微小RNA-126靶向调控血管内皮生长因子对新生大鼠缺氧-缺血性脑病神经元损伤的影响

doi:10.16098/j.issn.0529-1356.2021.06.006

摘要/Abstract

摘要：

目的探讨微小RNA-126（miR-126）靶向调控血管内皮生长因子（VEGF）对新生大鼠缺氧-缺血性脑病（HIE）神经元损伤的影响。方法选择清洁级新生7日龄SD雄性大鼠随机分为4组，假手术组（A组）、HIE组（B组）、HIE+阴性对照组（C组）和HIE+miR-126过表达组（D组），每组18只。建立HIE模型后进行大鼠神经功能缺损评分及脑组织含水量测定；采用HE染色光学显微镜下观察各组大鼠脑海马组织CA1区病理形态学改变；采用Real-time PCR检测大鼠脑组织miR-126和VEGF的表达水平；采用免疫组织化学法检测各组大鼠脑海马组织CA1区VEGF蛋白表达情况；采用双荧光素酶靶标实验验证miR-126与VEGF基因的靶向关系；采用流式细胞术检测脑海马组织神经元凋亡水平；采用Western blotting检测各组大鼠脑组织剪切Caspase-3（cleaved-Caspase-3）蛋白表达水平。结果 A组大鼠无神经功能损伤，神经行为学评分为0分，脑组织无损伤；B组和C组大鼠神经行为学评分分别为（2.50±0.55）分和（2.33±0.82）分，脑组织损伤明显；D组大鼠神经行为学评分为（1.50±0.55）分，脑组织损伤有改善；与A组相比，B组、C组和D组大鼠神经行为学评分（P<0.05）及脑组织含水量（P<0.05）升高；与B组相比，D组大鼠神经行为学评分（P<0.05）及脑组织含水量（P<0.05）降低。与A组相比，B组和C组大鼠脑组织miR-126的表达水平均显著降低,VEGF mRNA和蛋白表达水平、脑海马组织神经元凋亡率及脑组织cleaved-Caspase-3的表达水平均显著升高（P<0.05）；与B组相比，D组大鼠miR-126的表达水平均显著升高，VEGF mRNA和蛋白表达水平、脑海马组织神经元凋亡率及脑组织cleaved-Caspase-3的表达水平均显著降低（P<0.05）。荧光素酶报告基因实验结果显示，miR-126和VEGF能够靶向结合。结论过表达miR-126后可降低脑海马组织神经元凋亡水平，改善HIE的发展，其作用机制可能与miR-126靶向抑制VEGF基因表达有关。

关键词: 微小RNA-126, 血管内皮生长因子, 缺氧-缺血性脑病, 神经元, 实时定量聚合酶链反应, 大鼠

Abstract:

Objective To investigate the effect of targeting vascular endothelial growth factor (VEGF) by microRNA-126 (miR-126) on neuronal damage in neonatal rats with hypoxic-ischemic encephalopathy(HIE). Methods Newborn 7 days old SD male rats were randomly divided into four group, sham operation group(group A), HIE group(group B), HIE+negative control group(group C), and HIE+miR-126 overexpression group(group D), eighteen in each group. After modeling, neurological deficit score and brain water content were measured. HE staining was used to observe the pathological changes of CA1 area in hippocampus of brain in each group. Real-time PCR was used to detect the expression of miR-126 and VEGF. Immunohistochemistry was used to detect the expression of VEGF in CA1 area in hippocampus of brain. Double luciferase target experiment was used to verify the targeting relationship between miR-126 and VEGF gene. Flow cytometry was used to detect neuron apoptosis in hippocampus. Western blotting was used to detect the expression of cleaved-Caspase-3 protein in brain tissue of rats in each group. Results There was no neurobehavioral damage in group A, the neurobehavioral score was 0, and the brain tissue was not damaged; the neurobehavioral scores in group B and group C were (2.50±0.55) and (2.33±0.82) respectively, and the brain tissue damage was obvious; the neurobehavioral score in group D was (1.50±0.55), and the damage of brain tissue was improved. Compared with the group A, the neurobehavioral score (P<0.05) and brain water content of group B and group C increased significantly (P<0.05); Compared with the group B, the neurobehavioral score (P<0.05) and brain water content of group D (P<0.05) decreased. Compared with the group A, the expression level of miR-126, VEGF mRNA and protein, neuron apoptosis rate and cleavedCaspase-3 in brain tissue of group B and group C were all significantly lower (P<0.05). Compared with the group B, the expression level of miR-126, VEGF mRNA and protein, neuron apoptosis rate and cleaved-Caspase-3 in hippocampus of group D were all significantly higher (P<0.05). The result of luciferase reporter gene experiment showed that miR-126 and VEGF could be targetly binded. Conclusion Overexpression of miR-126 can reduce neuronal apoptosis in hippocampus of brain and improve the development of HIE. The mechanism may be related to the targeted inhibition of VEGF gene expression by miR-126.

Key words: MicroRNA-126, Vascular endothelial growth factor, Hypoxic-ischemic encephalopathy, Neuron, Real-time PCR, Rat

中图分类号:

R742

刘鑫霍世芳马中岭卢忠胜. 微小RNA-126靶向调控血管内皮生长因子对新生大鼠缺氧-缺血性脑病神经元损伤的影响[J]. 解剖学报, 2021, 52(6): 875-881.

LIU Xin HUO Shi-fang MA Zhong-ling LU Zhong-sheng. Effect of targeting vascular endothelial growth factor by microRNA-126 on neuronal damage in neonatal rats with hypoxic-ischemic encephalopathy[J]. Acta Anatomica Sinica, 2021, 52(6): 875-881.

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