miR-21 Reduces Hydrogen Peroxide-Induced Apoptosis in c-kit+ Cardiac Stem Cells In Vitro through PTEN/PI3K/Akt Signaling

The low survival rate of cardiac stem cells (CSCs) in the infarcted myocardium hampers cell therapy for ischemic cardiomyopathy. MicroRNA-21 (miR-21) and one of its target proteins, PTEN, contribute to the survival and proliferation of many cell types, but their prosurvival effects in c-kit+ CSC remain unclear. Thus, we hypothesized that miR-21 reduces hydrogen peroxide- (H2O2-) induced apoptosis in c-kit+ CSC and estimated the contribution of PTEN/PI3K/Akt signaling to this oxidative circumstance. miR-21 mimics efficiently reduced H2O2-induced apoptosis in c-kit+ CSC, as evidenced by the downregulation of the proapoptosis proteins caspase-3 and Bax and upregulation of the antiapoptotic Bcl-2. In addition, the gain of function of miR-21 in c-kit+ CSC downregulated the protein level of PTEN although its mRNA level changed slightly; in the meantime, miR-21 overexpression also increased phospho-Akt (p-Akt). The antiapoptotic effects of miR-21 were comparable with Phen (bpV), the selective inhibitor of PTEN, while miR-21 inhibitor or PI3K's inhibitor LY294002 efficiently attenuated the antiapoptotic effect of miR-21. Taken together, these results indicate that the anti-H2O2-induced apoptosis effect of miR-21 in c-kit+ CSC is contributed by PTEN/PI3K/Akt signaling. miR-21 could be a potential molecule to facilitate the c-kit+ CSC therapy in ischemic myocardium.


Introduction
Ischemic heart disease is still the leading cause of deaths worldwide. Despite advances in medicine, such as the catheter-based therapies, the 5-year mortality rate for myocardial infarction remains as high as 50% [1]. Alternative strategies, such as stem cell-based therapies, are urgently needed [2].
Stem cell-based therapies are promising in repairing cardiac damage due to ischemia-reperfusion (I/R) injury [3,4]. Among various types of stem cells being investigated, c-kit + cardiac stem cells (CSCs) appeared to be particularly promising because they are capable of differentiating into cardiomyocytes, smooth muscle cells, and endothelial cells [5]. In the past decade, studies demonstrated the abilities of human and rodent c-kit + CSCs to promote cardiac regeneration and attenuate myocardial infarction (MI) induced heart dysfunction and remodeling in various animal models [6][7][8][9][10][11][12][13]. A recent report showed the beneficial effects of c-kit + CSCs on ventricular remodeling and dysfunction sustained for more than one year in rats [7].
CSCs treatment of MI has demonstrated efficacy in the SCIPIO human Phase I trial [14]. After receiving CSCs, MI patients showed smaller infarct scars six months later. Despite the minimal cardiomyogenic potential of CSCs [7,15], many researches have displayed their potential of promoting angiogenesis as well as decreasing apoptosis and necrosis in vivo, either via differentiation towards vascular lineages [16] or by secretion of growth factors [17] and/or extracellular microRNAs (miRNAs) [18].
However, poor engraftment and viability of CSCs minimize the percentage of cell survival and hamper functional 2 Oxidative Medicine and Cellular Longevity improvements and cardiac outcomes [19]. The very poor survival of donor cells is one of the challenges that need to be overcome before CSC-based therapies become a clinical reality. It is reported that >90% of transplanted CSCs die within a week and >95% within 5 weeks in mice with MI [20,21]. The massive loss of CSCs limits their effectiveness as a therapy. Strategies to enhance cell survival after adoptive transfer would produce notable therapeutic implications in post-MI patients. Strategies to increase cell survival include preconditioning the cells with a variety of techniques, including heat shock of the cells prior to transplantation, forced expression of survival factors in the donor cells, and exposure of cells to prosurvival factors [22][23][24]. Hu et al. improved the engraftment of transplanted CSCs and therapeutic efficacy for treatment of ischemic heart disease using a miRNA prosurvival cocktail, which contained miR-21, miR-24, and miR-221 [19].
In this study, we provide evidence that miR-21 may protect c-kit + CSCs against H 2 O 2 -induced apoptosis through the PI3K/PTEN/Akt signaling. This suggests that miR-21 possesses the ability to protect c-kit + CSCs from oxidative injury and miR-21 could be a potential molecule to facilitate stem cell treatment of ischemic myocardium.

c-kit + Cells Isolation, Purification, and Identification.
CSCs were isolated [58] and purified [59] using previously published methods, with some modifications. Rats were deeply anesthetized with sevoflurane, and the atrial appendage was sliced and digested with 0.1% collagenase type II (Sigma, USA). After about 40 min digestion at 37 ∘ C, cells were collected by sedimentation at 1200 rpm for 5 minutes (min). Then, cells from the atrial appendage were incubated in a humidity chamber in Ham's F12 medium containing 10% FBS, 1% penicillin and streptomycin, 1% Lglutamine, 20 ng/mL human recombinant fibroblast growth factor, 20 ng/mL leukocyte inhibitory factor, and 10 ng/mL epidermal growth factor (EGF). When cells confluence reached >90%, they were suspended by trypsinization. Then, CSCs were incubated with rabbit anti-c-kit antibody (1 : 250 in F12 medium) for 1 hour (h) and sorted out with antirabbit secondary antibody conjugated 2.8 m magnetic beads (Dynal Biotech, Norway) in 30 min as instructed by the manufacturer's protocols. The purified c-kit + CSCs were cultured in the aforementioned F12 medium. Flow cytometry was used to confirm the expression patterns of CSCs markers. Cells were incubated with fluorochrome-conjugated primary antibodies: anti-CD34-PE, anti-CD45-PE, and anti-c-kit primary antibody and anti-c-kit IgG-allophycocyanin (APC) secondary antibody (all from BioLegend, USA).

H 2 O 2 -Induced Oxidative Stress Model and Its Effect on Apoptosis in CSCs.
Harvested CSCs were incubated in serum-free F12 medium for 24 h before being treated with H 2 O 2 (0, 50, 100, and 200 M) for 2 h. Early apoptosis and necrosis of CSCs were determined by flow cytometry using Annexin V-FITC/PI staining assay as reported elsewhere [34]. The phosphatidylserine level on CSC surface was estimated with Annexin V-FITC and propidium iodide (PI) apoptosis detection kit (Solarbio, China) according to the manufacturer's instructions. Apoptosis and necrosis of c-kit + CSCs were analyzed in a FACSCalibur flow cytometer (BD Biosciences, USA). Results were expressed as the percentage of apoptotic or necrotic cells from total cells. Flow cytometry was performed thrice using CSCs from three independent experiments.

Reverse Transcription and Real-Time PCR of miR-21 and
PTEN. mRNA and miRNA levels were determined by using quantitative RT-PCR as previously reported [60,61]. Briefly, RNAs from CSCs were isolated by the TRIzol (Invitrogen, USA) method. RT-PCR was performed on cDNA generated from 3 g of total RNA with a cDNA synthesis kit (TaKaRa, Japan) according to the manufacturer's protocol. RT-qPCR was performed with the CFX Connect Real-Time system (Bio-Rad, USA) using a SYBR Green PrimeScript RT kit (TaKaRa, Japan) based on the manufacturer's instructions. The PCR conditions included predenaturation at 95 ∘ C for 30 s followed by 40 cycles of denaturation at 95 ∘ C for 10 s and combined annealing/extension at 58 ∘ C for 30 s. All the mRNA expression levels were calculated based on the comparative quantification method (2 −ΔΔCT ). U6 and -actin were used as internal controls for miR-21 and PTEN mRNA quantitation, respectively.

miR-21 Mimics and Inhibitor Transfection and the Detection of miR-21's Effects on Apoptosis in CSCs.
For the miR-21 gain-of-function and loss-of-function experiments, miR-21 mimics, miR-21 inhibitor, and their control scrambles were added in 1.5 mL F12 medium in 6-well plates with 5 L transfection reagent Lipofectamine 2000 (Invitrogen, USA) and then incubated with c-kit + CSCs for 48 h according to the manufacturer's instructions. Early apoptosis and necrosis of CSCs were determined by flow cytometry using Annexin V-FITC/PI staining assay as previously mentioned. Flow cytometry was performed twice using c-kit + CSCs from three independent experiments.

Immunofluorescence of Caspase-3.
To characterize purity of isolated CSCs, immunocytochemistry was used to verify c-kit expression on purified cells as reported elsewhere [62]. Cells were fixed with 4% paraformaldehyde and then blocked with 10% goat serum before being incubated with anticaspase-3 antibody. c-kit + CSCs were subsequently incubated with FITC-conjugated secondary antibody. After washing, the nuclei were counterstained with DAPI. The immunofluorescence photos were taken with a fluorescence microscope (Olympus, Japan).

Western Blot.
Western blot analysis of total protein from c-kit + cell lysis was performed as described previously [63]. The protein extracts were separated by SDSpolyacrylamide gels electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After overnight blocking in nonfat milk solution, membranes were probed with anti-PTEN, anti-phospho-Akt, anti-Akt, anti-caspase-3, anti-Bax, anti-Bcl-2, anti--actin, or anti-GAPDH primary antibodies. PVDF membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h and then enhanced chemiluminescence (Amersham Biosciences, USA). Immunoreactivity was visualized by a ChemiDoc MP system (Bio-Rad, USA). Protein levels were normalized toactin or GAPDH.

Statistical
Analysis. Data are presented as mean ± SD. All data were analyzed by Student's -test or by one-way ANOVA followed by LSD or Dunnett's T3 post hoc test for multiple comparisons. A value of less than 0.05 was considered to be statistically significant. Data analyses were carried out using SPSS (v.19.0, IBM, USA).

Isolated c-kit +
CSCs. c-kit + CSCs were isolated from rat atrial appendage and purified using anti-rabbit secondary antibody conjugated magnetic beads. Flow cytometry showed that 90.2% of cells were c-kit positive after the purification. Purified cells were stained with anti-c-kit antibody and counterstained with DAPI to visualize the nuclei. The immunofluorescence staining also showed a high percentage of double staining of c-kit + and DAPI (Figure 1(a)).        The upregulation of miR-21 was stable in 72 h, and no difference was detected among 24, 48, and 72 h group (Figure 2(a)). We choose 48 h as the incubation time in the subsequent experiments. In addition, 48 h miR-21 inhibitor treatment decreased miR-21 expression compared with control or inhibitor scramble group (Figure 2(b)).

miR-21 Decreased H 2 O 2 -Induced Apoptosis in CSCs.
The antiapoptotic effect of miR-21 was detected with flow cytometry using the Annexin V-FITC/PI staining assay. We found that miR-21 and its inhibitor have a little effect on apoptosis in normal cultured CSC, but miR-21 mimics significantly decreased CSC apoptosis after H 2 O 2 insult (Figures 2(c) and 2(d)). Moreover, the caspase-3 and Bax expression was markedly decreased, while Bcl-2 apparently increased in the miR-21 mimics group, as determined by using immunofluorescence and western blot (Figure 3).

miR-21 Decreased PTEN Protein Expression.
Although PTEN was extensively reported as one of miR-21's target genes in many cell types, western blot was employed to verify miRmimic's effect on PTEN expression in c-kit + CSCs. mRNA level of PTEN did not change (Figure 4(a)), while PTEN protein was significantly downregulated in mimics group as compared with control or miR-21 scramble group ( < 0.05, Figure 4(b)).

Discussion
Stem cell therapy is promising for the prevention and treatment of ischemic cardiomyopathy [1]. c-kit + CSCs have emerged as one of the most potential CSCs [64]. Nevertheless, poor engraftment and viability limit the percentage of injected stem cells that contribute to cardiac functional improvements [19]. miRNAs hold the potential of improving engraftment and functional outcomes of cardiac progenitor cell transplantation [19,31]. Studies have shown that miR-21 protects the myocardium against ischemic injury [61]. miR-21 also protects cardiomyocytes and BMSCs from H 2 O 2induced cell apoptosis and death. However, it is unknown Inhibitor Mimics        miR-21 expression in c-kit + CSCs too. Although miR-21 was found to be downregulated by H 2 O 2 treatment in H9C2 cell line [65] and BMSCs [27], most of the cardiac (myocytes, fibroblasts) and vascular cells (smooth muscle cells) treated with H 2 O 2 showed increased miR-21 level [61,66]. These results indicate that miR-21 expression patterns after H 2 O 2 insult are cell specific. In addition, we should realize that miR-21 is a double-edged sword in ischemia-reperfusion injury, such as inflammation and angiogenesis [67]. This negative correlation between apoptosis and miR-21 expression indicates that miR-21 downregulation may aggravate apoptosis. This interaction was further confirmed by the gainof-function analyses of miR-21, in which miR-21 significantly decreased c-kit + CSC apoptosis, as well as the caspase-3 and Bax expression, and meanwhile increased Bcl-2 protein expression (Figures 3(a)-3(c)). These results indicate that miR-21 may be an antiapoptotic factor in c-kit + CSCs under oxidative stress.
Although PTEN has been extensively reported as one of miR-21's target genes, it is not confirmed in CSCs to our knowledge. In many cell types, such as hepatocytes, cardiomyocytes, and cancer cells, miR-21 mediates the expression of PTEN [27,49,57]. We hypothesized that PTEN is a target gene of miR-21 in CSCs too. PTEN expression was directly calculated (Figure 3) after upregulation of miR-21 and the results confirmed our assumption. miR-21 upregulation caused significant downregulation of PTEN protein expression although the mRNA level of PTEN did not change much (Figures 3(a)-3(c)).
The PI3K/Akt pathway constitutes an approach to inhibit apoptosis and promote cell proliferation [68]. The activation of Akt significantly protects cells from H 2 O 2 -induced cell apoptosis [69,70]. It has been reported that miR-21 affects the PI3K/Akt pathway through the expression of PTEN [49], but this effect has not been investigated in c-kit + CSCs. To study whether the PTEN/PI3K/Akt signaling is responsible for miR-21 mediated antiapoptotic effect, we blocked PTEN and PI3K with their inhibitors Phen and LY294002, respectively, and then tested phosphorylation of Akt. Just like the prosurvival effects of miR-21, Phen administration decreased H 2 O 2induced apoptosis in c-kit + CSCs (Figures 4(a) and 4(b)). For PI3K blocker LY294002, it partially reversed antiapoptotic effects of miR-21 mimics; at the same time, it attenuated miR-21's suppression of caspase-3 and Bax and the upregulation of Bcl-2 (Figures 6(c)-6(e)). Furthermore, both Phen and miR-21 mimics increased p-Akt level, while PI3K inhibitor LY294002 decreased p-Akt level drastically, which suggests that Akt is downstream of PI3K and PTEN. Although most literatures reported that Phen only downregulated the activity of PTEN but not its expression, some found that the mRNA and/or the protein expression was decreased after Phen administration [71,72] as our results showed. We deem that the time window and the dose could be two influence factors. There may exist some feedback between PTEN activity and PTEN expression.
We found that the PTEN and PI3K inhibitors did not completely offset the prosurvival effects of miR-21. This is reasonable because miR-21 targets more than one gene, and, besides PTEN and PI3K mediated pathway, there could be other survival pathways involved. For example, miR-21 protects cardiac myocytes from the H 2 O 2 -induced injury by targeting PDCD4 gene, which is upstream of activator protein 1 (AP-1) [61]. AP-1 has been proved to be a key signaling molecule that determines life or death cell fates in response to extracellular stimuli including ROS. In addition, miR-21 promotes glioma invasion by targeting RECK and TIMP3 genes, which are suppressors of malignancy and inhibitors of matrix metalloproteinases [73]. PTEN also enhances human multipotent cardiovascular progenitors therapeutic effects via miR-21 initiated PTEN/HIF-1 /VEGF-A signaling. miR-21 in hESC-derived stage-specific embryonic antigen 1 (SSEA-1) positive cells inhibited phosphatase and tensin homolog (PTEN), which resulted in the activation of HIF-1 and the systemic release of VEGF-A [39].
In conclusion, our data reveal that miR-21 prevents CSCs from H 2 O 2 -induced apoptosis partially through the PTEN/PI3K/Akt pathway. The present study demonstrates that miR-21 is a prosurvival molecule for stressed c-kit + CSCs. It also indicates that modification on miRNA expression may be able to enhance the clinical efficacy of cellular therapy.
We must confess some shortcomings of this study. In vivo studies are warranted to further confirm miR-21 and PTEN/PI3K/Akt pathway's effects on survival of c-kit + CSCs. Besides, the luciferase assay could be a more direct way to confirm that PTEN is the target gene of miR-21.