Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been used in experimental research and clinical trials for heart function restoration and cardiomyocyte regeneration. However, due to a hostile microenvironment created by ischemia, hypoxia and pro-inflammatory factors, the survival rate of implanted BM-MSCs remains low. Therefore, strategies that can promote BM-MSC survival and prevent apoptosis are required. Previous studies have reported that microRNA-16 (miR-16) can inhibit cell proliferation by targeting several proteins and signal pathway, not only by inducing apoptosis. In the present study, it was investigated whether inhibition of miR-16 reduced BM-MSC apoptosis in a model of ischemia. Flow cytometry analysis revealed that BM-MSCs underwent apoptosis in response to hypoxia/serum deprivation (SD). Additionally, in hypoxic/SD conditions, miR-16 expression increased and B-cell lymphoma (Bcl)-2 protein expression decreased in BM-MSCs. miR-16 did not affect Bcl-2 mRNA expression but downregulated Bcl-2 protein expression. miR-16 inhibitor transfection significantly increased Bcl-2 protein expression and the percentage of apoptotic BM-MSCs was reduced. The pro-apoptotic effects of miR-16 were partially elevated by knocking down of Bcl-2. Furthermore, it was demonstrated that miR-16 exerted its pro-apoptotic effects by activating the mitochondrial pathway of apoptosis via apoptotic protease activating factor-1/caspase-9/poly (ADP ribose) polymerase. Taken together, the results indicated that miR-16 downregulated Bcl-2 expression and promoted BM-MSC apoptosis, indicating that therapies targeting miR-16 may improve the effectiveness of BM-MSC transplantation therapy.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) are capable of self-renewal and multilineage differentiation into various cell types (
microRNAs (miRNAs/miRs) are endogenous RNAs ~22 nucleotides in length that negatively regulate gene expression by targeting mRNAs for cleavage or translational repression, which occurs primarily through base pairing to the 3′ untranslated region (UTR) of target mRNAs (
Among the known miRNAs, miR-16 has been reported to regulate apoptosis and the cell cycle by targeting cyclin D3 (CCND3), cyclin E1 (CCNE1), CDK6 and B-cell lymphoma (Bcl)-2 in tumor cells (
Accumulating evidence suggests that miR-16 targets Bcl-2 at the posttranscriptional level (
Male Sprague-Dawley rats weighing 60–80 g (8–10 weeks old) were housed under standardized conditions at 22°C in a 12-h light/dark cycle and fed a laboratory diet with water
BM-MSCs were cultured using the whole bone marrow adherent method, as previously reported (
Apoptosis was induced by hypoxia and SD
Small interfering RNAs (siRNAs) are small double-stranded RNAs that target mRNA to silence its expression. A Bcl-2 siRNA duplex was synthesized by Thermo Fisher Scientific, Inc. (sense, 5′-GCUGCACCUGACGCCCUUCTT-3′ and antisense, 3′-TTCGACGUGGACUGCGGGAAG-5′). Cells were transfected using X-tremeGENE™ siRNA Transfection Reagent (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) as previously described (
BM-MSC viability and proliferation was determined using an MTT assay (Sigma-Aldrich; Merck KGaA) and an EdU incorporation assay (Guangzhou RiboBio Co., Ltd., Guangzhou, China), respectively, according to the manufacturers' protocols. For the MTT assay, cells were seeded into a 96-well plate (3,000 cells/well), and viability was detected with the addition of 20 µl MTT (5 mg/ml), dissolved in DMSO, to the culture medium. The absorbance of each well was quantified at 490 nm using the Infinite M200 PRO plate reader (Tecan, Morrisville, NC, USA). All data were calculated from triplicate samples and are presented as the mean ± standard deviation.
For the EdU incorporation assay, BM-MSCs were cultured in 96-well plates at a density of 4×103 cells/well for 24 h at 37°C. Following this, 50 µM EdU was added to each well and cells were cultured for additional 2 h at 37°C. Cells were fixed with 4% formaldehyde for 15 min at room temperature and subsequently treated with 0.5% Triton X-100 for 20 min for permeabilization. Following three washes with PBS, 100 µl 1X Apollo reaction cocktail was added to each well and the cells were incubated for 30 min at room temperature prior to staining with 100 µl Hoechst 33342 (10 µg/ml) at room temperature (24°C) for 30 min and visualization under a fluorescent microscope (magnification, × 100; Leica Microsystems GmbH, Wetzlar, Germany). The positive staining rate (%) was counted as positive cells (green cells)/overall cells (blue cells). DAPI (50 µg/ml) stain was conducted in 37°C for 2 h. Cells were counted from 6 random fields in triplicate wells for each condition and expressed as percentage of total number of cells in the field. All experiments were performed in triplicate and three independent repeated experiments were performed.
Total RNA was extracted from the BM-MSCs with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA with the miRcute miRNA first-strand cDNA synthesis kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. PCR (Initial Denaturation, 98°C, 30 sec, 25–35 cycles at 98°C, 5–10 sec; 45–72°C, 10–30 sec; 72°C, 15–30 sec per kb. Final Extension, 72°C, 5–10 min. Hold, 4–10°C.) was performed to analyze the level of miR-16 using the miRcute miRNA qPCR Detection kit (SYBR Green; Tiangen Biotech Co., Ltd.). All primers for miR-16 and U6 for the TaqMan miRNA assays were purchased from Thermo Fisher Scientific, Inc. The miR-16 primer sequences were as follows: forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′. The relative expression level of miR-16 was normalized to that of the internal control U6 using the 2−∆∆Cq cycle threshold method (
Cells were washed twice with ice-cold PBS 72 h after transfection and harvested for further analysis. Total protein was obtained with RIPA protein extraction buffer (Thermo Fisher) and the concentration was analyzed using the bicinchoninic acid protein assay and 50 µg total protein per lane, extracted with ice-cold PBS was resolved via 12% SDS-PAGE and subsequently transferred onto polyvinylidene difluoride membranes. Non-specific binding was inhibited by incubating the membranes with 5% skim milk and Tris buffered saline with 0.5% Tween-20 (TBST) at 4°C for 12 h. Membranes were incubated with the following primary antibodies overnight at 4°C: Bcl-2, caspase-3 (1:1,000; cat no. ab49822), caspase-9 (1:1,000; cat. no. ab61789; both Abcam, Cambridge, UK), APAF-1 (1:1,000), cleaved PARP (1:1,000; Cell Signaling Technology; Danvers, MA, USA) and β-actin (1:1,000; OriGene Technologies, Inc., Beijing, China). The membranes were washed with TBS-T and subsequently incubated with horseradish peroxidase-conjugated Affinipure goat anti-rabbit IgG and anti-mouse IgG secondary antibodies (1:5,000; TA140003; OriGene Technologies, Inc.) for 1 h at 37°C. Specific complexes were visualized on an X-ray film via enhanced chemiluminescence (ECL) detection with BeyoECL Plus (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer's protocol. Densitometric analysis was performed using a GS-710 Imaging Densitometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and Image Lab™ Software (170–9690) to measure protein levels normalized to that of internal control β-actin. All data were obtained in triplicate, independent experiments.
Cell apoptosis was examined using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, cells were incubated with 5 µl Annexin V-FITC solution in the dark at room temperature for 15 min, followed by staining with 5 µl propidium iodide (PI) at room temperature for 5 min, according to the manufacturer's protocol. Stained cells were distinguished as viable (Annexin V/PI−), dead (Annexin V/PI+), early apoptotic (Annexin V+/PI−), or late apoptotic (Annexin V+/PI+) using a FACSCalibur flow cytometer. Data were analyzed with the BD FACSCanto II equipped with BD FACSDiva software version 8.0 (BD Biosciences). Experiments were done in triplicate.
The MMP was analyzed using the JC-1 MMP Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol (
All data are expressed as the mean ± standard deviation. Differences between the mean values were calculated with one way analysis of variance with Fisher's LSD used for the post-hoc multiple comparisons test. Statistical analyses were performed using SPSS software (version 22.0; IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.
To determine if miR-16 was involved in hypoxia/SD-induced apoptosis, BM-MSCs were exposed to hypoxic/SD conditions for different periods of time (3–24 h). Apoptosis was detected by Annexin V-FITC, which binds to phosphatidylserine, a phospholipid that is redistributed from the inner to the outer leaflet of the cell membrane in early apoptosis. Following membrane integrity loss, PI may also enter the cell and intercalate into DNA (
Previous studies have indicated that miRNAs regulate gene expression by decreasing mRNA translation, increasing mRNA degradation, or both (
To confirm whether elevated miR-16 expression decreased Bcl-2 expression in hypoxia/SD-induced BM-MSCs apoptosis, miR-16 mimics were transfected. As a control, BM-MSCs were transfected with a miR-16 inhibitor or were placed in hypoxic/SD conditions with no transfection. The miR-16 mimic increased miR-16 expression in a dose-dependent manner; no significant difference in Bcl-2 mRNA expression was detected (
To examine the potential role of miR-16 in regulating cell viability and proliferation, MTT and EdU assays were performed. Notably, it was determined that the number of EdU-positive cells (green) in the miR-16 mimic group were reduced compared with the inhibitor group (
To examine the role of miR-16 in apoptosis promotion, miR-16 expression was increased or decreased in BM-MSCs by transfecting cells with a miR-16 mimic, or a miR-16 inhibitor for 72 h, respectively. After 24 h of incubation under hypoxic/SD conditions, miR-16 led to a marked pro-apoptotic effect. Flow cytometry also demonstrated that there was a significant decrease in the percentage of apoptotic cells in the anti-miR-16 group (
Cytochrome c is a key mediator of apoptosis that is released from mitochondria, and its release is inhibited by the presence of Bcl-2 incorporated into the outer membrane of organelles (
To explore miR-16-mediated regulation of Bcl-2 in BM-MSCs apoptosis and growth, Bcl-2 siRNA was used to knockdown Bcl-2 expression. As presented in
The results of the present study revealed that miR-16 expression was significantly upregulated under hypoxic/SD conditions in BM-MSCs. Downregulation of miR-16 resulted in decreased apoptosis and enhanced cell proliferation. Bcl-2 was identified as a direct and functional target of miR-16. Further experiments indicated that miR-16 induced an intrinsic apoptosis pathway via APAF-1/caspase-9/PARP. The present study may lead to the discovery of a more optimized and effective target in MSC-based therapy for myocardial infarction. BM-MSC transplantation is a potential therapeutic approach to improve cardiac function in patients following myocardial infarction (
Recent evidence has revealed significant roles of miRNAs, including miR-16, in numerous forms of cardiovascular disease (
To elucidate the association between the upregulation of miR-16 expression under hypoxic/SD conditions and downregulation of its target Bcl-2 protein, RT-qPCR and western blot analysis was performed in the present study. Overexpression of miR-16 led to decreased Bcl-2 protein expression and increased apoptosis. In BM-MSCs transfected with miR-16 mimics, increased apoptosis, release of cytochrome c, and cleavage of pro-caspase-9 and PARP was observed, indicating that the reduction in Bcl-2 protein expression by miR-16 was sufficient to initiate the apoptotic process. These results suggest that miR-16 induced apoptosis in BM-MSCs through activation of the APAF-1/caspase-9/PARP pathway.
In conclusion, the present study indicated that exposure to hypoxic/SD conditions significantly increased the expression of miR-16 in BM-MSCs. miR-16 overexpression may promote apoptosis through downregulation of the anti-apoptotic Bcl-2 protein. Additionally, it was demonstrated that miR-16 suppressed the expression of Bcl-2 at the post-transcriptional level and induced apoptosis by activating the intrinsic APAF-1/caspase-9/PARP pathway. Furthermore, inhibition of miR-16 provided protection to BM-MSCs against hypoxia/SD-induced apoptosis. Therefore, miR-16 may be a potential therapeutic target for supporting cellular transplantation therapy in myocardial infarction.
The authors would like to thank Dr Wei Liu (The Key Laboratory of Myocardial Ischemia Mechanism and Treatment, Harbin Medical University, Ministry of Education, Harbin, Heilongjiang, China) for her excellent technical assistance and helpful discussions.
The present study was supported by the National Natural Science Foundation of China (grant no. 3001-30400432).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JR contributed to the experimental design, performed the molecular biology experiments and the statistical analysis, and drafted the manuscript. SF participated in the design of the study and performed the statistical analysis. YW revised the manuscript and performed some of the experiments. BL was responsible for MSC transfection and statistical analysis. BY participated in the design of the study. SL conceived the study and participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.
All experimental animal procedures were approved by the Ethical Committee on Animal Care and Use of Harbin Medical University (Harbin, China).
Not applicable.
The authors declare that they have no competing interests.
miR-16 expression in hypoxia/SD-induced apoptosis. (A) Cells were stained with Annexin V and PI and analyzed by flow cytometry. The percentage of cells in each group within the gated areas is indicated; the upper right panel represents cells undergoing late apoptosis, and the lower right panel represents cells undergoing early apoptosis. (B) Fold change in apoptosis. Data are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 vs. ctrl, +P<0.05 vs. the respective group at 3 h, #P<0.05 vs. the respective group at 9 h. (C) Quantitative analysis of miR-16 and Bcl-2 mRNA and protein expression by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Fold change compared with the expression levels in corresponding control cells are presented. Each data point represents the mean ± standard deviation of three independent experiments. *P<0.05 and #P<0.05 vs. Ctrl. SD, serum deprivation; PI, propidium iodide; Bcl-2, B-cell lymphoma 2; miR-16, microRNA-16; Ctrl, control.
Bcl-2 was confirmed a target of miR-16 and its expression was inversely proportional to miR-16 expression. (A) BM-MSCs were transfected with a miR-16 mimic or (B) miR-16 inhibitor (15–30 nM). Protein expression was calculated by densitometric analysis and experiments were performed in triplicate. *P<0.05 vs. Ctrl. (C) Reverse transcription-quantitative polymerase chain reaction analysis of miR-16 and Bcl-2 mRNA expression in BM-MSCs following transfection with miRNA mimics (0, 15, 20 and 30 nM) for 24 h. *P<0.05 vs. Ctrl group. (D) Protein expression of Bcl-2 following transfection with either a miR-16 mimic or inhibitor, or under hypoxic/SD conditions. Densitometric analysis was performed to calculate relative protein levels. All data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. Ctrl group. B-cell lymphoma 2; miR-16, microRNA-16; BM-MSC, bone marrow-derived mesenchymal stem cell; SD, serum deprivation; Ctrl, control.
Effects of miR-16 on proliferation and hypoxia/SD-induced apoptosis. (A) Representative images from the EdU incorporation assay. Magnification, ×100. (B) The proliferation rate of BM-MSCs transfected with miR-16 inhibitor was significantly higher compared with the other experimental groups. *P<0.05 vs. Ctrl group, #P<0.05 vs. the anti-miR-16 group. (C) BM-MSC viability following miR-16 mimic or inhibitor transfection. (D) Flow cytometry analysis of apoptotic cells following Annexin V/PI staining. (E) Fold change in cell apoptosis. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 and #P<0.05 vs. the respective Ctrl group. miR-16, microRNA-16; SD, serum deprivation; BM-MSC, bone marrow-derived mesenchymal stem cell; PI, propidium iodide; Ctrl, control.
miR-16 promoted apoptosis via activation of a mitochondrial pathway involving APAF-1/caspase-9/PARP. (A) Representative immunoblots and quantitative analysis of mitochondrial or cytosolic cytochrome c levels in BM-MSCs transfected with miR-16 mimics or inhibitor. The expression of (B) APAF-1 and caspase-9, and (C) cleaved caspase-3 and cleaved PARP was significantly increased in cells transfected with miR-16 mimics. (D) BM-MSCs (red) transfected with miR-16 mimics or inhibitor were then exposed to hypoxia/SD for 24 h, and mitochondrial membrane (green) potential was assessed using a JC-1 MMP Assay. Magnification, ×100. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 and #P<0.05 vs. the respective Ctrl group. miR-16, microRNA-16, APAF-1, apoptotic protease activating factor-1; PARP, poly (ADP ribose) polymerase; BM-MSC, bone marrow-derived mesenchymal stem cell; SD, serum deprivation; cytC, cytochrome c; mito, mitochondrial; cyto, cytoplasmic; Cl, cleaved; MMP, mitochondrial membrane potential.
Bcl-2 was involved in miR-16-induced apoptosis under hypoxic/SD conditions. (A) Silencing of Bcl-2 expression was confirmed by western blot analysis in BM-MSCs transfected with siBcl-2 or (B) co-transfected with siBcl-2 and miR-16 inhibitor. (C) BM-MSC proliferation and viability following transfection of miR-16 inhibitor and siBcl-2 was determined by EdU and MTT assays. (D) Flow cytometry analysis of apoptosis following miR-16 inhibitor and siBcl-2 transfection. (E) Fold change in cell apoptosis. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. ctrl, #P<0.05 vs. Hypoxia/SD, +P<0.01 vs. anti-miR-16. Bcl-2, B-cell lymphoma 2; miR-16, microRNA-16; BM-MSC, bone marrow-derived mesenchymal stem cell; siBcl-2, Bcl-2 small interfering RNA; SD, serum deprivation; Ctrl, control.