MicroRNA-124a regulates the differentiation of bone marrow mesenchymal stem cells into neurons

Abstract

Objective

This study investigated the effects of microRNA-124a on the differentiation of bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanism.

Methods

Flow cytometry was used for isolation and identification of BMSCs. Real-time polymerase chain reaction (RT-PCR) was used to detect gene mRNA expression. Apoptosis was detected using Annexin V-FITC/PI Apoptosis Detection Kit. Cell proliferation ability was tested using Cell Counting Kit-8 (CCK-8). The differentiation of BMSCs into neuron inducers β-thiol ethanol or baicalin formed the basis of the study.

Results

β-thiol ethanol markedly suppressed the microRNA-124a expression of BMSCs, baicalin markedly induced the microRNA-124a expression of BMSCs and β-thiol ethanol or baicalin promoted apoptosis and reduced the growth of BMSCs. Only the microRNA-124a inhibitor did not affect apoptosis or the differentiation of BMSCs, and it increased the effects of β-thiol ethanol or baicalin on the apoptosis of BMSCs.

Conclusion

β-thiol ethanol and baicalin treatment could affect microRNA-124a expression in BMSCs. We demonstrated that microRNA-124a promoted the differentiation of BMSCs into neurons.

Keywords:

• MicroRNA-124a
• BMSCs
• β-thiol ethanol
• baicalin

1. Introduction

Cerebrovascular disease is characterized by high incidence and disability rates as well as high mortality and can, accordingly, seriously affect people’s quality of life [1]. Furthermore, cerebral infarction represents 80% of cerebrovascular diseases and is caused by the sudden interruption of cerebral blood flow, leading to the ischemic necrosis of brain tissue and resulting in neurological damage [2]. In recent years, although great progress has been made in the use of recombinant tissue plasminogen activators for thrombolytic therapy, because the thrombolytic time window is too narrow, only a few patients benefit from this treatment; it is thus urgent that a new method to treat cerebral infarction is identified [3].

A large number of animal experiments have confirmed that bone marrow mesenchymal stem cells (BMSCs) can reach the lesion site through brain parenchyma and cerebrospinal fluid transplantation and differentiate into neural stem cells, which can safely and effectively promote the recovery of injured nerve function; however, the clinical application of this method is still in the exploratory stage [4–6]. Mesenchymal stem cells are non-hematopoietic stem cells that mainly exist in bone marrow and are early cells of mesoderm development [7,8]. The cells are isolated by adherent culture in vitro and differentiated into a variety of tissue and cell types other than hematopoietic cells, especially those derived from the mesoderm and ectoderm [9]. Because of the convenient source of BMSCs, relatively easy amplification and no immune rejection in autotransplantation [10,11]. There are also no ethical or moral issues, which may exist when using embryonic and neural stem cells, and, as such, it is not necessary to use a large number of immunosuppressants [12]. It is easy to transfect and express exogenous genes, and therefore, mesenchymal stem cells (MSCs) are expected to become ideal cells for treating nervous system diseases and ideal seed cells and tissue regeneration tools for gene therapy and continue to attract more attention in clinical applications [13].

In recent years, the important regulatory role of microRNAs in the biological behavior of MSCs has gradually emerged [14]. MicroRNAs can regulate genomes through transcription, post-transcription and epigenetic levels, as well as heterochromatin formation, and participate in the important biological processes of MSCs, including their proliferation, differentiation, signal transduction and death [15,16]. Among microRNAs in humans (miRBase, www.mirbase.org), microRNA-124 is the highest expressed miRNA in the human nervous system, accounting for approximately 5–48% of the total miRNAs in the mammalian cerebral cortex; it has three types: microRNA-124a, microRNA-124b and microRNA-124c [17]. In the process of embryonic neurogenesis, microRNA-124 is expressed at a low level in neural precursor cells but at a high level in neurons undergoing differentiation and maturation [17–19]. MicroRNA-124 can recognize and regulate hundreds of target mRNAs and plays a decisive role in the development of the nervous system [18]. Experimental studies show that microRNA-124 can participate in the regulation of the proliferation and neurogenesis of neural precursor cells by regulating Notch signaling pathway [20,21]. Notch signaling plays a critical role in the differentiation of MSCs. However, the role of microRNA-124 in MSC differentiation is currently unknown. This study aimed to investigate the effect of microRNA-124 on BMSCs and its underlying mechanism.

2. Materials and methods

2.1. Isolation and identification of BMSCs

Bone marrow MSCs were harvested from Sprague-Dawley rats, aged 10 weeks and weighing 180–250 g, housed in a controlled environment (24 ± 1 °C, 12 h light/12 h dark cycle) and allowed food and water ad libitum [22,23]. All procedures were conducted according to the guidelines of the Animal Care Committee. Bone marrow cells were flushed out and collected from the femur and tibia of rats, plated in T25 flasks and cultured overnight in a 37 °C incubator with 5% CO₂. Bone marrow MSCs were incubated with alpha minimum essential medium (Gibco, CA, USA) containing 20% fetal bovine serum (Gibco), 2 mM Glutamax and 1% penicillin and streptomycin (Gibco) in a 5% CO₂ atmosphere at 37 °C. The BMSCs were incubated with phycoerythrin (PE)-conjugated anti-CD29 (BD Biosciences, USA), anti-CD34 (BD Biosciences) and PE-conjugated anti-CD90 (BD Biosciences) and then analyzed via flow cytometry (BD Biosciences).

2.2. Cell culture and transfection

Bone marrow MSCs were incubated in a 5% CO₂ atmosphere at 37 °C. MicroRNA-124a inhibitor plasmid was transfected into BMSC cell lines using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Transfection was performed according to the manufacturer’s instructions. Specifically, the plasmid was mixed with Lipofectamine 2000 and allowed to rest for 30 min. Subsequently, it was added to the cells, and 24 h after transfection, the cells were cultured using a fresh medium. Transfection was completed 48 h later and cultured for subsequent experiments. The experiment was performed in triplicate. The cells used in this study were within 10 passages.

2.3. Real-time polymerase chain reaction

The total RNAs were isolated using an RNA isolator and total RNA extraction reagent (Takara, Tokyo, Japan), and cDNA was synthesized using PrimeScipt RT Master Mix (Takara, Tokyo, Japan). Real-time polymerase chain reaction (RT-PCR) was performed with the ABI Prism 7500 sequence detection system according to the Prime-Script™ RT detection kit. The reaction mixtures were incubated at 50 °C for 15 min, followed by 95 °C for 5 min; then, 35 PCR cycles were performed with the following temperature profiles: 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 1 min. The relative levels of the sample mRNA expression were calculated and expressed as 2^−ΔΔCT. The experiment was performed in triplicate.

2.4. Apoptosis analysis

The BMSCs were analyzed using the Annexin V-FITC/PI Apoptosis Detection Kit (BD, MA, USA). The cells were then collected and subjected to V-FITC and PI staining, in accordance with the reagent manufacturer’s instructions, and thereafter analyzed via flow cytometry (BD Biosciences). The experiment was performed in triplicate.

2.5. Proliferation assay

For the cell counting kit-8 (CCK-8), after 48 h of transfection, a total of approximately 2 × 10³ cells/well was seeded in a 96-well plate. After culturing at the indicated time (0, 1, 2, 3, and 4 days), cellular proliferation was detected using a CellTiter^®-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The experiment was performed in triplicate.

2.6. Statistical analysis

GraphPad Prism 6 was used to conduct the statistical analysis. Data were shown as mean ± SD. Comparisons of data between groups were performed using Student’s t-test (between-group comparison) or one-way analysis of variance (multi-group comparison), followed by Tukey’s post hoc test; p < 0.05 was considered statistically significant.

3. Results

3.1. β-Thiol ethanol and baicalin controlled microRNA-124a expression of BMSCs

This study collected BMSCs and analyzed them using flow cytometry. Cell markers CD29, CD34 and CD90 on the surface of BMSCs were used for the flow cytometry screening of BMSCs (Figure 1). For the differentiation of BMSCs into neuron inducers, β-thiol ethanol or baicalin was used. Additionally, a β-thiol ethanol or baicalin 0 h group was used as a control group. The results of RT-PCR showed that β-thiol ethanol markedly suppressed the microRNA-124a expression of BMSCs (Figure 2(A)), and baicalin markedly induced the microRNA-124a expression of BMSCs (Figure 2(B)).

Figure 1. Appraisal of BMSCs and analyzed using flow cytometry. Flow cytometry for sorting of BMSCs.

Figure 2. MicroRNA-124a mRNA expression of BMSCs. (A) β-Thiol ethanol reduced microRNA-124a expression of BMSCs. (B) Baicalin reduced microRNA-124a expression of BMSCs. *p < 0.05 compared with β-thiol ethanol at 0 h group or baicalin at 0 h group.

3.2. β-Thiol ethanol or baicalin controlled apoptosis of BMSCs

This study explored whether the function of β-thiol ethanol and baicalin regulated the apoptosis of BMSCs. Additionally, a β-thiol ethanol or baicalin 0-h group was used as a control group. Moreover, V-FITC and PI staining were used for apoptosis detection. The flow cytometry results showed that β-thiol ethanol markedly promoted the apoptosis of BMSCs (Figure 3(A)) and baicalin dramatically promoted the apoptosis of BMSCs, both in a time-dependent manner (Figure 3(B)).

Figure 3. β-thiol ethanol or baicalin controlled apoptosis of BMSCs. (A) β-Thiol ethanol reduced apoptosis of BMSCs. (B) Baicalin reduced apoptosis of BMSCs. *p < 0.05 compared with β-thiol ethanol at 0 h group or baicalin at 0 h group.

3.3. MicroRNA-124a is one target for the effects of β-thiol ethanol or baicalin on apoptosis of BMSCs

This study explored the mechanism of the effects of β-thiol ethanol or baicalin on the apoptosis of BMSCs based on the above results. The control and negative control (NC) groups were used as control groups. The results of RT-PCR showed that microRNA-124a inhibitor treatment could reduce microRNA-124a expression in BMSCs (Figure 4). Next, we used an MTT assay to detect the BMSCs cells viability. Inhibit NC was used as a control group. The results showed that β-thiol ethanol or baicalin reduced the growth of BMSCs. However, microRNA-124a did not affect the growth of BMSCs (Figure 5). Additionally, β-thiol ethanol or baicalin dramatically promoted the apoptosis of BMSCs (Figure 6(A)). Only the microRNA-124a inhibitor did not affect the apoptosis of BMSCs (Figure 6(B)), and it increased the effects of β-thiol ethanol on the apoptosis of BMSCs (Figure 6(B)). Moreover, the microRNA-124a inhibitor increased the effects of baicalin on the apoptosis of BMSCs (Figure 6(B)). The above results indicate microRNA-124a as a target for the effects of β-thiol ethanol or baicalin on the apoptosis of BMSCs.

Figure 4. MicroRNA-124a inhibitor reduced microRNA-124a expression in BMSCs. The RT-PCR results of microRNA-124a expression. *p < 0.05 compared with β-thiol ethanol at 0 h group or baicalin at 0 h group.

Figure 5. MicroRNA-124a is one target for the effects of β-thiol ethanol or baicalin on cell growth of BMSCs. The MTT results of BMSCs with different treatment. *p < 0.05 compared with inhibit NC.

Figure 6. MicroRNA-124a Is one target for the effects of β-thiol ethanol or baicalin on apoptosis of BMSCs. (A) Apoptosis rate of BMSCs. (B) Apoptosis assay using flow cytometry. *p < 0.05 compared with inhibit NC.

4. Discussion

The clinical treatment of cerebral infarction currently includes early thrombolysis, which improves cerebral blood circulation, promotes brain metabolism and uses antiplatelet drugs; however, the death of ischemic nerve cells after infarction is still unavoidable and is the main reason for the high disability and mortality rates of cerebral infarction [24–26]. Therefore, the repair of damaged brain tissue and the recovery of nerve function serve as ideal treatment approaches [27]. In recent years, with the advancement of stem cell engineering, stem cell therapy has become a new method for treating cerebrovascular diseases by replacing and repairing damaged nerve cells [28]. MicroRNAs play a key role in regulating physiological processes, such as differentiation, proliferation and the apoptosis of stem cells. However, the molecular mechanism of stem cell regulation by microRNA requires further study [15,16]. The above findings [15,16] are also consistent with the results of this study. Nazari et al. [29,30] reported that microRNAs 219 and miR-338 have potentially important roles in oligodendrocyte development and can influence oligodendrocyte development in the central nervous system. This further confirms the critical role of miRNAs in the nervous system. In this study, BMSCs were identified and isolated from rats. The effect of microRNA-124a expression on BMSCs was investigated, and the results showed that β-thiol ethanol significantly inhibited microRNA-124a expression in BMSCs. Baicalin significantly induced microRNA-124a expression in BMSCs, and elevated microRNA-124a expression promoted the apoptosis of BMSCs.

At present, autologous MSC transplantation is used to treat end-stage congestive heart failure, myocardial infarction, diabetes foot, lower limb and ischemic diseases as well as neurological diseases such as stroke, Alzheimer’s disease and spinal cord injury [31]. Notably, MSCs can secrete brain-derived neurotrophic factor, nerve growth factor, vascular endothelial growth factor, hepatocyte growth factor, interleukin (IL), and other regenerative factors, such as increasing the release of TNF, IL-10, and other factors in ischemic brain tissue to play an anti-inflammatory role [32]. Other studies have shown that MSCs can produce an extracellular matrix to protect neurons after transplantation [33–35]. These data reveal that β-thiol ethanol or baicalin markedly promote the apoptosis of BMSCs in a time-dependent manner. Sun et al. reported that miR-124a improved the effect of BMSCs in injured podocytes [36]. These data reveal that microRNA-124a promotes the differentiation of BMSCs into neurons.

When an intracerebral hemorrhage occurs, the total iron concentration increases, resulting in serious damage to neurons [37]. Heme and free iron released during hematoma absorption may be one of the important inducing factors of nerve cell death following an intracerebral hemorrhage [38]. Baicalin has a neuroprotective effect on experimental intracerebral hemorrhage in rats; however, the molecular mechanism is currently unknown. We found that β-thiol ethanol or baicalin reduced cell growth among BMSCs. MicroRNA-124a did not affect the growth of BMSCs but increased the effects of β-thiol ethanol and baicalin on the growth of BMSCs. The high expression of microRNA-124a can also promote the apoptosis of BMSCs.

This study has some limitations Firstly, the molecular mechanism of how microRNA-124a impacts apoptosis in BMSCs requires further investigation. Secondly, an in vitro cell line model was used in this study; experiments will subsequently be performed in vivo to validate the experiment conclusions.

In conclusion, our study showed that β-thiol ethanol decreased the expression of microRNA-124a, while baicalin treatment increased the expression of microRNA-124a and microRNA-124a promoted the differentiation of BMSCs into neurons, which was achieved by promoting apoptosis. MicroRNA-124a could thus be a clinical factor for the differentiation of BMSCs into neurons. The molecular mechanism by which microRNA-124a regulates apoptosis in BMSCs still requires additional investigation, and drug development targeting microRNA-124a remains a challenge.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was funded by Hainan Province Science and Technology Special Fund (No. ZDYF2021SHFZ112) of Hainan Province Clinical Medical Center.