MiRNA-424(322)-5p promotes corneal neovascularization in rats
via activating Raf/MEK/ERK pathways- Journal of Third Military Medical University Vol. 43, Issue 17, Pages: 1650-1657(2021)
- 作者机构：
- 作者简介：
- 基金信息：
DOI：10.16016/j.1000-5404.202103108
CLC：Received：12 March 2021，
Published：15 September 2021
- 稿件说明：
移动端阅览
Lang CAO, Xue LI, Shanbi ZHOU, et al. MiRNA-424(322)-5p promotes corneal neovascularization in rats
via activating Raf/MEK/ERK pathways[J]. Journal of Third Military Medical University, 2021, 43(17): 1650-1657.Lang CAO, Xue LI, Shanbi ZHOU, et al. MiRNA-424(322)-5p promotes corneal neovascularization in rats
via activating Raf/MEK/ERK pathways[J]. Journal of Third Military Medical University, 2021, 43(17): 1650-1657. DOI： 10.16016/j.1000-5404.202103108.
摘要
目的
2
探讨microRNA-424(322)-5p[miR-424(322)-5p]在大鼠角膜新生血管(corneal neovascularization
Cor NV)形成中的作用及其可能机制。
方法
2
20只雌性SD大鼠通过缝线法诱导构建大鼠Cor NV模型，左眼为实验组，右眼为正常对照组，收集建模第0、4、7、14天的角膜组织，通过HE染色、免疫组化(IHC)观察角膜结构、血管形成及蛋白表达；通过RT-PCR检测建模第4天的大鼠角膜组织中的miR-424(322)-5p及血管内皮生长因子(vascular endothelial growth factor，VEGF)mRNA的表达。通过脂质体转染人脐静脉内皮细胞(human umbilical vein endothelial cells，HUVECs)，分为miR-424(322)-5p模拟物[miR-424(322)-5p mimic]组、阴性对照[miR-424(322)-5p NC]组、正常空白(CON)组，用RT-PCR检测miR-424(322)-5p转染效率，CCK-8、划痕实验、Transwell实验、Matrigel胶成管实验，分别检测各组HUVECs的增殖、迁移、成管能力；蛋白免疫印迹法(Western blot)检测VEGF及Raf/MEK/ERK通路的蛋白相对表达量。
结果
2
成功构建了大鼠Cor NV模型，HE染色切片显示：与正常对照组比较，建模后实验组第4、7、14天的大鼠角膜组织结构紊乱，角膜水肿增厚，新生血管形成，炎性细胞浸润；免疫组化(IHC)结果显示：与正常对照组比较，实验组V
EGF的表达量明显增加，呈棕黄色；RT-PCR结果显示建模第4天后的大鼠角膜组织中miR-424(322)-5p及VEGF mRNA的表达显著增高(
P
<
0.01)。通过脂质体转染HUVECs后的miR-424(322)-5p mimic组miR-424(322)-5p的表达量显著高于NC组和CON组(
P
<
0.01)，CCK-8、划痕实验、Transwell实验及Matrigel胶成管实验结果表明miR-424(322)-5p促进HUVECs的增殖、迁移及成管能力(
P
<
0.01)；Western blot结果显示VEGF及Raf/MEK/ERK通路的蛋白相对表达量明显上调(
P
<
0.01)。
结论
2
miR-424(322)-5p可能通过激活VEGF及Raf/MEK/ERK通路，促进HUVECs的迁移、增殖及成管能力，从而参与Cor NV的形成。
Abstract
Objective
2
To explore the role and possible mechanism of microRNA-424(322)-5p (miR-424(322)-5p) in the formation of corneal neovascularization (Cor NV) in rats.
Methods
2
Corneal neovascularization model was established in 20 female rats by suturing. Their left eye was used as the experimental group and the right eye was used as the normal control group. On the 0th
4th
7th
14th days after modeling
corneal structure
blood vessel formation and protein expression in the corneal tissues were observed by HE staining and immunohistochemistry (IHC). On the 4th day after modeling
the mRNA expression of miR-424(322)-5p and vascular endothelial growth factor (VEGF) in the corneal tissue was detected by RT-PCR. Human umbilical vein endothelial cells (HUVECs) were transfected with liposomes and divided into miR-424(322)-5p mimic group
negative control (miR-424(322)-5p NC) group and blank control (CON) group
respectively. The transfection efficiency of miR-424(322)-5p was tested by RT-PCR. CCK-8 assay
scratch assay
Transwell assay and Matrigel tube assay were performed to measure the proliferation
migration
and tube forming ability of HUVECs in each group
respectively. The levels of VEGF and Raf/MEK/ERK pathway proteins were detected by Western blotting.
Results
2
The rat Cor NV model was successfully established. HE staining showed that the rat corneal tissues o
f the experimental group were in disordered arrangement
corneal edema and thickening
neovascularization
and inflammatory cell infiltration on the 4th
7th and 14th day after modeling. The results of IHC indicated that the expression of VEGF was enhanced significantly after modeling when compared with the normal control group. RT-PCR displayed that the expression of miR-424(322)-5p and VEGF mRNA in rat cornea tissues was significantly enhanced on the 4th day after modeling (
P
<
0.01). The expression level of miR-424(322)-5p in the mimic group after HUVECs transfected with liposomes was significantly higher than that in the NC group and the CON group (
P
<
0.01). The results of CCK-8 assay
scratch assay
migration assay and Matrigel tube assay displayed that miR-424(322)-5p promoted the migration
proliferation and tube forming ability of HUVECs (
P
<
0.01). Western blotting showed that the protein levels of VEGF and Raf/MEK/ERK pathway proteins were significantly up-regulated (
P
<
0.01).
Conclusion
2
Overexpression of miR-424(322)-5p promotes the migration
proliferation and tube formation of HUVECs by activating VEGF and Raf/MEK/ERK pathways
and thereby participates in the formation of Cor NV.
关键词
Keywords
references
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