Jianfeng Wang , Peibang He , Qi Tian Yu Luo, Yan He, Chengli Liu Pian Gong Yujia Guo Qingsong Ye, , Mingchang Li

Abstract Human dental pulp stem cells (hDPSCs) promote recovery after ischemic stroke; however, the therapeutic efficacy is limited by the poor survival of transplanted cells. For in vitro experiments in the present study, we used oxygen-glucose deprivation/reoxygenation in hDPSCs to mimic cell damage induced by ischemia/reperfusion. We found that miRNA-34a-5p (miR-34a) was elevated under oxygen-glucose deprivation/reoxygenation conditions in hDPSCs. Inhibition of miR-34a facilitated the proliferation and antioxidant capacity and reduced the apoptosis of hDPSCs. Moreover, dual-luciferase reporter gene assay showed WNT1 and SIRT1 as the targets of miR-34a. In miR-34a knockdown cell lines, WNT1 suppression reduced cell proliferation, and SIRT1 suppression decreased the antioxidant capacity. Together, these results indicated that miR-34a regulates cell proliferation and antioxidant stress via targeting WNT1 and SIRT1, respectively. For in vivo experiments, we injected genetically modified hDPSCs (anti34a-hDPSCs) into the brains of mice. We found that anti34a-hDPSCs significantly inhibited apoptosis, reduced cerebral edema and cerebral infarct volume, and improved motor function in mice. This study provides new insights into the molecular mechanism of the cell proliferation and antioxidant capacity of hDPSCs, and suggests a potential gene that can be targeted to improve the survival rate and efficacy of transplanted hDPSCs in brain after ischemic stroke.

Key Words: antioxidant capacity; HO-1; human dental pulp stem cells; ischemic stroke; miR-34a; Nrf2; proliferation; SIRT1; WNT1; β-catenin

Introduction

Cerebral ischemic stroke is a prevalent neurological disorder caused by the occlusion of cerebral vessels, and has a high rate of mortality and morbidity. It also can lead to disability in some patients despite appropriate therapy (Barthels and Das, 2020; Kuriakose and Xiao, 2020). Recombinant tissue plasminogen activator thrombolysis and intravascular thrombectomy are the only approved medical treatments for this condition. Thus, exploring a novel therapeutic method to further improve the prognosis of patients is necessary.

Stem cell transplantation for ischemic stroke has made rapid progress in recent years. Most animal studies have shown that stem cells recover neurological function, modulate the immune response, and promote angiogenesis and nerve regeneration (Isakson et al., 2015; Cao et al., 2017; Saffari et al., 2021, 2022). Reports have demonstrated the efficacy of stem cell transplantation in treating ischemic stroke (Chen et al., 2001; Song et al., 2017; Wen et al., 2021). Human dental pulp stem cells (hDPSCs) are a subtype of mesenchymal stem cells (MSCs) that originate from the neural crest and express MSC markers (Kato et al., 2020). hDPSCs have been shown to promote functional recovery and reduce infarct size following transient middle cerebral artery occlusion (tMCAO) in mice, and have the advantages of minor ethical controversy and easy access (Barros et al., 2015; Song et al., 2017).The release of inflammatory factors and production of reactive oxygen species (ROS) in the cerebral infarction area reduce the survival rate of stem cells, leading to a remarkable decrease in the effectiveness of stem cell therapy (Swanger et al., 2005; Hicks et al., 2009; Park et al., 2018; Kang et al., 2019). Therefore, increasing the survival rate of stem cellsin vivohas become the key to their clinical application. Genetic modification can improve thein vivosurvival rate and the inherent function of stem cells (Korshunova et al., 2020).

MicroRNAs (miRNAs) are single-stranded RNA molecules composed of 21–23 nucleotides. When mature, miRNAs regulate gene expression by binding to the 3′ untranslated region (UTR) of the target gene (van Rooij et al., 2012; Simkin et al., 2014). MiR-34a, a member of the miR-34 family (miR-34a/b/c), was the first miRNA discovered to be directly regulated by p53, a tumor suppressor gene (Bommer et al., 2007). Initially, miR-34a was reported to have an inhibitory effect on tumor cell apoptosis. In recent years, studies on the function of miR-34a in stem cells have gradually increased. Previous studies have shown that miR-34a enhances osteoblastic differentiation from bone marrow MSCs (bMSCs) (Liu et al., 2019), and the inhibition of miR-34a promotes bMSCs survival under oxidative stress (Liu et al., 2018b). In addition, miR-34a plays proapoptotic and prosenescence roles in bMSCs (Zhang et al., 2015). MiR-34a inhibitor protects bMSCs from hyperglycemic injury through the autophagy pathway (Pi et al., 2021). Thus, inhibiting miR-34a has been shown to improve stem cell survivalin vivoandin vitro. However, the explicit mechanisms have not been clarified.

The WNT signaling pathway is essential for normal cell growth and development, including canonical WNT/β-catenin, noncanonical planar cell polarity, and WNT/Ca2+pathways (Gelebart et al., 2008; Vijayakumar et al., 2011; Wang et al., 2011). The WNT/β-catenin signaling pathway regulates stem cell pluripotency and renewal and maintains stem cell quantity (Han et al., 2017; Kinosada et al., 2021). WNT1 is one of the members of the WNT family proteins that trigger the WNT/β-catenin signaling pathway (Xu et al., 2020). An activated WNT1/β-catenin signaling axis likely enhances stem cell proliferation. SIRT1, a deacetylase, plays a vital role in oxidative stress injury by regulating various target genes and proteins. SIRT1 overexpression promotes resistance to oxidative stress (Kowluru et al., 2014; Gu et al., 2016; Conti et al., 2018; Liao et al., 2020). SIRT1 can also regulate nuclear factor erythroid 2-related factor 2 (Nrf2), which binds to antioxidative response elements to control various antioxidative genes against oxidative stress (Gu et al., 2016; Xue et al., 2016). Heme oxygenase-1 (HO-1) is a common and vital antioxidant enzyme regulated by Nrf2 (Paiva et al., 2012; Su et al., 2020). The SIRT1/Nrf2/HO-1 signaling axis is one of the essential endogenous antioxidative stress pathways. Activation of the WNT1/β-catenin and SIRT1/Nrf2/HO-1 signaling axes may strengthen the survival of hDPSCsin vivoand enhance their efficacy.

We hypothesized that inhibiting miR-34a would enhance the survival and reduce the apoptosis of hDPSCs. We also hypothesized that miR-34a inhibits stem cell proliferation by directly binding to WNT1 and consequently downregulates the WNT1/β-catenin signaling axis, and that miR-34a directly binds to SIRT1 and consequently downregulates the SIRT1/Nrf2/HO-1 signaling axis, thus reducing the antioxidant capacity of hDPSCs. In this study, we also aimed to verify whether hDPSCs with miR-34a knockdown have superior efficacy in tMCAO injury to unaltered hDPSCs.

Methods

Isolation, culture, and identification of hDPSCs

Discarded and healthy impacted human third molars were obtained from patients between 18 and 30 years old after receiving informed consent. The use of these hDPSCs in our experiments was approved by the Ethics Committee of the Renmin Hospital of Wuhan University (approval No. WDRY2022-K025) on February 28, 2022. hDPSCs were isolated, cultured, and identified as previously described (Li et al., 2021). The tooth surface was disinfected with 70% v/v ethanol, and the dental pulp tissues were removed using the dental handpiece, collected, and washed three times with 2.5% streptomycin/penicillin (S/P; Gibco, Grand Island, NY, USA) in phosphate-buffered saline (PBS). The pulp tissues were then minced and digested in a solution of 3 mg/mL collagenase type I (Gibco) and 4 mg/mL dispase (MilliporeSigma, Burlington, MA, USA) at 37°C for 30 minutes. The cell suspension was cultured in α-modified Eagle’s medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% S/P at 37°C with 5% CO2. After 5 days, the culture medium was removed and then replaced with fresh medium every 2 days. The hDPSCs in passages three, four, and five were used in the experiments.

Flow cytometry and immunofluorescence staining were performed to identify hDPSCs as previously described (Nam et al., 2011; Li et al., 2021). For flow cytometry, the antibodies of human CD45 (BD Pharmingen, San Diego, CA, USA, Cat# 555748, RRID: AB_1727488) and CD44 (BD Pharmingen, Cat# 555476, RRID: AB_2076224) were used in accordance with the manufacturers’ protocols. Flow cytometric analysis was performed using a BD FACSCalibur flow cytometer (BD Pharmingen). For immunostaining, the hDPSCs were fixed with 4% paraformaldehyde (PFA) for 10 minutes, followed by permeabilization with 0.2% Triton X-100 (Servicebio, Wuhan, China) in PBS for 30 minutes at room temperature. The cells were then incubated with hDPSC markers, rabbit polyclonal anti-CD45 (1:200; Servicebio, Cat# GB113885), rabbit polyclonal anti-CD105 (1:200; Servicebio, Cat# GB113377), rabbit polyclonal anti-CD90 (1:200; Servicebio, Cat# GB11182), rabbit polyclonal anti-CD34 (1:200; Servicebio, Cat# GB111693), rabbit polyclonal anti-CD44 (1:200; Servicebio, Cat# GB113500), and rabbit polyclonal anti-CD14 antibody (1:200; Servicebio, Cat# GB11254) at 4°C overnight. The hDPSCs were washed with PBS three times and then incubated with goat anti-rabbit secondary antibody conjugated with Cy3 (1:200; Servicebio, Cat# GB21403) and goat anti-rabbit secondary antibody conjugated with FITC (1:200; Servicebio, Cat# GB22303) for 1 hour at room temperature. Nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI; Servicebio) for 5 minutes at room temperature. Images were acquired with an FSX100 microscope (Olympus, Tokyo, Japan).

Oxygen-glucose deprivation/reoxygenation induction

For oxygen-glucose deprivation (OGD) induction, the culture medium was replaced by a glucose-free Dulbecco’s Modified Eagle Medium (Solarbio, Beijing, China). The hDPSCs were incubated in a hypoxic incubator (37°C, 1% O2, 5% CO2, and 94% N2; Thermo Fisher Scientific, Waltham, MA, USA) at 37°C. After being cultured in the hypoxic incubator for 6, 12, 18, and 24 hours, the cells were returned to normal incubation conditions (37°C, 95% air and 5% CO2) with the regular culture medium for reoxygenation (R) for 6 hours. The timeline for the cell experiments is shown in Additional Figure 1.

Lentiviral transduction and RNA interference of hDPSCs

The hDPSCs were cultured until they reached approximately 30–50% confluence. The cells were then transduced with the four purchased lentiviral particles (GeneChem, Shanghai, China) for 10–16 hours in accordance with the manufacturer’s protocol. The lentiviral particles were miR-34a overexpression (miR-34a), lentiviral vector of miR-34a (miR-NC), miR-34a knockdown (anti-34a), and lentiviral vector of anti-34a (anti-NC). The cells were divided into four groups: miR-34a, miR-NC, anti-34a, and anti-NC. At 72 hours after transduction, transduction efficiency was monitored under a fluorescence microscope (Olympus), and the miR-34a level of the four groups was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).

siRNA transfection was used to decrease WNT1 and SIRT1 expression. Prior to transfection, the cells were plated in six-well plates overnight and then incubated with siRNA (GeneChem) and siRNA transfection reagent (GeneChem) in Opti-medium (Gibco) for 6 hours. Transfection efficiency was tested at 48 hours after transfection by western blot assay. Cells were divided into three groups as follows: siRNA WNT1 group, siRNA SIRT1 group, and negative control siRNA group (siRNA NC).

Western blot assay

Total protein of hDPSCs were extracted using radioimmunoprecipitation assay lysis buffer (Servicebio) containing 1% phenylmethanesulfonyl fluoride (Cell Signaling Technology, Danvers, MA, USA), and their concentrations were determined using the bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). The proteins were loaded into 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (Servicebio) and then transferred onto polyvinylidene fluoride membranes (MilliporeSigma), which were then probed with primary antibodies recognizing WNT1 (1:1000; Proteintech, Wuhan, China, Cat#27935-1-AP, RRID: AB_2881013), β-catenin (1:1000; Proteintech, Cat# 51067-2-AP, RRID: AB_2086128), SIRT1 (1:1000; Proteintech, Cat#13161-1-AP, RRID: AB_10646436), Nrf2 (1:1000; Proteintech, Cat#16396-1-AP, RRID: AB_2782956), HO-1 (1:1000; Proteintech, Cat# 10701-1-AP, RRID: AB_2118685), Bcl-2 (1:1000; Proteintech, Cat# 26593-1-AP, RRID: AB_2818996), Bax (1:1000; Proteintech, Cat# 60267-1-Ig, RRID: AB_2848213), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436). The membranes were then incubated with appropriate horse radish peroxidaseconjugated secondary antibodies (1:1000; Proteintech, Cat# SA00001-1, RRID: AB_2722565 and Cat# SA00001-2, RRID: AB_2722564) for 1–2 hours. Protein signals were detected by Immobilon Western Chemiluminescent HRP substrate (Millipore, Billerica, MA, USA) and quantified using Image-Pro Plus 7.0 (Media Cybernetics, Rockville, MD, USA). The protein expression was normalized to GAPDH expression.

qRT-PCR

Total RNA of hDPSCs was collected using Trizol reagent (Servicebio), precipitated with chloroform and isopropyl alcohol, washed with ethanol, dissolved in nuclease-free water, and reverse transcribed into cDNA using a SweScript RT II First Strand cDNA Synthesis Kit (Servicebio). qPCR was performed using the SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan) for cDNA amplification of specific target genes. The primer pairs used for qPCR are presented in Table 1. The relative expression levels of hsa-miR-34a-5p (miR-34a) were calculated using the 2–ΔΔCtmethod. GAPDH was used as the internal control. The qRT-PCR conditions were as follows: 94°C for 3 minutes initial denaturation, followed by 35 cycles of 94°C denaturation for 15 seconds and 65°C annealing for 30 seconds, and 72°C extension for 1 minute.

Table 1 ｜The sequences of gene primers for quantitative reverse transcriptionpolymerase chain reaction

Cell viability and proliferation assay

Cell viability was assessed by using the cell counting kit-8 (CCK-8) assay (Beyotime). The hDPSCs were seeded in 96-well plates and grown to 80–90% confluency. The cells were incubated with CCK-8 reagent at 10 μL per well for 2 hours at the end of treatment, and absorbance was read with a wavelength of 450 nm using a microplate reader (PerkinElmer, Waltham, MA, USA). Cell proliferation was analyzed by 5-ethynyl-2′-deoxyuridine (EdU) assay (Beyotime). The hDPSCs were treated with 20 mM EdU for 24 hours, fixed with 4% PFA, and permeabilized with 0.2% Triton X-100. Finally, the cells were incubated with EdU reaction cocktail and counterstained with DAPI.

Assessment of oxidative stress

Dihydroethidium (DHE) staining was performed to detect the superoxide anion production of hDPSCs. The cells were incubated with 5 μM DHE (Beyotime) at 37°C for 10–15 minutes. DHE signals were detected using a fluorescence microscope (Olympus). Images were analyzed with ImageJ software (version 2.0.0, National Institutes of Health, MD, USA). Superoxide dismutase (SOD) activity was measured using a SOD assay kit (Beyotime) in accordance with the manufacturer’s instructions. Absorbance was read on a microplate reader (PerkinElmer) at 450 nm with a reference wavelength of 655 nm.

Cell apoptosis assay

The infarct brain tissue was cut in 20 μm coronal sections. For cultured hDPSCs and brain tissue sections, apoptosis was detected by TUNEL Apoptosis Detection Kit (Beyotime) in accordance with the manufacturer’s instructions. Cells and tissue sections were fixed with 4% PFA, permeabilized with 0.2% Triton X-100 at room temperature, and incubated with the TUNEL assay reaction mixture at 37°C for 1.5 hours. Nuclear counterstaining was performed with DAPI. The number of TUNEL-positive nuclei per field was counted and expressed as a percentage of the total cell number.

Dual-luciferase reporter assay

The wild-type (WT) and mutation-type (MUT) 3′-UTRs of WNT1 and SIRT1 genes were created. pmirGLO dual-luciferase vectors were constructed by GeneChem. In brief, human 293T cells (ATCC, Shanghai, China, CAT# CRL-3216, RRID: CVCL-0063) were plated into 24-well plates overnight and subsequently co-transfected with WT or MUT plasmid (WNT1, SIRT1) 3′-UTR vectors and miR-34a mimic (Ribobio, Shanghai, China; miR10000255-1-5) or mimic NC (Ribobio; miR1N0000001-1-5) using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific). Renilla and firefly luciferase activities were measured using Dual-Luciferase Reporter Assay System (Yeasen, Shanghai, China) after 48 hours in accordance with the manufacturer’s instructions. Relative luciferase activity was analyzed by dual-luciferase assay (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to Renilla luciferase activity for the calculations.

Animals

All animal ex periments were approved and supervised by the Ethics Committee of the Renmin Hospital of Wuhan University (approval No. WDRY2022-K025) on February 28, 2022. All experiments were designed and reported according to the Animal Research: Reporting ofIn VivoExperiments (ARRIVE) guidelines (Percie du Sert et al., 2020). Adult C57BL/6 mice (n= 96, WT, 8 weeks of age, weighing 22–25 g) were purchased from the Beijing Vital River Laboratory Animal Technology (Beijing, China; license No. SYXK (Jing) 2022-0018). All mice were housed in a specific-pathogen-free laboratory under controlled conditions (12-hour light/dark cycle, humidity 60 ± 10%, 24 ± 2°C).

Animal model of cerebral ischemia/reperfusion injury

Cerebral ischemia was induced by tMCAO. The mice were anesthetized with 2% isoflurane for induction and maintained with 1.5% isoflurane in a mixture of N2(70%) and O2(30%) via a face mask, and then a median incision was made in the neck. The left common carotid artery (CCA), external carotid artery, and internal carotid artery were exposed. The origin of the external carotid artery and CCA was ligated by a 6–0 silk suture, and arteriotomy was performed in the CCA. A silicone rubber-coated 6–0 nylon monofilament was inserted into the CCA and advanced about 9–10 mm along the internal carotid artery to the bifurcation of the MCA. Reperfusion was achieved by removing the suture after 1 hour of occlusion. The mice were warmed with an electric heating blanket throughout the experiment.

At 6 hours after reperfusion, the mice were randomly administered with PBS (ischemia/reperfusion [I/R] + PBS), hDPSCs with miR-34a knockdown (I/R + anti34a), or its vector control anti-NC cell lines (I/R + anti-NC) via stereotactic injection. hDPSCs were transplanted as previously reported (Sakata et al., 2012). The cells were mixed with PBS to form single-cell suspensions (1 × 105cells per μL) and then injected into the cortex along the anterior–posterior axis at the following three coordinates (2 μL per location; mm): (1) anterior–posterior: +1.0, medial–lateral: +2.0, dorsal–ventral: –1.0; (2) anterior–posterior: –0.5, medial–lateral: +2.5, dorsal–ventral: –1.0; and (3) anterior–posterior: –2.0, medial–lateral: +2.5, dorsal–ventral: –1.0. The three coordinates were as previously reported (Sakata et al., 2012). For the sham group (sham), the mice were subjected to the same surgical procedures without MCA occlusion. The timeline and design for the animal experiments is shown in Additional Figures 1 and 2.

Assessment of brain edema

Brain tissue from both hemispheres was sampled on day 3 after the MCAO operation. Wet weight was measured by weighing fresh brain tissue on an electronic balance (Sartorius, G?ttingen, Germany). Dry weight was recorded after the brain tissue was dried at 100°C for 48 hours. The percentage of brain water content was calculated as follows:

Neurological deficit assessment

For the assessment of neurological changes, modified neurological severity score (mNSS) (Goldstein and Davis, 1990) was measured by two investigators in a blind method on day 7 after surgery. Motion, sensation and reflex, muscle mass, abnormal behavior, vision, touch, and balance were evaluated. The score was graded on a scale of 0 to 18 (0, normal score; 18, maximal deficit score).

Histopathological examination

Hematoxylin-eosin (HE) staining was used to assess the histopathological damage in the injured cerebral cortex. Brains were collected on day 7 after surgery, fixed with 4% PFA, embedded in paraffin and sliced at a thickness of 20 μm in the coronal orientation using a microtome (Leica, Nussloch, Germany). The brain sections were stained with HE (Servicebio) in accordance with the manufacturer’s protocol, and images were captured and observed under a light microscope (Olympus).

Infarct volume measurement

On day 7 after surgery, the brain tissue was cut into coronal sections 2.0 mm thick. The sections were stained at 37°C for 20 minutes using 2% 2,3,5-triphenyltetrazolium chloride (MilliporeSigma). Then, the sections were immersed in 4% PFA overnight. The infarct tissue and non-infarct tissue were distinguished and the total infarct volume was calculated by integrating the lesion area of all sections. Then, the ratio of infarct volume to total volume was calculated.

Rotarod test

Rotarod test was used to assess motor performance from the day of surgery to post-operative day 7. The time that each mouse spent on a rotarod was measured by using an accelerating rotarod. Before tMCAO, each mouse was trained for 3 days. The time to fall off the rotarod was recorded. The times were calculated by averaging the times recorded for each mouse in three experiments.

Statistical analysis

No statistical methods were used to predetermine sample sizes; however, our sample sizes were similar to those reported in previous publications (Itokazu et al., 2018). GraphPad Prism 8.3.0 software (GraphPad Software, San Diego, CA, USA, www.graphpad.com) was used for data analysis. The data are presented as mean ± standard deviation. Statistical comparisons between two groups were performed using Student’st-test.P< 0.05 was considered statistically significant.

Results

Identification and characterization of hDPSCs

The hDPSCs were adherent and had a typical spindle-like and elongated fibroblastic shape (Figure 1A). Flow cytometry showed that hDPSCs strongly expressed the hDPSC marker CD44, and expressed CD45, the surface antigen of hematopoietic stem cells, at a very low level (0.33%) (Figure 1B). Immunofluorescence staining showed that hDPSCs were positive for CD90, CD105, and CD44, and negative for CD14, CD34, and CD45 (Figure 1C).

Figure 1｜Identification and characterization of hDPSCs.

Oxygen-glucose deprivation/reoxygenation induces hDPSC apoptosis and miR-34a upregulation

Compared with that in the normal group, cell viability in the hDPSCs with OGD decreased in a time-dependent manner at 12, 18, and 24 hours. We observed no significant difference between the normal group and the hDPSCs that underwent OGD for 6 hours (P> 0.05; Figure 2A). Moreover, we found that miR-34a expression in hDPSCs gradually increased with the extension of OGD treatment time (Figure 2B). To explore the role of miR-34a in hDPSCs, we constructed four cell groups with lentiviral transfection: miR-34a, miRNC, anti-34a, and anti-NC. The virus vector carried the green fluorescent protein, so successfully transfected cells showed green fluorescence under a fluorescence microscope (Figure 2C). We detected the expression level of miR-34a by RT-qPCR. Compared with that in the anti-NC group, the miR-34a expression level in the anti-34a group was significantly decreased (P< 0.01; Figure 2D). Compared with that in the miR-NC group, the miR-34a expression level in the miR-34a group was significantly elevated (P< 0.01; Figure 2D).

Figure 2｜MiR-34a expression levels following OGD/R in human dental pulp stem cells and construction of cell groups with miR-34a overexpression and knockdown.

Inhibition of miR-34a suppresses hDPSC apoptosis

On the basis of previous reports (Zhang et al., 2015; Liu et al., 2018b, 2019) and the above results, we speculated that miR-34a is involved in hDPSC apoptosis. Thus, we detected the expression level of apoptosis-related proteins (Bcl-2 and Bax) in the four cell lines (anti-NC, anti-34a, miR-NC, and miR-34a). The expression level of Bcl-2, an anti-apoptotic protein, was the highest in the anti-34a group and the lowest in the miR-34a group. The expression of Bax, a pro-apoptotic protein, was significantly the lowest in the anti-34a group and the highest in the miR-34a group (Figure 3A). We used TUNEL staining to evaluate the apoptosis rate of the four cell groups. The apoptosis rate was the lowest in the anti-34a group and the highest in the miR-34a group (Figure 3B). Thus, we concluded that hDPSC apoptosis was suppressed by inhibiting miR-34a and aggravated by overexpressing miR-34a.

Figure 3｜Effects of miR-34a on human dental pulp stem cell apoptosis.

Inhibition of miR-34a improves the proliferation and antioxidant capacity of hDPSCs

To further explore how miR-34a regulates apoptosis, we detected the cell proliferation and viability of the four cell groups using EdU staining and CCK8 assay. In addition, we used SOD assay and DHE staining to evaluate the antioxidant stress ability of the four cell lines. The proliferation rate and cell activity was significantly increased in the anti-34a group (P< 0.01,vs. anti-NC group) and decreased in the miR-34a group (P< 0.01,vs. miR-NC group) (Figure 4A). Cell activity was significantly increased in the anti-34a group (P< 0.01,vs. anti-NC group) and decreased in the miR-34a group (P< 0.001,vs. miR-NC group) (Figure 4B). SOD activity was significantly increased in the anti-34a group (P< 0.05,vs. anti-NC group) and significantly decreased in the miR-34a group (P< 0.01,vs. miR-NC group). DHE staining showed that the intracellular ROS was significantly decreased in the anti-34a group (P< 0.01,vs.anti-NC group) and significantly increased in the miR-34a group (P< 0.01,vs.miR-NC group) (Figure 4C and D). These results showed that miR-34a inhibition enhanced the proliferation and antioxidant capacity of hDPSCs.

Inhibition of miR-34a upregulates the WNT1/β-catenin axis and SIRT1/Nrf2/HO-1 axis via targeting WNT1 and SIRT1, respectively

To investigate the intrinsic mechanism of miR-34a regulation on the pathophysiological processes of hDPSCs, we used western blot to explore the expression of intracellular proteins. We detected the protein levels of WNT1, β-catenin, SIRT1, Nrf2, and HO-1, and found that WNT1 and β-catenin expression were increased with miR-34a inhibition and decreased with miR-34a overexpression. The same results were observed for SIRT1, Nrf2, and HO-1 (Figure 5A). By combining the miRbase and TargetScan databases, we predicted the binding sites of miR-34a to the WNT1 and SIRT1 mRNAs (Figure 5B and C). We used dual-luciferase reporter genes to further investigate the mechanism. Luciferase activity was significantly reduced in the group co-transfected with the WT WNT1 3′-UTR plasmid and miR-34a mimic (P< 0.01,vs.the group co-transfected with the WT WNT1 3′-UTR plasmid and NC mimic), and was not significantly altered in the group co-transfected with the MUT 3′-UTR plasmid and miR-34a mimic (P> 0.05;vs. the group cotransfected with the MUT WNT1 3′-UTR plasmid and NC mimic; Figure 5B). Similar to WNT1, SIRT1 showed a significant decrease in luciferase activity in the group co-transfected with the WT SIRT1 3′-UTR plasmid and miR-34a mimic (P< 0.01,vs. the group co-transfected with the WT SIRT1 3′-UTR plasmid and NC mimic), and exhibited no significant change in the group cotransfected with the MUT SIRT1 3′-UTR plasmid and miR-34a mimic (P> 0.05;vs.the group co-transfected with the MUT SIRT1 3′-UTR plasmid and NC mimic; Figure 5C).

WNT1 or SIRT1 suppression promotes hDPSC apoptosis with miR-34a knockdown

To verify whether the miRNA-34a inhibition exerts an antiapoptotic effect on hDPSCs by targeting WNT1 and SIRT1, we suppressed WNT1 and SIRT1 expression by siRNA in the miR-34a knockdown (anti-34a) cell line. WNT1 protein level was downregulated in the siRNA WNT1 group compared with that in the siRNA NC group (Figure 6A). SIRT1 protein expression was decreased in the siRNA SIRT1 group compared with that in the siRNA NC group (Figure 6B). TUNEL staining showed that the apoptosis rates in the anti-34a + siRNA WNT1 and anti-34a + siRNA SIRT1 groups were elevated compared with that in the anti-34a + siRNA NC group (P< 0.05; Figure 6C). These results indicated that miR-34a inhibition reduced hDPSC apoptosis by targeting WNT1 and SIRT1.

WNT1 suppression reduces cell proliferation and SIRT1 suppression weakens resistance to oxidative stress in miR-34a knockdown cell line

Our previous results showed that miR-34a inhibition promoted the cell proliferation and antioxidant capacity of hDPSCs. Cell proliferation and viability were decreased in the anti34a + siRNA WNT1 group compared with those in the anti34a + siRNA NC group (P< 0.05; Figure 7A and B), indicating that WNT1 suppression partially counteracted the pro-proliferative effect. Similarly, SOD activity was significantly decreased in the anti-34a + siRNA SIRT1 group compared with that in the anti-34a + siRNA NC group (P< 0.01; Figure 7C), indicating that SIRT1 suppression partially counteracted the antioxidative effect. DHE staining showed that intracellular ROS was significantly increased in the anti-34a + siRNA SIRT1 group compared with that in the anti-34a + siRNA NC group (P< 0.05; Figure 7D). These results indicated that miR-34a inhibition promoted cell proliferation by upregulating WNT1 and enhanced the antioxidative capacity by upregulating SIRT1.

Figure 4｜Effects of miR-34a on the proliferation and antioxidant capacity of hDPSCs.

Figure 5｜Effects of miR-34a on the WNT1/β-catenin axis and SIRT1/Nrf2/HO-1 axis in hDPSCs.

Transplantation of hDPSCs with miR-34a knockdown has better protective effects on mice against cerebral ischemic injury than normal hDPSCs

To further investigate whether miR-34a knockdown could optimize hDPSC therapy for ischemic stroke in mice, we injected normal hDPSCs and hDPSCs with miR-34a knockdown into the ischemic cortex of mice by stereotactic injection, and then assessed the efficacy of the cellular therapy using HE staining (histopathological damage), TUNEL staining, neurological deficit score, brain water content (brain edema), rotarod test, and infarct volume. HE staining showed that in the sham group, cell nuclei were arranged in an orderly manner. In the I/R + PBS group, cell nuclei were disorganized and lysed, and tissue edema was severe, indicating that the tMCAO model was successfully constructed. The injection of hDPSCs, especially hDPSCs with miR-34a knockdown, significantly alleviated the tissue edema and reduced the lysis of cell nuclei (Figure 8A). In the I/R + PBS group, the apoptosis rate was significantly higher than that in the sham group, I/R + anti-NC group, and I/R + anti-34a group (P< 0.01 for all comparisons). Furthermore, the apoptosis rate of the I/R + anti-34a group was significantly lower than that of the I/R + anti-NC group (P< 0.05; Figure 8B and C). The mNSS score was the highest in the I/R + PBS group, indicating that the neurological injury was the most serious in this group. Compared with that in the I/R + PBS group, the mNSS scores in the I/R + anti-NC group and I/R + anti-34a group were significantly decreased (P< 0.05; Figure 8D). The mNSS score of the I/R + anti-34a group was lower than that of the I/R + anti-NC group (P< 0.05; Figure 8D). As shown in Figure 8E, the mice subjected to tMCAO had severe brain edema, which was clearly relieved by the transplantation of hDPSCs, especially hDPSCs with miR-34a knockdown. At 7 days post-operative observation, the rotarod test showed that the time spent on the rotating rod was shorter in the I/R + PBS group than in the Sham group, and time spent on the rod was longer in the I/R + anti-34a and I/R + anti-NC groups than in the I/R + PBS group. The I/R + anti-34a group stayed on the rotating rod longer than the I/R + anti-NC group (Figure 8F). The I/R + PBS group had a larger infarct area compared with the Sham group, and the I/R + PBS group had a larger infarct area compared with the I/R + anti-NC group and I/R + anti-34a group (Figure 8G). The I/R + anti-34a group had a significantly smaller infarct volume than the I/R + anti-NC group (P< 0.05; Figure 8H). These results indicated that hDPSCs ameliorated histopathological damage, neurological deficit, brain edema, and sensorimotor function, and reduced the infarct volume. These effects were further enhanced using the hDPSCs with miR-34a knockdown.

Figure 6｜Effects of WNT1 or SIRT1 suppression on apoptosis in miR-34a knockdown cell line.

Figure 8｜Transplanted human dental pulp stem cells exerted neuroprotective effects against ischemic stroke in mice.

Discussion

Our results showed that miR-34a expression was markedly increased in hDPSCs under the OGD/R condition. Inhibition of miR-34a in hDPSCs facilitated cell viability, cell proliferation, and antioxidant and antiapoptotic capacities of hDPSCs. The findings indicate that miR-34a suppression within hDPSCs promoted the proliferation and antioxidant capacity of these cells through the WNT1/β-catenin and SIRT1/Nrf2/HO-1 signaling pathways by directly targeting WNT1 and SIRT1. Subsequent animal experiments showed that the transplantation of hDPSCs with miR-34a knockdown had superior effects to normal hDPSCs on ischemic stroke. These results suggest that miR-34a inhibition enhanced the survival of hDPSCs. Thus, we conclude that miR-34a suppression may be a promising strategy to enhance the clinical application of hDPSC transplantation for cerebral ischemic stroke.

To address the issue of stem cell survivalin vivo, we focused on a number of genes that affect cell death, including long noncoding RNA, mRNA, and noncoding RNA, and proteins. MiRNA binds primarily to mRNA 3′-UTR and degrades target mRNA while terminating protein translation (Ambros, 2004). Most oncology studies of miR-34a have reported that miR-34a overexpression has a significant inhibitory effect on cell proliferation, migration, and invasion ability (Wang and Wang, 2017). Given that miR-34a is a negative regulator of cell survival, it has potential to solve the problem of stem cell survival. MiR-34a has been less studied in stem cells, and its regulation of the pathophysiological processes of stem cells remains unclear. Our study provides insights into the underlying mechanisms of the pathophysiological processes of stem cells.The WNT/β-catenin signaling pathway regulates proliferation and maintains the self-renewal of stem cells (Koval et al., 2018; Zeng et al., 2018; Li et al., 2019). Numerous miRNA molecules, such as miR-155, miR-638, and miR-27a, have been implicated in the regulation of the WNT/β-catenin signaling pathway (Jafari and Abediankenari, 2017; Kong et al., 2017; Wei et al., 2017; Liu et al., 2018a). However, whether miR-34a regulates WNT/β-catenin remains unknown. Our study is the first to show the pro-proliferative effects of miR-34a suppression on hDPSCs by targeting WNT1. Additional in depth investigations of the specific regulation relationship between miR-34a and WNT/β-catenin are required. Our results suggested that the proproliferative effect of miR-34a suppression is only partially regulated through the WNT1/β-catenin signaling pathway, thus indicating the existence of other downstream regulatory processes. SIRT1 regulates cell oxidative stress, proliferation, apoptosis, senescence, and DNA repair (Carafa et al., 2016). SIRT1 activates Nrf2, which leads to Nrf2 nuclear translocation, and thus promotes the expression of antioxidant genes such as HO-1 (Tang et al., 2014). MiR-34a plays proapoptotic and prosenescence roles in MSCs by targeting SIRT1 (Zhang et al., 2015; Pi et al., 2021). We used a dual-luciferase reporter gene assay to investigate the relationship between miR-34a and SIRT1, and found that the binding site is different from the results of the study of Zhang et al. (2015). We found that miR-34a suppression also played an antioxidant role in hDPSCs by targeting SIRT1, which differs from the findings of Zhang et al. (2015). Our findings indicate that the antioxidative effect of miR-34a inhibition on hDPSCs was also partly related to the SIRT1/Nrf2/HO-1 signaling axis. These findings support our hypothesis that SIRT1, a direct target of miR-34a, is involved in several physiological and pathological processes of hDPSCs.hDPSCs, one type of MSCs, possess MSC-like features such as multidifferentiation potential and MSC-like markers expression. bMSCs are the most widely used in previous animal studies. Because of their easy accessibility without invasive surgical procedures or ethical concerns and the high proliferation ratein vitro, hDPSCs have more clinical advantages than bMSCs (Pereira et al., 2019; Albashari et al., 2021). We chose hDPSCs for this study; however, additional experiments are needed to explore the advantages and disadvantages of both hDPSCs and bMSCs, such as their efficacy for different diseases, degrees of enrichment in the damaged area, and side effects. Evidence from previous studies of ischemic stroke in animal models suggests that stem cell-based therapy is beneficial for neurological recovery (Oki et al., 2012; Du et al., 2020; Feng et al., 2020). For tMCAO treatment in mice, stem cells can be implanted in many ways, including stereotactic injection, tail vein injection, and intranasal administration (Du et al., 2020). In the present study, we chose stereotactic injection, which has the advantage of allowing hDPSCs to accumulate directly in the damaged area; however, this method also makes the stem cells highly susceptible to death due to the inflammatory response and ROS release, which reduces the treatment efficacy. Stem cells have multiple effects, including modulation of immunity and inflammation, and promotion of vascular and neural regeneration (Zhang et al., 2014; Muffat et al., 2016). However, many issues in stem cell therapy remain to be resolved, such as tumorigenicity, uncontrollability, immunosuppressive effect, and the inferior survival rate of stem cellsin vivo. Our findings indicate that inhibiting miR-34a through genetic modification may solve the problem of low survival of stem cells.

In the present study, we demonstrated that hDPSCs improved the neurological function and reduced the infarct volume of mice that underwent tMCAO. Moreover, miR-34a inhibition in hDPSCs had positive effects on the tMCAO injury of mice, and thus enhanced the survival of transplanted hDPSCs in the lesion area. Different approaches have been suggested to address this issue (Xu et al., 2019). Gene modification of stem cells is an important part of the present study. Our results provide a candidate gene to address the issue of stem cell survival. However, ourin vivostudy had limitations. After stem cell transplantation, we had a short follow-up period and did not observe the proliferation, apoptosis, and resistance to oxidative stress of the stem cellsin vivo.

Conclusions

In summary, our findings indicated that miR-34a suppression enhanced the proliferation and antioxidant capacity of hDPSCs by directly binding to WNT1 and SIRT1 to upregulate the WNT1/β-catenin and SIRT1/Nrf2/HO-1 signaling pathways, respectively. Furthermore, hDPSC transplantation, especially with hDPSCs with miR-34a knockdown, had neuroprotective effects against ischemic stroke in mice. Therefore, miR-34a is a promising candidate for gene modification within stem cells for transplantation in ischemic stroke treatment.

Author contributions:ML, QY, and JW conceived and designed the study. JW, PH, YH, YL, QT, and CL performed the experiments. JW, PG, and YG analyzed the data. JW wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Conflicts of interest:The authors declare no conflict of interest.

Data availability statement:All relevant data are within the paper and its Additional files.

Open access statement:This is an open access journal, and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Additional files:

Additional Figure 1: The timelines of the cell and animal experiments.

Additional Figure 2: The study flow of the animal experiment.