Clinical and Experimental Pharmacology and Physiology (2014) 41, 351–357
doi: 10.1111/1440-1681.12227
ORIGINAL ARTICLE
MicroRNA-130a regulates autophagy of endothelial progenitor cells through Runx3 Quanfu Xu,*§ Shu Meng,*§ Bo Liu,*§ Mao-Quan Li,† Yeting Li,* Lu Fang‡ and Yi-Gang Li* *Department of Cardiology, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, †Department of Interventional Radiology, Tenth People’s Hospital of Tongji University, Shanghai, China and ‡Vascular Pharmacology Laboratory, Baker Heart and Diabetes Institute, Melbourne, Vic., Australia
SUMMARY Dysfunction of endothelial progenitor cells (EPC) contribute to diabetic vascular disease. MicroRNAs (miRNAs) are key regulators of diverse cellular processes, including angiogenesis. We recently reported that downregulated miR-130a in patients with Type 2 diabetes mellitus (DM) results in EPC dysfunction, including increased apoptosis, likely via its target runtrelated transcription factor 3 (Runx3). However, whether miR-130a affects the autophagy of EPC is unknown. The aim of the present study was to explore the effects of miR-130a on the autophagy and cell death of EPC, as well as their expression of Beclin 1 (BECN1; an initiator of autophagosome formation) and the anti-apoptotic protein Bcl2 (which binds to and inactivates BECN1), and the role of Runx3 in mediating these effects. The EPC were cultured from peripheral blood mononuclear cells of diabetic patients and non-diabetic controls. Cells were transfected with an miR-130a inhibitor, or mimic-miR-130a or mimic-miR-130a plus lentiviral vector expressing Runx3 to manipulate miR-130a and/or Runx3 levels. The number of autophagosomes was counted under transmission electron microscopy and cell death was examined by flow cytometry. The mRNA expression of Beclin1 was measured by real-time polymerase chain reaction and the protein expression of Beclin1 and Bcl2 was determined by western blotting. Both the number of autophagosomes and Beclin1 expression were increased in EPC from patients with DM. Inhibition of miR-130a increased the number of autophagosomes and Beclin1 expression, but attenuated Bcl2 expression. Overexpression of miR-130a decreased the number of autophagosomes, cell death and Beclin1 expression, but promoted Bcl2 expression; these effects were mediated by Runx3. In conclusion, miR-130a is important for maintaining normal autophagy levels and promoting the survival of EPC via regulation of Bcl-2 and Beclin1 expression, via Runx3. MiR-130a
Correspondence: Yi-Gang Li, Department of Cardiology, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Kongjiang Road 1665, Shanghai 200092, China. Email: drliyigang@outlook. com §
These authors contributed equally to this article.
Received 24 November 2013; revision 8 March 2014; accepted 11 March 2014. © 2014 Wiley Publishing Asia Pty Ltd
may be a regulator linking apoptosis and the autophagy of EPC. Key words: autophagy, Beclin1, diabetes mellitus, endothelial progenitor cells, miR-130a.
INTRODUCTION Endothelial dysfunction is an important factor in the pathogenesis of vascular disease in diabetes mellitus (DM).1 Endothelial progenitor cells (EPC) play an important role in vascular repair.2 It is known that patients with DM have a reduced number of EPC, which exhibit impaired function, which subsequently results in a reduction in the ability of EPC to perform endothelial repair.3–6 Apoptosis, a process of programmed cell death, is increased in EPC from patients with DM, and represents an underlying mechanism for the reduction of EPC numbers in DM. Recently, growing evidence suggests that autophagy may be associated with an alternative form of programmed cell death.7 Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and damaged organelles that maintains cellular homeostasis.8 Autophagy is initiated by the formation of double membrane-bound vesicles known as autophagosomes, which then fuse with the lysosome to degrade the sequestered cargo and to recycle nutrients and membranes.9 Autophagy is principally a cell protection and cell cleaning mechanism. Autophagy levels are very low under physiological conditions, but are upregulated in response to stress, such as nutrient deprivation, hypoxia, mitochondrial dysfunction and infection.7,8,10 Upregulation of autophagy is an adaptive response to survival, whereas excessive autophagy may cause self-digestion and promote cell death through excessive degradation of cellular constituents depending on the cellular and environmental context.11 Altered autophagy has been implicated in diseases such as cancer, neurodegenerative disorders, cardiovascular diseases and DM.12,13 However, whether autophagy is dysregulated in EPC from patients with DM is unknown. Autophagy is regulated by many autophagy-related genes (Atg), which are involved in autophagosome formation.14 Of these Atg, Beclin 1 (BECN1), the mammalian orthologue of yeast Atg6, interacts with Class III phosphatidylinositol 3-kinase (PI3K) and plays an important role in the initiation of autophagosome formation in autophagy.15 Binding of Bcl2 to BECN1 inhibits BECN1-mediated autophagy via sequestration of BECN1 away from Class III PI3-K, whereas disruption of the association
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between BECN1 and Bcl2 induces the initiation of autophagy by freeing BECN1 to bind to Class III PI3-K.16 Thus, in addition to inhibiting apoptotic cell death, Bcl2 inhibits autophagic cell death by binding to and inactivating BECN1. MicroRNAs (miRNAs) are a class of endogenously expressed, short, non-coding RNAs that post-transcriptionally regulate gene expression by inhibiting mRNA stability and/or protein translation.17 Single miRNAs can simultaneously regulate a multitude of targets and biological network.18,19 MicroRNAs are implicated in a broad range of biological processes, including cell proliferation, differentiation, apoptosis and stress response, linking them to numerous human diseases.18 In a recent study, we reported that miR-130a is downregulated in EPC derived from patients with Type 2 DM20; furthermore, miR-130a plays an important role in maintaining normal EPC function.21 Decreased miR-130a in EPC from patients with DM contributes to increased apoptosis and impaired function of EPC, mediated by its target runt-related transcription factor 3 (Runx3).21 However, how miR-130a influences autophagy of EPC has not been explored. Both autophagy and apoptosis play important roles in mammalian development, cellular homeostasis and oncogenesis, and they are highly interconnected and share many key regulators.22,23 In addition, several miRNAs have recently been implicated in the regulation of autophagy.24–26 So, we hypothesized that miR-130a, which decreases apoptosis in EPC, would also play an important role in regulating autophagy in EPC. Thus, the aims of the present study were to examine: (i) change of autophagy in EPC from patients with DM; (ii) how miR-130a regulates autophagy, cell death and BECN1 and Bcl2 expression in EPC; and (iii) whether Runx3 mediates the effects of miR130a on the autophagy and BECN1 and Bcl2 expression of EPC.
(a)
(b)
(c)
Fig. 1 Altered autophagy in endothelial progenitor cells (EPC) isolated from diabetic patients or non-diabetic controls. (a) Representative transmission electron microscopy images showing the autophagosomes (arrows) in EPC from diabetic and non-diabetic samples. (b) The number of autophagosomes per cell was increased in EPC from diabetic samples. (c) Beclin 1 (BECN1) mRNA levels were measured using SYBR green real-time polymerase chain reaction and are normalized against b-actin. Data are the mean SD (n = 3 in each group). *P < 0.05.
Effects of miR-130a on autophagy
RESULTS
Diabetic patients and non-diabetic controls were matched by age, gender, smoking history, blood pressure and cardiovascular risk factors (see Table S1 available as Supplementary Material to this paper). Consistent with previous results,21 miR-130a was significantly reduced, whereas Runx3, a direct target of miR-130a, was significantly increased in EPC derived from DM patients compared with those from non-diabetic controls (see Fig. S1a,b). After transfection with miR-130a or its controls, EPC retained high expression of the EPC markers CD34 and CD133, as determined by flow cytometry (Fig. S1c).
Previously, we reported that miR-130a regulated EPC functions21; in the present study we investigated whether miR-130a has any effect on the autophagy of EPC. Manipulation of miR130a was achieved by transfecting EPC with mimic-miR-130a or miR-130a inhibitor to overexpress or suppress miR-130a expression, respectively (see Fig. S2a). Compared with vector transfection, miR-130a inhibitor induced autophagosome accumulation in EPC from DM patients, although the effect did not reach statistical significance (P = 0.065; Fig. 2a). In contrast, a significant reduction in the number of autophagosomes was observed in EPC from DM patients overexpressing miR-130a; this was blocked by Runx3, a target of miR-130a (Fig. 2a).
Enhanced autophagy in EPC from DM patients
Regulation of BECN1 by miR-130a via Runx3
Autophagic activity was evaluated by counting the number of autophagosomes in EPC using transmission electron microscopy (TEM). The mean number of autophagosomes per cell, which was quite low in EPC from non-diabetic controls, was significantly increased in EPC from DM patients Fig. 1a,b). Because BECN1 plays an important role in the initiation of autophagosome formation in autophagy,15 we further measured gene expression of BECN1 in both diabetic and non-diabetic EPC using real-time polymerase chain reaction (PCR). Expression of BECN1 mRNA was significantly upregulated in EPC from DM patients compared with non-diabetic controls (Fig. 1c).
We further evaluated whether miR-130a modulated BECN1 and whether Runx3 was involved in such modulation. Consistent with previous results,21 miR-130a negatively regulated its target Runx3 in EPC (see Fig. S2b–g). Overespression of miR-130a significantly reduced, whereas miR-130a inhibition significantly increased, BECN1 mRNA expression in EPC from both controls and DM patients (Fig. 2c). Moreover, suppression of BECN1 expression at the mRNA and protein levels by miR-130a overexpression was completely rescued by cotransfection of mimicmiR-130a and lenti-Runx3 into EPC (Fig. 2d–g). These results suggest that miR-130a inhibits BECN1 through its target Runx3.
Subject characteristics and EPC
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MiR-130a regulates autophagy of EPC Effects of miR-130a on apoptosis and Bcl2 Because anti-apoptotic Bcl2 inhibits BECN1-dependent autophagy,27 and Runx3 has been reported to downregulate Bcl2 in cancer cells,28 we assumed that the miR-130a/Runx3 axis regulates both apoptosis and autophagy through Bcl2 and Bcl2– BECN1 interactions, respectively. Consistent with our previous study,21 we confirmed that apoptotic EPC were significantly increased in DM patients compared with non-diabetic controls and that miR-130a overexpression significantly inhibited EPC apoptosis by suppressing Runx3 (see Fig. S3a–c). We then examined the effect of miR-130a on antiapoptotic protein Bcl2. Inhibition of miR-130a significantly reduced Bcl2 protein expression in EPC from both groups. Overexpression of miR-130a significantly increased protein expression of Bcl2, which was counteracted by cotransfection of mimic-miR-130a and lenti-Runx3 (Fig. 3a,b), suggesting that miR-130a increases Bcl2 expression by repressing expression of Runx3. Effects of miR-130a on necrotic cell death We have demonstrated that miR-130a suppressed both apoptosis and autophagy in EPC. We further determined the role of miR130a in necrotic cell death. Compared with non-diabetic controls, cell death was significantly increased for EPC from DM patients (Fig. 2b). Overexpression of miR-130a protected EPC from cell death; however, Runx3 abolished such protection (Fig. 2b).
DISCUSSION In the present study we made several important observations. First, autophagosome accumulation and increased BECN1 expression were observed in EPC from DM patients. Next, miR-130a overexpression decreased, whereas miR-130a inhibition increased, the number of autophagosomes and BECN1 expression in EPC. In addition, miR-130a positively regulated Bcl2 expression in EPC, as well as protecting EPC against cell death. Furthermore, Runx3, a target of miR-130a, counteracted the effects of miR130a on autophagosome accumulation, cell death and BECN1 and Bcl-2 expression. Together, these observations suggest that miR-130a plays an important role in maintaining normal autophagy levels and promoting the survival of EPC in DM by regulating Bcl2 and BECN1 expression, via its target Runx3. Emerging evidence supports a role for autophagy in the pathophysiology of DM.13 However, how autophagy is altered in EPC in DM is unknown. Patients with DM have a reduced number of EPC, which exhibit impaired function.3–6 The reduction in the number of EPC in DM could be explained by increased apoptosis of EPC.21 In the present study, we found that autophagy was dysregulated in EPC from diabetic patients, as indicated by increases in the number of autophagosomes and BECN1 expression. Altered autophagy may contribute to the decreased number and impaired function of EPC in DM. In addition, a previous study suggested that altered autophagy may lead to the loss of islet b-cell mass in patients with Type 2 DM.29 Recent studies have implicated novel roles for miRNAs in the regulation of the autophagy process. For example, miR-885-39 may have a role in the regulation of early autophagy induction.24 In addition, miR-30a negatively regulates BECN1 expression and
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inhibits autophagic activity,25 and miR-376b is another miRNA that regulates BECN1 expression.26 We recently reported that miR130a was downregulated in EPC from DM patients,20 and that downregulation of miR-130a increased EPC apoptosis and impaired EPC function, via Runx3.21 In the present study, we found that miR-130a also inhibits autophagosome accumulation and BECN1 expression in EPC, an effect mediated by Runx3. Thus, downregulated miR-130a in EPC from DM patients causes dysregulated autophagy and autophagosome accumulation, which may contribute to excessive autophagic cell death and impaired function of the EPC. Thus, miR-130a is essential for protecting EPC against excessive apoptotic and autophagic cell death and maintaining normal EPC number and function. Similarly, miR130a was recently reported to inhibit autophagy by reducing autophagosome formation in human lymphocytic leukaemia.30 It is well established that endothelial dysfunction leads to the earliest events in atherogenesis and DM has been associated with accelerated atherosclerosis. Thus, the defect in EPC number and function due to downregulated miR-130 in DM, which leads to an impaired endothelial repair capacity of EPC, may play an important role in promoting atherogenesis in DM.31 Restoring miR-130 and subsequently enhancing EPC survival could improve endothelial repair and attenuate atherosclerosis in DM patients. Notably, it has been recognized that the striking accumulation of autophagic vacuoles in cells likely reflects an imbalance between the rates of autophagic sequestration and completion of the degradation process. That is, these cells can be thought of as undergoing ‘autophagic stress’.32 Patients and animals with Type 2 DM show autophagic vaculoe accumulation in cells due to defective autophagic flux,29,33 and therapies that induce autophagosome formation without increasing autophagic flux may cause more harm than good, whereas inhibitors that block the early steps of autophagy may be cytoprotective.10 Our results show that miR-130a is likely to decrease autophagosome formation by inhibiting BECN1 and protect the EPC against cell death, but whether miR-130 restores altered autophagic flux and regulates autophagic flux pathways in EPC from DM patients warrants further investigation. Both apoptotic and autophagic response machineries share common parthways.34 Apoptosis and autophagy may coexist in the same cell.35 The molecular regulators of both pathways are interconnected. The anti-apoptotic protein Bcl-2 acts as an antiautophagy protein via its inhibitory interaction with BECN1.27 Because Runx3 was reported to downregulate Bcl2 in cancer cells,28 we speculated that the miR-130a/Runx3 axis would regulate Bcl2 in EPC. Indeed, we found that inhibition of miR-130a significantly reduced, whereas overexpression of miR-130a significantly increased, protein expression of Bcl2 in EPC and that this effect was mediated by Runx3. These results suggest a cross-talk between apoptosis and autophagy in EPC in DM and that miR130a may be an important mediator of such cross-talk by regulating Runx3. In summary, in EPC, miR-130a suppresses its target Runx3 and subsequently upregulates Bcl2, which not only inhibits apoptosis, but also binds and inactivates BECN1, thereby inhibiting the initiation of autophagy. In addition, miR-130a may reduce BECN1 levels by suppressing Runx3 and inhibit autophagy in a Bcl2-independent manner. However, the cross-talk between apoptosis and autophagy is quite complex and involves a lot of regulators.22,36 Further studies are needed to investigate
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MiR-130a regulates autophagy of EPC
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Fig. 2 MicroRNA (miR)-130a reduced autophagy by suppressing Beclin 1 (BECN) via runt-related transcription factor 3 (Runx3). Endothelial progenitor cells (EPC) isolated from diabetic patients (■) or non-diabetic controls (□) were transfected with: (a) empty vector (Vector), anti-miR-130a, mimic-miR-130a (miR-130a) or both mimic-miR-130 and lentiviral Runx3 (miR-130 + Runx3); (c) mimic-miR-130a (miR-130a) or negative control (mimic-con), or miR-130a inhibitor (Anti-miR-130a) or negative control (Anti-con); or (b, d–g) empty vector (Vector), mimic-miR-130a (miR-130a) or both mimic-miR-130 and lentiviral Runx3 (miR-130 + Runx3). (a) The number of autophagosomes was counted under transmission electron microscopy. (b) Cell death, defined as Annexin-V- and propidium iodide-positive cells, was measured by flow cytometry. (c,d) Beclin 1 (BECN1) mRNA levels were measured by SYBR green real-time polymerase chain reaction and normalized against b-actin. (e–g) Expression of Runx3 and BECN1 protein was determined by western blotting. (e,f) Quantification of Runx3 and BECN1 bands. (g) representative bands from western blotting analysis. Data are the mean SD (n = 3 in each group). *P < 0.05.
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In conclusion, in addition to reducing apoptosis, miR-130a decreases autophagy and cell death in EPC by increasing antiapoptotic Bcl2 and decreasing the autophagosome initiator BECN1 via suppression of Runx3 (Fig. 4). Thus, miR-130a is essential for maintaining normal autophagy levels and normal EPC number and function.
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The EPC used in the present study were obtained from patients with Type 2 DM or non-diabetic controls. The characteristics of the EPC donors are gvien in Table S1. The study protocol was approved by the Ethics Review Board of the Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University. The study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each subject.
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Fig. 3 MicroRNA (miR)-130a upregulated antiapoptotic Bcl2 via runtrelated transcription factor 3 (Runx3). Endothelial progenitor cells (EPC) isolated from diabetic patients (■) or non-diabetic controls (□) were transfected with miR-130a inhibitor (Anti-miR-130a), negative control (Anticon), empty vector (Vector), mimic-miR-130a (miR-130a) or both mimicmiR-130 and lentiviral Runx3 (miR-130 + Runx3). Expression of Bcl2 protein was determined by western blotting analysis. (a) Quantification of Bcl2 bands. (b) Representative bands from western blotting analysis. Data are the mean SD (n = 3 in each group). *P < 0.05.
the effects of miR-130a on other regulators involved in the crosstalk between apoptosis and autophagy. The current interpretation of our data is limited by the fact that the EPC were pooled from five individuals in the diabetic or control groups, thus precluding sufficient assessment of association between clinical data and the biochemical outcomes.
Isolation and culture of EPC The EPC were cultured as described previously.20 Briefly, peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Isopaque Plus (Histopaque-1077; Sigma, St Louis, MO, USA) density gradient centrifugation of peripheral blood. The CD133 cells were selected from PBMC using CD133-coupled magnetic microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The CD133positive cells were then seeded onto fibronectin-coated six-well plates and cultured in endothelial basal medium (EBM; Lonza, Basel, Switzerland) supplemented with vascular endothelial growth factor (Peprotech, Rocky Hill, NJ, USA), human
miR-130a
Runx3
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Autophagy Fig. 4 Proposed mechanisms by which miR-130a regulates autophagy and apoptosis in endothelial progenitor cells (EPC). In EPC, miR-130a suppresses its target runt-related transcription factor 3 (Runx3) and subsuquently upregulates Bcl2, which not only inhibits apoptosis, but also binds and inactivates Beclin 1, thereby inhibiting the initiation of autophagy. In addition, miR-130a may reduce Beclin 1 levels by suppressing Runx3 and inhibiting autophagy in a Bcl2-independent manner.
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recombinant long insulin-like growth factor-1, ascorbic acid, cortisol and 20% fetal calf serum at 37°C in a 5% CO2 incubator. After 4 days, the medium was refreshed and after 7 days of culture early EPC developed an elongated spindle-shaped morphology. After 7 days culture, the EPC were used in the studies described below. The EPC from five donors each in the control or DM groups were pooled for real-time polymerase chain reaction (PCR) and western blotting analysis.
onto a 10% sodium dodecyl sulphate polyacrylamide gel. The separated proteins were transferred to polyvinylidene fluoride membranes and probed with primary antibodies against BECN1, Runx3, Bcl2 and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. b-Actin was used as loading control. Protein bands were detected using Odyssey (LI-COR Biosciences, Lincoln, NE, USA) and semiquantitative analysis was performed using Quantity one (Bio-Rad, Hercules, CA, USA).
Manipulation of miR-130a in EPC
Transmission electron microscopy
To inhibit or enhance miR-130a expression, am miR-130a inhibitor (MH12274; Ambion, Carlsbad, CA, USA) or a negative control (AM17010; Ambion), or a mimic-miR-130a (MC12274; Ambion) or a negative control (4464058; Ambion) was transfected into EPC using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.20 Cells were harvested 24 h after transfection and miR-130a expression was determined by quantitative real-time PCR.
For TEM, EPC were fixed with 2.5% glutaraldehyde in phosphate buffer and stored at 4°C overnight. The EPC were further fixed with 1% osmium tetroxide and stained with 1% uranyl acetate, followed by a gradient dehydration step using ethanol and acetone. The EPC were then embedded in araldite. Ultrathin sections were obtained (50 nm) and placed on uncoated copper grids. Images were examined with an H-7500 transmission electron microscope (Hitachi, Tokyo, Japan). Autophagic vacuoles (autophagosomes) from three sections per cell and three cells per group were counted.
Lentiviral constructs, packaging and transduction Expression plasmids for Runx3 were created using PCR amplification with human genomic DNA as the template. The primers used are given in Table S2. The PCR product of Runx3 was cloned into lentiviral expression plasmid pCDH-EF1-MCS-T2APuro (CD520A-1; System Biosciences, Mountain View, CA, USA). All constructs were confirmed by sequencing. To produce lentivirus, the plasmid DNA was transfected into 293T cells using psPAX2, a pMD2G packaging construct and Lipofectamine Plus reagent (Invitrogen) according to the manufacturer’s instructions. After 6 h, the medium was refreshed and viral supernatant was collected 48 h later. The EPC were seeded onto six-well plates (5 9 105 cells/well) and cells were transfected with lentiviral vectors at a multiplicity of infection (MOI) of 10, as described previously.20 Forty-eight hours after transfection, cells were selected and cloned by culture in the presence of puromycin (2 lg/mL) for 1 week. Isolation and quantification of RNA Total RNA was isolated from EPC using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Real-time PCR was performed to measure the expression levels of miRNA and mRNA on a Roche Light Cycler 480 system (Roche, Basel, Switzerland). For detection of miRNAs, TaqMan miRNA assay kits (Applied Biosystems, Carlsbad, CA, USA) were used in accordance with the manufacturer’s instructions. For measurement of mRNA, SYBR green was used. With each run, a reaction product melting curve was performed to provide evidence for a single PCR product. Levels of miRNA were normalized against those of small nucleolar RNA U6 (RNU6) expression, whereas mRNA levels were normalized against b-actin.
Flow cytometry analysis of EPC cell death Cell death was quantified by flow cytometry using an Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cells positive for both Annexin-V and propidium iodide were considered to be dead cells. Statistical analysis Data are expressed as the mean SD. Student’s t-test or the Wilcoxon rank-sum test was used to compare continuous variables between two groups depending on data distribution. Fisher’s exact test was used for comparisons of categorical variables. One-way ANOVA with Tukey’s post hoc test was used to compare differences among multiple groups when appropriate. Two-tailed P < 0.05 was considered significant. All experiments were performed at least three times. All statistical analyses were performed using IBM SPSS statistics for Windows 19.0 (SPSS, Chicago, IL, USA).
ACKNOWLEDGEMENTS This project was supported by grants from the Shanghai Committee of Science and Technology of China (12ZR1419500, 10JC1 412700), Shanghai’s Health Bureau Funding (ZYSNXD-CC-ZD YJ029) and National Natural Science Foundation of China (81270207).
DISCLOSURE The authors declare no conflicts of interest.
REFERENCES
Western blotting 20
Western blotting was performed as described previously. Briefly, EPC were lysed with 100 mmol/L phenylmethylsulphonyl fluoride and the protein extracts were denatured and loaded
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20. Meng S, Cao JT, Zhang B, Zhou Q, Shen CX, Wang CQ. Downregulation of microRNA-126 in endothelial progenitor cells from diabetes patients, impairs their functional properties, via target gene Spred-1. J. Mol. Cell. Cardiol. 2012; 53: 64–72. 21. Meng S, Cao J, Zhang X et al. Downregulation of microRNA-130a contributes to endothelial progenitor cell dysfunction in diabetic patients via its target Runx3. PLoS ONE 2013; 8: e68611. 22. Gump JM, Thorburn A. Autophagy and apoptosis: What is the connection? Trends Cell Biol. 2011; 21: 387–92. 23. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing. Crosstalk between autophagy and apoptosis. Nat. Rev. Mol. Cell Biol. 2007; 8: 741–52. 24. Huang Y, Chuang AY, Ratovitski EA. Phospho-DeltaNp63alpha/ miR-885-3p axis in tumor cell life and cell death upon cisplatin exposure. Cell Cycle 2011; 10: 3938–47. 25. Zhu H, Wu H, Liu X et al. Regulation of autophagy by a beclin 1-targeted microRNA, miR-30a, in cancer cells. Autophagy 2009; 5: 816–23. 26. Korkmaz G, le Sage C, Tekirdag KA, Agami R, Gozuacik D. miR376b controls starvation and mTOR inhibition-related autophagy by targeting ATG4C and BECN1. Autophagy 2012; 8: 165–76. 27. Pattingre S, Tassa A, Qu X et al. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 2005; 122: 927–39. 28. Guo C, Ding J, Yao L et al. Tumor suppressor gene Runx3 sensitizes gastric cancer cells to chemotherapeutic drugs by downregulating Bcl-2, MDR-1 and MRP-1. Int. J. Cancer 2005; 116: 155–60. 29. Masini M, Bugliani M, Lupi R et al. Autophagy in human type 2 diabetes pancreatic beta cells. Diabetologia 2009; 52: 1083–6. 30. Kovaleva V, Mora R, Park YJ et al. miRNA-130a targets ATG2B and DICER1 to inhibit autophagy and trigger killing of chronic lymphocytic leukemia cells. Cancer Res. 2012; 72: 1763–72. 31. Liao YF, Feng Y, Chen LL et al. Coronary heart disease risk equivalence in diabetes and arterial diseases characterized by endothelial function and endothelial progenitor cell. J. Diabetes Complicat. 2014; 28: 214–18. 32. Chu CT. Autophagic stress in neuronal injury and disease. J. Neuropathol. Exp. Neurol. 2006; 65: 423–32. 33. Marsh BJ, Soden C, Alarcon C et al. Regulated autophagy controls hormone content in secretory-deficient pancreatic endocrine betacells. Mol. Endocrinol. 2007; 21: 2255–69. 34. Maiuri MC, Le Toumelin G, Criollo A et al. Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin1. EMBO J. 2007; 26: 2527–39. 35. Kunchithapautham K, Rohrer B. Apoptosis and autophagy in photoreceptors exposed to oxidative stress. Autophagy 2007; 3: 433–41. 36. Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A. Life and death partners. Apoptosis, autophagy and the cross-talk between them. Cell Death Differ. 2009; 16: 966–75.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Fig. S1 Downregulation of miR-130a and upregulation of Runx3 in diabetic EPC. Cell markers of EPC were kept after transfection. Fig. S2 Expression of miR-130a and Runx3 in the EPC groups regulated by miR-130 inhibitor and mimic. Fig. S3 miR-130a decreases apoptosis via Runx3 in EPC. Table S1 Baseline characteristics of the study subjects. Table S2 Oligonucleotides for cloning Runx3. Data S1 Methods.
© 2014 Wiley Publishing Asia Pty Ltd
doi: 10.1111/1440-1681.12227
ORIGINAL ARTICLE
MicroRNA-130a regulates autophagy of endothelial progenitor cells through Runx3 Quanfu Xu,*§ Shu Meng,*§ Bo Liu,*§ Mao-Quan Li,† Yeting Li,* Lu Fang‡ and Yi-Gang Li* *Department of Cardiology, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, †Department of Interventional Radiology, Tenth People’s Hospital of Tongji University, Shanghai, China and ‡Vascular Pharmacology Laboratory, Baker Heart and Diabetes Institute, Melbourne, Vic., Australia
SUMMARY Dysfunction of endothelial progenitor cells (EPC) contribute to diabetic vascular disease. MicroRNAs (miRNAs) are key regulators of diverse cellular processes, including angiogenesis. We recently reported that downregulated miR-130a in patients with Type 2 diabetes mellitus (DM) results in EPC dysfunction, including increased apoptosis, likely via its target runtrelated transcription factor 3 (Runx3). However, whether miR-130a affects the autophagy of EPC is unknown. The aim of the present study was to explore the effects of miR-130a on the autophagy and cell death of EPC, as well as their expression of Beclin 1 (BECN1; an initiator of autophagosome formation) and the anti-apoptotic protein Bcl2 (which binds to and inactivates BECN1), and the role of Runx3 in mediating these effects. The EPC were cultured from peripheral blood mononuclear cells of diabetic patients and non-diabetic controls. Cells were transfected with an miR-130a inhibitor, or mimic-miR-130a or mimic-miR-130a plus lentiviral vector expressing Runx3 to manipulate miR-130a and/or Runx3 levels. The number of autophagosomes was counted under transmission electron microscopy and cell death was examined by flow cytometry. The mRNA expression of Beclin1 was measured by real-time polymerase chain reaction and the protein expression of Beclin1 and Bcl2 was determined by western blotting. Both the number of autophagosomes and Beclin1 expression were increased in EPC from patients with DM. Inhibition of miR-130a increased the number of autophagosomes and Beclin1 expression, but attenuated Bcl2 expression. Overexpression of miR-130a decreased the number of autophagosomes, cell death and Beclin1 expression, but promoted Bcl2 expression; these effects were mediated by Runx3. In conclusion, miR-130a is important for maintaining normal autophagy levels and promoting the survival of EPC via regulation of Bcl-2 and Beclin1 expression, via Runx3. MiR-130a
Correspondence: Yi-Gang Li, Department of Cardiology, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Kongjiang Road 1665, Shanghai 200092, China. Email: drliyigang@outlook. com §
These authors contributed equally to this article.
Received 24 November 2013; revision 8 March 2014; accepted 11 March 2014. © 2014 Wiley Publishing Asia Pty Ltd
may be a regulator linking apoptosis and the autophagy of EPC. Key words: autophagy, Beclin1, diabetes mellitus, endothelial progenitor cells, miR-130a.
INTRODUCTION Endothelial dysfunction is an important factor in the pathogenesis of vascular disease in diabetes mellitus (DM).1 Endothelial progenitor cells (EPC) play an important role in vascular repair.2 It is known that patients with DM have a reduced number of EPC, which exhibit impaired function, which subsequently results in a reduction in the ability of EPC to perform endothelial repair.3–6 Apoptosis, a process of programmed cell death, is increased in EPC from patients with DM, and represents an underlying mechanism for the reduction of EPC numbers in DM. Recently, growing evidence suggests that autophagy may be associated with an alternative form of programmed cell death.7 Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and damaged organelles that maintains cellular homeostasis.8 Autophagy is initiated by the formation of double membrane-bound vesicles known as autophagosomes, which then fuse with the lysosome to degrade the sequestered cargo and to recycle nutrients and membranes.9 Autophagy is principally a cell protection and cell cleaning mechanism. Autophagy levels are very low under physiological conditions, but are upregulated in response to stress, such as nutrient deprivation, hypoxia, mitochondrial dysfunction and infection.7,8,10 Upregulation of autophagy is an adaptive response to survival, whereas excessive autophagy may cause self-digestion and promote cell death through excessive degradation of cellular constituents depending on the cellular and environmental context.11 Altered autophagy has been implicated in diseases such as cancer, neurodegenerative disorders, cardiovascular diseases and DM.12,13 However, whether autophagy is dysregulated in EPC from patients with DM is unknown. Autophagy is regulated by many autophagy-related genes (Atg), which are involved in autophagosome formation.14 Of these Atg, Beclin 1 (BECN1), the mammalian orthologue of yeast Atg6, interacts with Class III phosphatidylinositol 3-kinase (PI3K) and plays an important role in the initiation of autophagosome formation in autophagy.15 Binding of Bcl2 to BECN1 inhibits BECN1-mediated autophagy via sequestration of BECN1 away from Class III PI3-K, whereas disruption of the association
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between BECN1 and Bcl2 induces the initiation of autophagy by freeing BECN1 to bind to Class III PI3-K.16 Thus, in addition to inhibiting apoptotic cell death, Bcl2 inhibits autophagic cell death by binding to and inactivating BECN1. MicroRNAs (miRNAs) are a class of endogenously expressed, short, non-coding RNAs that post-transcriptionally regulate gene expression by inhibiting mRNA stability and/or protein translation.17 Single miRNAs can simultaneously regulate a multitude of targets and biological network.18,19 MicroRNAs are implicated in a broad range of biological processes, including cell proliferation, differentiation, apoptosis and stress response, linking them to numerous human diseases.18 In a recent study, we reported that miR-130a is downregulated in EPC derived from patients with Type 2 DM20; furthermore, miR-130a plays an important role in maintaining normal EPC function.21 Decreased miR-130a in EPC from patients with DM contributes to increased apoptosis and impaired function of EPC, mediated by its target runt-related transcription factor 3 (Runx3).21 However, how miR-130a influences autophagy of EPC has not been explored. Both autophagy and apoptosis play important roles in mammalian development, cellular homeostasis and oncogenesis, and they are highly interconnected and share many key regulators.22,23 In addition, several miRNAs have recently been implicated in the regulation of autophagy.24–26 So, we hypothesized that miR-130a, which decreases apoptosis in EPC, would also play an important role in regulating autophagy in EPC. Thus, the aims of the present study were to examine: (i) change of autophagy in EPC from patients with DM; (ii) how miR-130a regulates autophagy, cell death and BECN1 and Bcl2 expression in EPC; and (iii) whether Runx3 mediates the effects of miR130a on the autophagy and BECN1 and Bcl2 expression of EPC.
(a)
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Fig. 1 Altered autophagy in endothelial progenitor cells (EPC) isolated from diabetic patients or non-diabetic controls. (a) Representative transmission electron microscopy images showing the autophagosomes (arrows) in EPC from diabetic and non-diabetic samples. (b) The number of autophagosomes per cell was increased in EPC from diabetic samples. (c) Beclin 1 (BECN1) mRNA levels were measured using SYBR green real-time polymerase chain reaction and are normalized against b-actin. Data are the mean SD (n = 3 in each group). *P < 0.05.
Effects of miR-130a on autophagy
RESULTS
Diabetic patients and non-diabetic controls were matched by age, gender, smoking history, blood pressure and cardiovascular risk factors (see Table S1 available as Supplementary Material to this paper). Consistent with previous results,21 miR-130a was significantly reduced, whereas Runx3, a direct target of miR-130a, was significantly increased in EPC derived from DM patients compared with those from non-diabetic controls (see Fig. S1a,b). After transfection with miR-130a or its controls, EPC retained high expression of the EPC markers CD34 and CD133, as determined by flow cytometry (Fig. S1c).
Previously, we reported that miR-130a regulated EPC functions21; in the present study we investigated whether miR-130a has any effect on the autophagy of EPC. Manipulation of miR130a was achieved by transfecting EPC with mimic-miR-130a or miR-130a inhibitor to overexpress or suppress miR-130a expression, respectively (see Fig. S2a). Compared with vector transfection, miR-130a inhibitor induced autophagosome accumulation in EPC from DM patients, although the effect did not reach statistical significance (P = 0.065; Fig. 2a). In contrast, a significant reduction in the number of autophagosomes was observed in EPC from DM patients overexpressing miR-130a; this was blocked by Runx3, a target of miR-130a (Fig. 2a).
Enhanced autophagy in EPC from DM patients
Regulation of BECN1 by miR-130a via Runx3
Autophagic activity was evaluated by counting the number of autophagosomes in EPC using transmission electron microscopy (TEM). The mean number of autophagosomes per cell, which was quite low in EPC from non-diabetic controls, was significantly increased in EPC from DM patients Fig. 1a,b). Because BECN1 plays an important role in the initiation of autophagosome formation in autophagy,15 we further measured gene expression of BECN1 in both diabetic and non-diabetic EPC using real-time polymerase chain reaction (PCR). Expression of BECN1 mRNA was significantly upregulated in EPC from DM patients compared with non-diabetic controls (Fig. 1c).
We further evaluated whether miR-130a modulated BECN1 and whether Runx3 was involved in such modulation. Consistent with previous results,21 miR-130a negatively regulated its target Runx3 in EPC (see Fig. S2b–g). Overespression of miR-130a significantly reduced, whereas miR-130a inhibition significantly increased, BECN1 mRNA expression in EPC from both controls and DM patients (Fig. 2c). Moreover, suppression of BECN1 expression at the mRNA and protein levels by miR-130a overexpression was completely rescued by cotransfection of mimicmiR-130a and lenti-Runx3 into EPC (Fig. 2d–g). These results suggest that miR-130a inhibits BECN1 through its target Runx3.
Subject characteristics and EPC
© 2014 Wiley Publishing Asia Pty Ltd
MiR-130a regulates autophagy of EPC Effects of miR-130a on apoptosis and Bcl2 Because anti-apoptotic Bcl2 inhibits BECN1-dependent autophagy,27 and Runx3 has been reported to downregulate Bcl2 in cancer cells,28 we assumed that the miR-130a/Runx3 axis regulates both apoptosis and autophagy through Bcl2 and Bcl2– BECN1 interactions, respectively. Consistent with our previous study,21 we confirmed that apoptotic EPC were significantly increased in DM patients compared with non-diabetic controls and that miR-130a overexpression significantly inhibited EPC apoptosis by suppressing Runx3 (see Fig. S3a–c). We then examined the effect of miR-130a on antiapoptotic protein Bcl2. Inhibition of miR-130a significantly reduced Bcl2 protein expression in EPC from both groups. Overexpression of miR-130a significantly increased protein expression of Bcl2, which was counteracted by cotransfection of mimic-miR-130a and lenti-Runx3 (Fig. 3a,b), suggesting that miR-130a increases Bcl2 expression by repressing expression of Runx3. Effects of miR-130a on necrotic cell death We have demonstrated that miR-130a suppressed both apoptosis and autophagy in EPC. We further determined the role of miR130a in necrotic cell death. Compared with non-diabetic controls, cell death was significantly increased for EPC from DM patients (Fig. 2b). Overexpression of miR-130a protected EPC from cell death; however, Runx3 abolished such protection (Fig. 2b).
DISCUSSION In the present study we made several important observations. First, autophagosome accumulation and increased BECN1 expression were observed in EPC from DM patients. Next, miR-130a overexpression decreased, whereas miR-130a inhibition increased, the number of autophagosomes and BECN1 expression in EPC. In addition, miR-130a positively regulated Bcl2 expression in EPC, as well as protecting EPC against cell death. Furthermore, Runx3, a target of miR-130a, counteracted the effects of miR130a on autophagosome accumulation, cell death and BECN1 and Bcl-2 expression. Together, these observations suggest that miR-130a plays an important role in maintaining normal autophagy levels and promoting the survival of EPC in DM by regulating Bcl2 and BECN1 expression, via its target Runx3. Emerging evidence supports a role for autophagy in the pathophysiology of DM.13 However, how autophagy is altered in EPC in DM is unknown. Patients with DM have a reduced number of EPC, which exhibit impaired function.3–6 The reduction in the number of EPC in DM could be explained by increased apoptosis of EPC.21 In the present study, we found that autophagy was dysregulated in EPC from diabetic patients, as indicated by increases in the number of autophagosomes and BECN1 expression. Altered autophagy may contribute to the decreased number and impaired function of EPC in DM. In addition, a previous study suggested that altered autophagy may lead to the loss of islet b-cell mass in patients with Type 2 DM.29 Recent studies have implicated novel roles for miRNAs in the regulation of the autophagy process. For example, miR-885-39 may have a role in the regulation of early autophagy induction.24 In addition, miR-30a negatively regulates BECN1 expression and
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inhibits autophagic activity,25 and miR-376b is another miRNA that regulates BECN1 expression.26 We recently reported that miR130a was downregulated in EPC from DM patients,20 and that downregulation of miR-130a increased EPC apoptosis and impaired EPC function, via Runx3.21 In the present study, we found that miR-130a also inhibits autophagosome accumulation and BECN1 expression in EPC, an effect mediated by Runx3. Thus, downregulated miR-130a in EPC from DM patients causes dysregulated autophagy and autophagosome accumulation, which may contribute to excessive autophagic cell death and impaired function of the EPC. Thus, miR-130a is essential for protecting EPC against excessive apoptotic and autophagic cell death and maintaining normal EPC number and function. Similarly, miR130a was recently reported to inhibit autophagy by reducing autophagosome formation in human lymphocytic leukaemia.30 It is well established that endothelial dysfunction leads to the earliest events in atherogenesis and DM has been associated with accelerated atherosclerosis. Thus, the defect in EPC number and function due to downregulated miR-130 in DM, which leads to an impaired endothelial repair capacity of EPC, may play an important role in promoting atherogenesis in DM.31 Restoring miR-130 and subsequently enhancing EPC survival could improve endothelial repair and attenuate atherosclerosis in DM patients. Notably, it has been recognized that the striking accumulation of autophagic vacuoles in cells likely reflects an imbalance between the rates of autophagic sequestration and completion of the degradation process. That is, these cells can be thought of as undergoing ‘autophagic stress’.32 Patients and animals with Type 2 DM show autophagic vaculoe accumulation in cells due to defective autophagic flux,29,33 and therapies that induce autophagosome formation without increasing autophagic flux may cause more harm than good, whereas inhibitors that block the early steps of autophagy may be cytoprotective.10 Our results show that miR-130a is likely to decrease autophagosome formation by inhibiting BECN1 and protect the EPC against cell death, but whether miR-130 restores altered autophagic flux and regulates autophagic flux pathways in EPC from DM patients warrants further investigation. Both apoptotic and autophagic response machineries share common parthways.34 Apoptosis and autophagy may coexist in the same cell.35 The molecular regulators of both pathways are interconnected. The anti-apoptotic protein Bcl-2 acts as an antiautophagy protein via its inhibitory interaction with BECN1.27 Because Runx3 was reported to downregulate Bcl2 in cancer cells,28 we speculated that the miR-130a/Runx3 axis would regulate Bcl2 in EPC. Indeed, we found that inhibition of miR-130a significantly reduced, whereas overexpression of miR-130a significantly increased, protein expression of Bcl2 in EPC and that this effect was mediated by Runx3. These results suggest a cross-talk between apoptosis and autophagy in EPC in DM and that miR130a may be an important mediator of such cross-talk by regulating Runx3. In summary, in EPC, miR-130a suppresses its target Runx3 and subsequently upregulates Bcl2, which not only inhibits apoptosis, but also binds and inactivates BECN1, thereby inhibiting the initiation of autophagy. In addition, miR-130a may reduce BECN1 levels by suppressing Runx3 and inhibit autophagy in a Bcl2-independent manner. However, the cross-talk between apoptosis and autophagy is quite complex and involves a lot of regulators.22,36 Further studies are needed to investigate
© 2014 Wiley Publishing Asia Pty Ltd
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Fig. 2 MicroRNA (miR)-130a reduced autophagy by suppressing Beclin 1 (BECN) via runt-related transcription factor 3 (Runx3). Endothelial progenitor cells (EPC) isolated from diabetic patients (■) or non-diabetic controls (□) were transfected with: (a) empty vector (Vector), anti-miR-130a, mimic-miR-130a (miR-130a) or both mimic-miR-130 and lentiviral Runx3 (miR-130 + Runx3); (c) mimic-miR-130a (miR-130a) or negative control (mimic-con), or miR-130a inhibitor (Anti-miR-130a) or negative control (Anti-con); or (b, d–g) empty vector (Vector), mimic-miR-130a (miR-130a) or both mimic-miR-130 and lentiviral Runx3 (miR-130 + Runx3). (a) The number of autophagosomes was counted under transmission electron microscopy. (b) Cell death, defined as Annexin-V- and propidium iodide-positive cells, was measured by flow cytometry. (c,d) Beclin 1 (BECN1) mRNA levels were measured by SYBR green real-time polymerase chain reaction and normalized against b-actin. (e–g) Expression of Runx3 and BECN1 protein was determined by western blotting. (e,f) Quantification of Runx3 and BECN1 bands. (g) representative bands from western blotting analysis. Data are the mean SD (n = 3 in each group). *P < 0.05.
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In conclusion, in addition to reducing apoptosis, miR-130a decreases autophagy and cell death in EPC by increasing antiapoptotic Bcl2 and decreasing the autophagosome initiator BECN1 via suppression of Runx3 (Fig. 4). Thus, miR-130a is essential for maintaining normal autophagy levels and normal EPC number and function.
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The EPC used in the present study were obtained from patients with Type 2 DM or non-diabetic controls. The characteristics of the EPC donors are gvien in Table S1. The study protocol was approved by the Ethics Review Board of the Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University. The study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each subject.
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Fig. 3 MicroRNA (miR)-130a upregulated antiapoptotic Bcl2 via runtrelated transcription factor 3 (Runx3). Endothelial progenitor cells (EPC) isolated from diabetic patients (■) or non-diabetic controls (□) were transfected with miR-130a inhibitor (Anti-miR-130a), negative control (Anticon), empty vector (Vector), mimic-miR-130a (miR-130a) or both mimicmiR-130 and lentiviral Runx3 (miR-130 + Runx3). Expression of Bcl2 protein was determined by western blotting analysis. (a) Quantification of Bcl2 bands. (b) Representative bands from western blotting analysis. Data are the mean SD (n = 3 in each group). *P < 0.05.
the effects of miR-130a on other regulators involved in the crosstalk between apoptosis and autophagy. The current interpretation of our data is limited by the fact that the EPC were pooled from five individuals in the diabetic or control groups, thus precluding sufficient assessment of association between clinical data and the biochemical outcomes.
Isolation and culture of EPC The EPC were cultured as described previously.20 Briefly, peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Isopaque Plus (Histopaque-1077; Sigma, St Louis, MO, USA) density gradient centrifugation of peripheral blood. The CD133 cells were selected from PBMC using CD133-coupled magnetic microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The CD133positive cells were then seeded onto fibronectin-coated six-well plates and cultured in endothelial basal medium (EBM; Lonza, Basel, Switzerland) supplemented with vascular endothelial growth factor (Peprotech, Rocky Hill, NJ, USA), human
miR-130a
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Autophagy Fig. 4 Proposed mechanisms by which miR-130a regulates autophagy and apoptosis in endothelial progenitor cells (EPC). In EPC, miR-130a suppresses its target runt-related transcription factor 3 (Runx3) and subsuquently upregulates Bcl2, which not only inhibits apoptosis, but also binds and inactivates Beclin 1, thereby inhibiting the initiation of autophagy. In addition, miR-130a may reduce Beclin 1 levels by suppressing Runx3 and inhibiting autophagy in a Bcl2-independent manner.
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recombinant long insulin-like growth factor-1, ascorbic acid, cortisol and 20% fetal calf serum at 37°C in a 5% CO2 incubator. After 4 days, the medium was refreshed and after 7 days of culture early EPC developed an elongated spindle-shaped morphology. After 7 days culture, the EPC were used in the studies described below. The EPC from five donors each in the control or DM groups were pooled for real-time polymerase chain reaction (PCR) and western blotting analysis.
onto a 10% sodium dodecyl sulphate polyacrylamide gel. The separated proteins were transferred to polyvinylidene fluoride membranes and probed with primary antibodies against BECN1, Runx3, Bcl2 and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. b-Actin was used as loading control. Protein bands were detected using Odyssey (LI-COR Biosciences, Lincoln, NE, USA) and semiquantitative analysis was performed using Quantity one (Bio-Rad, Hercules, CA, USA).
Manipulation of miR-130a in EPC
Transmission electron microscopy
To inhibit or enhance miR-130a expression, am miR-130a inhibitor (MH12274; Ambion, Carlsbad, CA, USA) or a negative control (AM17010; Ambion), or a mimic-miR-130a (MC12274; Ambion) or a negative control (4464058; Ambion) was transfected into EPC using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.20 Cells were harvested 24 h after transfection and miR-130a expression was determined by quantitative real-time PCR.
For TEM, EPC were fixed with 2.5% glutaraldehyde in phosphate buffer and stored at 4°C overnight. The EPC were further fixed with 1% osmium tetroxide and stained with 1% uranyl acetate, followed by a gradient dehydration step using ethanol and acetone. The EPC were then embedded in araldite. Ultrathin sections were obtained (50 nm) and placed on uncoated copper grids. Images were examined with an H-7500 transmission electron microscope (Hitachi, Tokyo, Japan). Autophagic vacuoles (autophagosomes) from three sections per cell and three cells per group were counted.
Lentiviral constructs, packaging and transduction Expression plasmids for Runx3 were created using PCR amplification with human genomic DNA as the template. The primers used are given in Table S2. The PCR product of Runx3 was cloned into lentiviral expression plasmid pCDH-EF1-MCS-T2APuro (CD520A-1; System Biosciences, Mountain View, CA, USA). All constructs were confirmed by sequencing. To produce lentivirus, the plasmid DNA was transfected into 293T cells using psPAX2, a pMD2G packaging construct and Lipofectamine Plus reagent (Invitrogen) according to the manufacturer’s instructions. After 6 h, the medium was refreshed and viral supernatant was collected 48 h later. The EPC were seeded onto six-well plates (5 9 105 cells/well) and cells were transfected with lentiviral vectors at a multiplicity of infection (MOI) of 10, as described previously.20 Forty-eight hours after transfection, cells were selected and cloned by culture in the presence of puromycin (2 lg/mL) for 1 week. Isolation and quantification of RNA Total RNA was isolated from EPC using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Real-time PCR was performed to measure the expression levels of miRNA and mRNA on a Roche Light Cycler 480 system (Roche, Basel, Switzerland). For detection of miRNAs, TaqMan miRNA assay kits (Applied Biosystems, Carlsbad, CA, USA) were used in accordance with the manufacturer’s instructions. For measurement of mRNA, SYBR green was used. With each run, a reaction product melting curve was performed to provide evidence for a single PCR product. Levels of miRNA were normalized against those of small nucleolar RNA U6 (RNU6) expression, whereas mRNA levels were normalized against b-actin.
Flow cytometry analysis of EPC cell death Cell death was quantified by flow cytometry using an Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cells positive for both Annexin-V and propidium iodide were considered to be dead cells. Statistical analysis Data are expressed as the mean SD. Student’s t-test or the Wilcoxon rank-sum test was used to compare continuous variables between two groups depending on data distribution. Fisher’s exact test was used for comparisons of categorical variables. One-way ANOVA with Tukey’s post hoc test was used to compare differences among multiple groups when appropriate. Two-tailed P < 0.05 was considered significant. All experiments were performed at least three times. All statistical analyses were performed using IBM SPSS statistics for Windows 19.0 (SPSS, Chicago, IL, USA).
ACKNOWLEDGEMENTS This project was supported by grants from the Shanghai Committee of Science and Technology of China (12ZR1419500, 10JC1 412700), Shanghai’s Health Bureau Funding (ZYSNXD-CC-ZD YJ029) and National Natural Science Foundation of China (81270207).
DISCLOSURE The authors declare no conflicts of interest.
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Western blotting 20
Western blotting was performed as described previously. Briefly, EPC were lysed with 100 mmol/L phenylmethylsulphonyl fluoride and the protein extracts were denatured and loaded
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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Fig. S1 Downregulation of miR-130a and upregulation of Runx3 in diabetic EPC. Cell markers of EPC were kept after transfection. Fig. S2 Expression of miR-130a and Runx3 in the EPC groups regulated by miR-130 inhibitor and mimic. Fig. S3 miR-130a decreases apoptosis via Runx3 in EPC. Table S1 Baseline characteristics of the study subjects. Table S2 Oligonucleotides for cloning Runx3. Data S1 Methods.
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