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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 induces apoptosis by modifying SIRT1 and FOXO3a expression. a, b Apoptosis was analyzed by measuring Annexin V+/PI– cells using flow cytometry in cultures of siRNA-SIRT1, siRNA-NT, or siRNA-SIRT1 cotransfected with miR-34a inhibitor-treated MSCs, under normal and H/SD conditions (MSCs were transfected for 72 hours and exposure to H/SD and maintained as such for 6 hours). *P <0.05 vs. normal siRNA-NT, △P < 0.05 vs. H/SD siRNA-NT. c, d MSCs were transfected with miR-34a mimic, NC mimic, siRNA-SIRT1, or siRNA-NT for 72 hours, respectively, and then CASP3 and PARP1 activity was measured using western blot. *P <0.05 vs. NC mimic, △P <0.05 vs. siRNA-NT. e, f Western blot analysis of SIRT1, FOXO3a, Bim, CASP3, and PARP1 protein expression in cultures of siRNA-NT, siRNA-SIRT1, miR-34a inhibitor, or siRNA-SIRT1 cotransfected with miR-34a inhibitor-treated MSCs, under normal and H/SD conditions (MSCs were transfected for 72 hours and exposure to H/SD and maintained as such for 6 hours). β-actin was used as the internal control. Each column represents mean ± SD from three independent experiments. *P <0.05 vs. normal scramble, △P <0.05 vs. H/SD scramble. CASP3 caspase 3, FOXO3a forkhead box O transcription factor 3a, H/SD hypoxia and serum deprivation, miRNA microRNA, NC negative control, PARP1 polyADP-ribose polymerase 1, PI propidium iodide, SIRT1 silent information regulator 1, siRNA small interfering RNA, siRNA-NT scrambled siRNA
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Fig3: miR-34a induces apoptosis by modifying SIRT1 and FOXO3a expression. a, b Apoptosis was analyzed by measuring Annexin V+/PI– cells using flow cytometry in cultures of siRNA-SIRT1, siRNA-NT, or siRNA-SIRT1 cotransfected with miR-34a inhibitor-treated MSCs, under normal and H/SD conditions (MSCs were transfected for 72 hours and exposure to H/SD and maintained as such for 6 hours). *P <0.05 vs. normal siRNA-NT, △P < 0.05 vs. H/SD siRNA-NT. c, d MSCs were transfected with miR-34a mimic, NC mimic, siRNA-SIRT1, or siRNA-NT for 72 hours, respectively, and then CASP3 and PARP1 activity was measured using western blot. *P <0.05 vs. NC mimic, △P <0.05 vs. siRNA-NT. e, f Western blot analysis of SIRT1, FOXO3a, Bim, CASP3, and PARP1 protein expression in cultures of siRNA-NT, siRNA-SIRT1, miR-34a inhibitor, or siRNA-SIRT1 cotransfected with miR-34a inhibitor-treated MSCs, under normal and H/SD conditions (MSCs were transfected for 72 hours and exposure to H/SD and maintained as such for 6 hours). β-actin was used as the internal control. Each column represents mean ± SD from three independent experiments. *P <0.05 vs. normal scramble, △P <0.05 vs. H/SD scramble. CASP3 caspase 3, FOXO3a forkhead box O transcription factor 3a, H/SD hypoxia and serum deprivation, miRNA microRNA, NC negative control, PARP1 polyADP-ribose polymerase 1, PI propidium iodide, SIRT1 silent information regulator 1, siRNA small interfering RNA, siRNA-NT scrambled siRNA

Mentions: After identifying SIRT1 as a direct target of miR-34a, we investigated whether knockdown of SIRT1 by siRNA (siRNA-SIRT1) induces apoptosis in MSCs. Similar to miR-34a mimic treatment, suppression of SIRT1 expression promoted apoptosis, revealed by flow cytometric analysis of the percentage of cells that were Annexin V+/PI– (Fig. 3a, b). CASP3 is a well-studied mediator of apoptosis, because it is either partially or totally responsible for the cleavage of many key proteins, such as PARP1 [18]. In this study, increased activities of the cleaved CASP3 and cleaved PARP1 were observed when SIRT1 was knocked down or miR-34a was overexpressed (Fig. 3b, d). These findings suggest that knockdown of SIRT1 or treatment with miR-34a mimic acts similarly in the regulation of apoptosis.Fig. 3


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 induces apoptosis by modifying SIRT1 and FOXO3a expression. a, b Apoptosis was analyzed by measuring Annexin V+/PI– cells using flow cytometry in cultures of siRNA-SIRT1, siRNA-NT, or siRNA-SIRT1 cotransfected with miR-34a inhibitor-treated MSCs, under normal and H/SD conditions (MSCs were transfected for 72 hours and exposure to H/SD and maintained as such for 6 hours). *P <0.05 vs. normal siRNA-NT, △P < 0.05 vs. H/SD siRNA-NT. c, d MSCs were transfected with miR-34a mimic, NC mimic, siRNA-SIRT1, or siRNA-NT for 72 hours, respectively, and then CASP3 and PARP1 activity was measured using western blot. *P <0.05 vs. NC mimic, △P <0.05 vs. siRNA-NT. e, f Western blot analysis of SIRT1, FOXO3a, Bim, CASP3, and PARP1 protein expression in cultures of siRNA-NT, siRNA-SIRT1, miR-34a inhibitor, or siRNA-SIRT1 cotransfected with miR-34a inhibitor-treated MSCs, under normal and H/SD conditions (MSCs were transfected for 72 hours and exposure to H/SD and maintained as such for 6 hours). β-actin was used as the internal control. Each column represents mean ± SD from three independent experiments. *P <0.05 vs. normal scramble, △P <0.05 vs. H/SD scramble. CASP3 caspase 3, FOXO3a forkhead box O transcription factor 3a, H/SD hypoxia and serum deprivation, miRNA microRNA, NC negative control, PARP1 polyADP-ribose polymerase 1, PI propidium iodide, SIRT1 silent information regulator 1, siRNA small interfering RNA, siRNA-NT scrambled siRNA
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Fig3: miR-34a induces apoptosis by modifying SIRT1 and FOXO3a expression. a, b Apoptosis was analyzed by measuring Annexin V+/PI– cells using flow cytometry in cultures of siRNA-SIRT1, siRNA-NT, or siRNA-SIRT1 cotransfected with miR-34a inhibitor-treated MSCs, under normal and H/SD conditions (MSCs were transfected for 72 hours and exposure to H/SD and maintained as such for 6 hours). *P <0.05 vs. normal siRNA-NT, △P < 0.05 vs. H/SD siRNA-NT. c, d MSCs were transfected with miR-34a mimic, NC mimic, siRNA-SIRT1, or siRNA-NT for 72 hours, respectively, and then CASP3 and PARP1 activity was measured using western blot. *P <0.05 vs. NC mimic, △P <0.05 vs. siRNA-NT. e, f Western blot analysis of SIRT1, FOXO3a, Bim, CASP3, and PARP1 protein expression in cultures of siRNA-NT, siRNA-SIRT1, miR-34a inhibitor, or siRNA-SIRT1 cotransfected with miR-34a inhibitor-treated MSCs, under normal and H/SD conditions (MSCs were transfected for 72 hours and exposure to H/SD and maintained as such for 6 hours). β-actin was used as the internal control. Each column represents mean ± SD from three independent experiments. *P <0.05 vs. normal scramble, △P <0.05 vs. H/SD scramble. CASP3 caspase 3, FOXO3a forkhead box O transcription factor 3a, H/SD hypoxia and serum deprivation, miRNA microRNA, NC negative control, PARP1 polyADP-ribose polymerase 1, PI propidium iodide, SIRT1 silent information regulator 1, siRNA small interfering RNA, siRNA-NT scrambled siRNA
Mentions: After identifying SIRT1 as a direct target of miR-34a, we investigated whether knockdown of SIRT1 by siRNA (siRNA-SIRT1) induces apoptosis in MSCs. Similar to miR-34a mimic treatment, suppression of SIRT1 expression promoted apoptosis, revealed by flow cytometric analysis of the percentage of cells that were Annexin V+/PI– (Fig. 3a, b). CASP3 is a well-studied mediator of apoptosis, because it is either partially or totally responsible for the cleavage of many key proteins, such as PARP1 [18]. In this study, increased activities of the cleaved CASP3 and cleaved PARP1 were observed when SIRT1 was knocked down or miR-34a was overexpressed (Fig. 3b, d). These findings suggest that knockdown of SIRT1 or treatment with miR-34a mimic acts similarly in the regulation of apoptosis.Fig. 3

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