mimic, miR-34a inhibitor, or NC inhibitor for 48 hours, respectively. The expression of miR-34a was determined by qRT-PCR. *P <0.05. b, c Effects of miR-34a on the vitality of MSCs were examined by CCK-8 assay. *P <0.05 vs. NC mimic (b), *P <0.05 vs. NC inhibitor (c). d, e Flow cytometric analysis of apoptotic cells in normal and H/SD conditions, in cultures of miR-34a mimic, NC mimic, miR-34a inhibitor, or NC inhibitor treated (MSCs were transfected for 48 hours and exposure to H/SD and maintained as such for 6 hours). Each column represents mean ± SD from three independent experiments. *P <0.05. H/SD hypoxia and serum deprivation, miRNA microRNA, NC negative control, PI propidium iodide
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Fig1: miR-34a expression increases under H/SD, and correlates with decreased cell survival and increased apoptosis. a Rat MSCs were cultured in normal condition or exposed to H/SD for 6 hours and were transiently transfected with miR-34a mimic, NC mimic, miR-34a inhibitor, or NC inhibitor for 48 hours, respectively. The expression of miR-34a was determined by qRT-PCR. *P <0.05. b, c Effects of miR-34a on the vitality of MSCs were examined by CCK-8 assay. *P <0.05 vs. NC mimic (b), *P <0.05 vs. NC inhibitor (c). d, e Flow cytometric analysis of apoptotic cells in normal and H/SD conditions, in cultures of miR-34a mimic, NC mimic, miR-34a inhibitor, or NC inhibitor treated (MSCs were transfected for 48 hours and exposure to H/SD and maintained as such for 6 hours). Each column represents mean ± SD from three independent experiments. *P <0.05. H/SD hypoxia and serum deprivation, miRNA microRNA, NC negative control, PI propidium iodide

Mentions: qRT-PCR results showed that miR-34a was expressed in normal MSCs and that expression increased significantly when exposed to atmospheric conditions representing H/SD for 6 hours (Fig. 1a). We then used CCK-8 to evaluate the role of miR-34a in MSC survival, and found that overexpression of miR-34a reduced cell survival, while inhibition of miR-34a expression showed the opposite effect (Fig. 1b, c). Apoptosis has been identified as a major mechanism that reduces the survival rate after transplantation of MSCs into the harsh microenvironment of infarcted myocardium [26, 28]. To further determine the role of miR-34a in MSCs under conditions of H/SD, Annexin V–FITC/PI staining was performed. The results showed that miR-34a mimic-treated MSCs were significantly more apoptotic than the NC mimic group both in normal and H/SD conditions (Fig. 1d, e). However, when miR-34a was inhibited, MSCs showed better resistance to H/SD than the NC inhibitor group (Fig. 1d, e). This supports our hypothesis that the decreased cell survival and increased apoptosis in MSCs are associated with overexpression of miR-34a.Fig. 1


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)

miR-34a expression increases under H/SD, and correlates with decreased cell survival and increased apoptosis. a Rat MSCs were cultured in normal condition or exposed to H/SD for 6 hours and were transiently transfected with miR-34a mimic, NC mimic, miR-34a inhibitor, or NC inhibitor for 48 hours, respectively. The expression of miR-34a was determined by qRT-PCR. *P <0.05. b, c Effects of miR-34a on the vitality of MSCs were examined by CCK-8 assay. *P <0.05 vs. NC mimic (b), *P <0.05 vs. NC inhibitor (c). d, e Flow cytometric analysis of apoptotic cells in normal and H/SD conditions, in cultures of miR-34a mimic, NC mimic, miR-34a inhibitor, or NC inhibitor treated (MSCs were transfected for 48 hours and exposure to H/SD and maintained as such for 6 hours). Each column represents mean ± SD from three independent experiments. *P <0.05. H/SD hypoxia and serum deprivation, miRNA microRNA, NC negative control, PI propidium iodide
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: miR-34a expression increases under H/SD, and correlates with decreased cell survival and increased apoptosis. a Rat MSCs were cultured in normal condition or exposed to H/SD for 6 hours and were transiently transfected with miR-34a mimic, NC mimic, miR-34a inhibitor, or NC inhibitor for 48 hours, respectively. The expression of miR-34a was determined by qRT-PCR. *P <0.05. b, c Effects of miR-34a on the vitality of MSCs were examined by CCK-8 assay. *P <0.05 vs. NC mimic (b), *P <0.05 vs. NC inhibitor (c). d, e Flow cytometric analysis of apoptotic cells in normal and H/SD conditions, in cultures of miR-34a mimic, NC mimic, miR-34a inhibitor, or NC inhibitor treated (MSCs were transfected for 48 hours and exposure to H/SD and maintained as such for 6 hours). Each column represents mean ± SD from three independent experiments. *P <0.05. H/SD hypoxia and serum deprivation, miRNA microRNA, NC negative control, PI propidium iodide
Mentions: qRT-PCR results showed that miR-34a was expressed in normal MSCs and that expression increased significantly when exposed to atmospheric conditions representing H/SD for 6 hours (Fig. 1a). We then used CCK-8 to evaluate the role of miR-34a in MSC survival, and found that overexpression of miR-34a reduced cell survival, while inhibition of miR-34a expression showed the opposite effect (Fig. 1b, c). Apoptosis has been identified as a major mechanism that reduces the survival rate after transplantation of MSCs into the harsh microenvironment of infarcted myocardium [26, 28]. To further determine the role of miR-34a in MSCs under conditions of H/SD, Annexin V–FITC/PI staining was performed. The results showed that miR-34a mimic-treated MSCs were significantly more apoptotic than the NC mimic group both in normal and H/SD conditions (Fig. 1d, e). However, when miR-34a was inhibited, MSCs showed better resistance to H/SD than the NC inhibitor group (Fig. 1d, e). This supports our hypothesis that the decreased cell survival and increased apoptosis in MSCs are associated with overexpression of miR-34a.Fig. 1

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