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MiRNA-142-5p在大鼠角膜新生血管中的作用及机制研究
Role of miRNA-142-5p in regulation of rat corneal neovascularization and related pathways
- 第三军医大学学报 2018年40卷第8期 页码：699-704
- 作者机构：
1.401331 重庆，重庆医科大学附属大学城医院眼科，重庆市眼科重点实验室
2.550002 贵阳，贵州省人民医院眼科
- 作者简介：
[周善璧，E-mail：zhoushanbi@aliyun.comZHOU Shanbi, E-mail:zhoushanbi@aliyun.com
- 基金信息：重庆市医学科研计划资助项目(2013-1-033)
DOI：10.16016/j.1000-5404.201711061
中图分类号： R364.33;R394.3;R772.2收稿：2017-11-09，
修回：2018-01-14，
纸质出版：2018-04-30
- 稿件说明：
移动端阅览
李化, 李彦, 周善璧. MiRNA-142-5p在大鼠角膜新生血管中的作用及机制研究[J]. 第三军医大学学报, 2018,40(8):699-704.
Hua LI, Yan LI, Shanbi ZHOU. Role of miRNA-142-5p in regulation of rat corneal neovascularization and related pathways[J]. Journal of Third Military Medical University, 2018, 40(8): 699-704.
李化, 李彦, 周善璧. MiRNA-142-5p在大鼠角膜新生血管中的作用及机制研究[J]. 第三军医大学学报, 2018,40(8):699-704. DOI： 10.16016/j.1000-5404.201711061.
Hua LI, Yan LI, Shanbi ZHOU. Role of miRNA-142-5p in regulation of rat corneal neovascularization and related pathways[J]. Journal of Third Military Medical University, 2018, 40(8): 699-704. DOI： 10.16016/j.1000-5404.201711061.
摘要
目的
2
观察microRNA-142-5p(miRNA-142-5p)对大鼠角膜新生血管(corneal neovascularization，CorNV)的作用及其与VEGF/PI3K/AKT通路的关联情况。
方法
2
角膜缝线法诱导SD大鼠右眼CorNV作为实验组，左眼作为对照组。取缝线后第7天的角膜进行实时荧光定量聚合酶反应(Real-time PCR，RT-PCR)检测miRNA-142-5p以及VEGF mRNA的表达水平。将miRNA-142-5p mimic转染人脐静脉内皮细胞(human umbilical vein endothelial cells，HUVECs)
RT-PCR检测miRNA-142-5p的表达量来验证转染是否成功。并通过RT-PCR和蛋白免疫印迹法(Western blot)检测HUVECs中VEGF及PI3K/AKT mRNA和蛋白的表达量。分别用Transwell、CCK-8法及Matrigel胶管腔形成实验检测HUVECs的迁徙、增殖及管腔形成能力。
结果
2
成功建立CorNV模型。RT-PCR结果显示7 d组miRNA-142-5p(6.799±1.683)和VEGF(3.297±0.334)的表达量较正常角膜组(normal)显著升高(
P
<
0.01)。转染miRNA-142-5p mimic后的miRNA-142-5p的表达量显著高于空白对照及阴性对照组(
P
<
0.01)。Transwell、CCK-8及Matrigel胶管腔形成实验结果示miRNA-142-5p提高了HUVECs的迁徙、增殖及管腔形成能力(
P
<
0.01)。转染miRNA-142-5p mimic后HUVECs中VEGF及PI3K/AKT的mRNA及相应蛋白的表达均明显上升(
P
<
0.05)。
结论
2
CorNV中miRNA-142-5p及VEGF的表达明显上调，过表达miRNA-142-5p可以显著提高HUVECs中VEGF及PI3K/AKT通路各主要成员mRNA和蛋白的表达水平，进而促进HUVECs的迁徙、增殖及管腔形成能力。表明miRNA-142-5p可通过PI3K/AKT通路参与CorNV的调控。
Abstract
Objective
2
To investigate the role of microRNA-142-5p (miRNA-142-5p) in corneal neovascularization (CorNV) in rats and its association with VEGF/PI3K/AKT pathway.
Methods
2
The right eyes of SD rats were used to establish models of CorNV induced by suture (experimental group)
and the untreated left eyes served as the control group. The corneal expression of miRNA-142-5p and vascular endothelial growth factor (VEGF) mRNA was detected using real-time PCR (RT-PCR) in the rats at 7 days after modeling. A miRNA-142-5p mimic was transfected into human umbilical vein endothelial cells (HUVECs) and RT-PCR was used to detect miRNA-142-5p expression to verify the success of transfection. The expressions of VEGF and PI3K/AKT mRNA and protein in the transfected cells were detected using RT-PCR and Western blotting
and the changes of the cell migration
proliferation and tube formation following the transfection were assessed using Transwell
CCK-8 and Matrigel assays
respectively.
Results
2
The CorNV model was successfully established. Compared with normal rat cornea
the cornea with CorNV showed significantly increased expression of miRNA-142-5p and VEGF mRNA at 7 d after the modeling (
P
<
0.01). HUVECs transfected with the miRNA-142-5p mimic showed significantly increased miRNA-142-5p expression compared with the control cells and the cells transfected with the negative control sequence (
P
<
0.01). Transwell
CCK-8 and Matrigel assays showed that transfection with miRNA-142-5p mimic significantly promoted the migration
proliferation and tube formation of HUVECs (
P
<
0.01)
and also significantly increased the expression of VEGF and PI3K/AKT at both mRNA and protein levels in the cells (
P
<
0.05).
Conclusion
2
The corneal expressions of miRNA-142-5p and VEGF are significantly up-regulated in rats with CorNV. The overexpression of miRNA-142-5p significantly increases the mRNA and protein expressions of VEGF and PI3K/AKT pathway in HUVECs and promotes the cell migration
proliferation and tube forming ability
suggesting the involvement of miRNA-142-5p in the regulation of CorNV through PI3K/AKT pathway.
关键词
Keywords
references
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