MiR-191-5p Disturbed the Angiogenesis in a Mice Model of Cerebral Infarction by Targeting Inhibition of BDNF
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.333459
Source of Support: None, Conflict of Interest: None
Keywords: Angiogenesis, brain-derived neurotrophic factor, cerebral infarction, miRNA-191-5p
Cerebral infarction is mainly caused by cerebral artery occlusion and interruption of blood supply. The death toll of cerebral infarction in China is about 1.6 million per year, which is one of the main causes of death and disability. Several common risk factors, including dyslipidemia, hypertension, and diabetes, have been identified to be associated with the pathogenesis of cerebral infarction. Ischemia irreversibly results in substantial neuronal degeneration and necrosis and causes severe neurological deficits. It has been proved that promoting angiogenesis in ischemic brain to increase the number of new collateral circulation will increase the blood supply and improve the ischemic brain function. However, research to date has not yet determined the underlying mechanisms that regulate angiogenesis.
Angiogenesis can provide nutritional support for nerve repair and is an important reason to promote the recovery of nerve function after cerebral infarction. There is a growing number of studies that acknowledge the involvement of microRNAs (miRNAs) in the regulation of angiogenesis in ischemic diseases. Changes in miRNA expression profiles of acute ischemic stroke (AIS) patients, middle cerebral artery occlusion (MCAO) rats, and oxygen–glucose deprivation/reoxygenation (OGD/R) cells have been detected, which indicates that several miRNAs, related to angiogenesis, such as miR-210, miR-31, and miR-191 have changed.,, However, to date, there has been little clear evidence on the mechanism of action of miRNAs involved in angiogenesis in cerebral infarction.
Brain-derived neurotrophic factor (BDNF) is a protein synthesized by brain tissue and is involved in the growth and development of neurons. Extensive research has shown that BDNF not only plays a key role in the physiological function of normal neurons but also regulates synaptic plasticity and function in the mature nervous system. After effective treatment of acute cerebral infarction in mice, the level of BDNF was significantly increased in serum, which in turn promoted the recovery of neurological function in mice. Moreover, BDNF is involved in angiogenesis in the heart and skeletal muscle., However, its function in cerebral infarction needs to be further studied. TargetScan database revealed a site in the BDNF 3' untranslated region (UTR) that binds to miR-191-5p. Therefore, the aim of this investigation was to determine whether the interaction of miR-191-5p targeting BDNF might be involved in the cerebral angiogenesis of MCAO mice.
Animal and experimental design
Fifty-three male C57BL/6 mice weighing 20–25 g were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (No: SYXK (Sichuan) 2019-189). Mice were randomly distributed into two groups—experimental group 1: sham operation group (sham, n = 6), MCAO-24h (n = 6), MCAO-48h (n = 6) and experimental group 2: sham (n = 7), MCAO (n = 7), MCAO + miR-191-5p negative control group (MCAO + NC-antagomir, n = 7), MCAO + miR-191-5p antagomir group (MCAO + miR-191-5p-antagomir, n = 7) and MCAO + BDNF overexpression group (MCAO + pcDNA-BDNF, n = 7). NC-antagomir, miR-191-5p antagomir, and the construction and identification of plasmid pcDNA-BDNF were synthesized by Sangon Biotech, Shanghai, China. All experimental protocols of this study were approved by the Animal Ethics Committee of Southwest Medical University (no, 20210306-3), and all efforts were made to minimize animal suffering and to reduce the number of animals used.
Establishment of mice MCAO model
MCAO mice model was established according to the modified Longa methods as described before. The mice were anesthetized by intraperitoneal injection of pentobarbital sodium 45 mg/kg. After shaving and disinfecting the neck, a longitudinal incision was made at the neck. The cerebral artery was ligated proximal to the heart with nylon thread (Beijing Shadong Biotechnology Co., Ltd.) and sutured. After 2-h ischemia, the thread was pulled out to achieve reperfusion. In the sham operation group, only the cerebral artery was exposed without ligation. If nerve defect appeared in the MCAO group within 24 h after operation, the model was considered to be successful. The mice could obtain food and water freely.
NC-antagonist, miR-191-5p-antagonist (5 nmol in a total volume of 5 μL PBS), and pcDNA-BDNF (0.002 nmol dissolved in 4 μL PBS) were administered by intracerebroventricular injection (ICV) 2 h before MCAO. The injections were performed as previously described. Mice were anesthetized and positioned lying prone in a stereotactic head frame. A scalp incision was made along the midline and a burr hole was drilled into the right side of the skull (0.5 mm posterior and 1.0 mm lateral to the bregma). NC-antagonist, miR-191-5p-antagonist, or pcDNA-BDNF were injected into right lateral ventricle with 0.2 μL/min using a Hamilton syringe (2.5 mm vertical) with a microinfusion pump (KDS 310, KD Scientific), and the incision was closed.
2, 3, 5-triphenyltetrazolium hydrochloride (TTC) staining
The brain was collected and sectioned in coronal section every 1 mm, stained with TTC for 20 min at 37°C. The volume of cerebral infarction (mm3) of brain tissue was calculated using the Image-Proplus image analysis and processing system (Motic Med 6.0 System).
2.5 Detection of vascular density by FITC-dextran
The mice were anesthetized by intraperitoneal injection of pentobarbital sodium 45 mg/kg and injected slowly through a femoral vein with FITC-dextran (0.2 ml, 50 mg/ml, Sigma). And 10 min later, brain tissues were soaked in 4% paraformaldehyde solution for 24 hr at 4%. Next, tissues were embedded with OCT (optimucutting temperature) and sliced into sections of 100 μm in thickness at –20 °C with a cryotome. At last, the five visual fields of the section were observed using the TCS SP5 laser confocal scanning microscope, and the image software was used to analyze the number of blood vessels and calculate the mean value.
Neurological function scores were performed according to an established graded scoring system, where 0 points: no signs of neurological impairment, 1 point: cannot extend the opposite front paw, 2 points: turn in circles to the hemiplegia side, 3 points: tipping toward the hemiplegia side when walking, and 4 points: inability to walk spontaneously, loss of consciousness.
The mice were placed on a horizontal grid floor (metal grid size: 50 × 40 cm, size of each grid: 3 × 3 cm, metal diameter: 0.4 cm) elevated above the surface and were allowed to walk for 2 min. Foot fault occurred when the mouse's foot did not touch the grid and then fell through the opening between the grids. The absences of all limbs were observed. The total number of foot faults was recorded for statistical analysis of the motor coordination of mice.
Real-time quantitative polymerase chain reaction (PCR)
The mRNA expressions of miR-191-5p and BDNF were detected according to the manufacturer's instruction (TaKaRa Biotechnology Co., Ltd, Dalian, China). The primer sequences (Sangon Biotech Co., Ltd, Shanghai, China) are listed below: GAPDH, 5'-TCGAGTCTACTGGCGTCTT-3' (Forward) and 5'-ATGAGCCCTTCCACGAT-3' (Reverse); miR-191-5p, 5'-TGCGCCAACGGAATCCCAAAAGC-3' (Forward) and 5'- CCAGTGCAGGGTCCGAGGTATT-3' (Reverse); BDNF, 5'-TCACAGCGGCAGATAAAA AGAC-3' (Forward) and 5'-TAAGGGCCCGAACATACGAT-3' (Reverse). The conditions of RT-qPCR were as follows: initial denaturation at 95°C for 10 min, denaturation at 95°C for 10 s, annealing at 60°C for 10 s, extension at 72°C for 10 s, reaction of 40 cycles, CT values were recorded, and relative expression levels were analyzed by 2−ΔΔCT. GAPDH was used as endogenous normalization control.
Western blot analysis
Total proteins of brain tissues were extracted by RIPA buffer (P1003B, Beyotime, Wuhan, China). Proteins were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the immunoblots were incubated overnight at 4°C with the following primary antibodies: BDNF (1:1000, ab203573, abcam) or β-actin (1:1000; 4967S, Cell Signaling Technology). After washed in Tris-buffered saline and Tween (TBS-T) for 3 times, horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (1:5000, Cell Signaling, Danvers, MA, USA) was added and incubated at room temperature for 2 h. Protein bands were determined by imaging analysis system with enhanced chemiluminescence (ECL) imaging (Shanghai Beyotime Biotechnology Co., Ltd. Shanghai, China).
Luciferase reporter assay
The BDNF gene 3′-UTR fragment containing the miR-191-5p targeting site was amplified, and the BDNF-WT, BDNF-mut with miR-191-5p mimics or miR-191-5p NC were co-transfected into 293 T cells using LipofectamineTM2000. At 48 h after transfection, the dual luciferin signal was measured in each group according to the instructions of the dual luciferase reporter gene assay kit (E1910, Promega, USA).
Statistical evaluation was performed using SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA). All the data were presented as the means ± standard deviation. One-way variance analysis was used to determine significantly different groups. P < 0.05 was considered as significant difference.
Expression of miR-191-5p and BDNF in mice cerebral tissue following MCAO
RT-qPCR results showed that the expression of miR-191-5p was significantly increased at 24 and 48 h following MCAO compared with the sham group [[Figure 1]a; P < 0.05 and P < 0.01, respectively]. However, western blot analysis demonstrated that the protein expression level of BDNF was significantly reduced at 24 and 48 h following MCAO compared with the sham group [[Figure 1]b; P < 0.01]. Together, these results suggested that miR-191-5p was highly expressed in MCAO mice, while BDNF was low expressed in MCAO mice.
Administration of exogenous miR-191-5pantagomir reduced infarction volume and improved neurological deficits of MCAO mice
To investigate the effects of miR-191-5p and BDNF in infarct volume and improvement of neurological deficits, MCAO mice were injected intracerebroventricularly with miR-191-5pantagomir or pcDNA-BDNF, respectively. The results revealed that the MCAO group showed a higher neurobehavioral score and the number of foot-faults than the sham group, while downregulation of miR-191-5p or upregulation of BDNF improved the neurobehavioral function scores and the performance of foot-fault [[Figure 2]a and [Figure 2]b; P < 0.01]. TTC staining showed white ischemic areas in the brain tissue of MCAO mice [Figure 2]c. Compared with the sham group, MCAO caused a significant brain infarction, but miR-191-5p antagomir treatment effectively reduced the infarct volume of MCAO mice [[Figure 2]c and [Figure 2]d; P < 0.01]. In addition, over-expression of BDNF also reduced the infarct volume of MCAO mice [[Figure 2]c and [Figure 2]d; P < 0.01]. These results showed that inhibition of miR-191-5p or increase of BDNF can promote the recovery of neurologic function, improve the performance of foot-fault test, and reduce the infarct volume after MCAO, implying its potential role in cerebral infarction.
Administration of exogenous miR-191-5pantagomir can promote vessel volume of MCAO mice
To investigate the role of miR-191-5p in the angiogenesis of MACO, FITC-dextran was used to detect cerebral blood vessel density. Compared with the sham group, the microvessel density of the MCAO group was significantly decreased [[Figure 3]a and [Figure 3]b; P < 0.01], but the microvessel density in miR-191-5pantagomir and pcDNA-BDNF groups were higher than that in the MCAO group [[Figure 3]a and [Figure 3]b; P < 0.01], indicating that inhibition of miR-191-5p or increase of BDNF promoted the microangiogenesis in cerebral ischemic area.
BDNF is the direct target of miR-191-5p
The expression of miR-191-5p in brain tissue of MCAO mice was significantly reduced by miR-191-5pantagomir detected by RT-qPCR [[Figure 4]a; P < 0.01]. On the contrary, the BDNF expression of brain tissue of MACO mice was increased by miR-191-5p antagomir treatment detected by RT-qPCR and western blot [[Figure 4]b and [Figure 4]c; P < 0.01]. TargetScan database was used to predict the interaction of miR-191-5p and BDNF, which indicated that BDNF is the direct target of miR-191-5p. The potential targeting relationship between miR-191-5p and BDNF was confirmed by dual luciferase reporter. MiR-191-5p mimic and BDNF-wt or mut were co-transfected into 293 T cells. We found that miR-191-5p mimic significantly decreased luciferase activity compared to the mimic control group [[Figure 4]d; P < 0.05]. These results suggested that administration of exogenous miR-191-5pantagomir greatly increased the level of BDNF in the brain tissue, and miR-191-5p might be involved in the inhibition of cerebral infarct angiogenesis through direct downregulation of BDNF.
In the present study, our results demonstrated that inhibition of miR-191-5p to increase BDNF expression has protective on MCAO mice. First, we found that miR-191-5p antagomir reduced the infarct volume and improved neurological function in MCAO mice. We further demonstrated that inhibiting miR-191-5p can promote angiogenesis in the brain tissue of MCAO mice through directly upregulating BDNF. These observations indicated that miR-191-5p is involved in the dysfunction of angiogenesis after cerebral infarction by targeting BDNF.
Angiogenesis is the basis of nerve repair after cerebral ischemia. As mentioned in the literature, angiogenesis has beneficial effects on the long-term survival of patients with cerebral ischemia. Evidence has demonstrated that several miRNAs had been involved in the regulation of angiogenesis. Gu et al. demonstrated that miR-191 was preferentially expressed in endothelial cells compared to other types of human cells and displayed an antiangiogenic effect. In the present study, we found that the expression of miR-191-5p was significantly promoted in the brain tissue of cerebral infarction area in MCAO mice, and the administration of antagomir-191-5p can promote the recovery of neurologic function, improve the performance of foot-fault test, reduce the infarct volume, and promote microangiogenesis in the cerebral ischemic area after MCAO. Our results complement nicely with a previous report showing that knockout of miR-191 reduced hepatic ischemia-reperfusion injury through inhibiting inflammatory responses and cell death. These results indicate that lowing miR-191 might be a potential therapy for ischemia diseases. However, although we found that levels of miR-191 were increased in mice with MCAO, the exact role of miR-191 in cerebral infarction requires further studies with larger samples.
Mechanistically, prior studies have noted that BDNF can modulate angiogenesis through direct or indirect mechanisms., For example, deletion of the BDNF gene can lead to impaired survival of capillary endothelial cells. Additionally, BDNF increased the formation of angiogenic tubes in HUVEC endothelial cells through ERK and Akt signal transduction pathways. Moreover, reduced BDNF affects inflammation and angiogenesis after myocardial infarction by regulating VEGF-A levels. These findings indicate that BDNF exhibits a direct role in the angiogenic process. In this study, we found that cerebral infarction can inhibit the expression of BDNF, and luciferase reporter assay validated that BDNF is a direct target of miR-191-5p. To further prove that miR-191-5p inhibits angiogenesis by targeted downregulation of BDNF, we measured the expression levels of the targets BDNF after miR-191-5p interference. Our data showed that administration of exogenous miR-191-5p greatly increased the expression level of BDNF in the brain tissue of MCAO mice. Therefore, miR-191-5p might disturb the angiogenesis of cerebral infarction by inhibiting BDNF.
In summary, our data revealed a novel role of miR-191-5p in promoting cerebral infarction by disturbing angiogenesis by inhibiting BDNF, and the identification of the miR-191-5p regulated BDNF may provide new insight into the potential molecular mechanisms of cerebral infarction.
Financial support and sponsorship
This work was supported by the grants (2019) 119 from the 2019 Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University-13 Fund Project.
Conflicts of interest
There are no conflicts of interest.
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