[1]曹浪,李雪,周善璧,等.miRNA-424(322)-5p通过激活Raf/MEK/ERK通路促进大鼠角膜新生血管形成[J].第三军医大学学报,2021,43(17):1650-1657.
 CAO Lang,LI Xue,ZHOU Shanbi,et al.MiRNA-424(322)-5p promotes corneal neovascularization in rats via activating Raf/MEK/ERK pathways[J].J Third Mil Med Univ,2021,43(17):1650-1657.
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miRNA-424(322)-5p通过激活Raf/MEK/ERK通路促进大鼠角膜新生血管形成(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
43卷
期数:
2021年第17期
页码:
1650-1657
栏目:
基础医学
出版日期:
2021-09-15

文章信息/Info

Title:
MiRNA-424(322)-5p promotes corneal neovascularization in rats via activating Raf/MEK/ERK pathways
作者:
曹浪李雪周善璧王豪
重庆医科大学附属第一医院眼科,眼科学重庆市重点实验室;贵州省人民医院眼科;重庆医科大学附属大学城医院眼科;重庆市涪陵中心医院眼科
Author(s):
CAO Lang LI Xue ZHOU Shanbi WANG Hao
Department of Ophthalmology, Chongqing Key Laboratory of Ophthalmology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016; 2Department of Ophthalmology, Guizhou Provincial People’s Hospital, Guiyang, Guizhou Province, 550002; 3Department of Ophthalmology, University-Town Hospital of Chongqing Medical University, Chongqing, 401331; 4Department of Ophthalmology, Fuling Central Hospital of Chongqing, Chongqing, 408000, China
 
关键词:
miR-424(322)-5p角膜新生血管Raf/MEK/ERK通路人脐静脉内皮细胞
Keywords:
miR-424(322)-5p corneal neovascularization human umbilical vein endothelial cells Raf/MEK/ERK pathway
分类号:
R363.21; R394.3; R772.23
文献标志码:
A
摘要:

目的探讨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VEGF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:

ObjectiveTo 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. MethodsCorneal 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. ResultsThe rat Cor NV model was successfully established. HE staining showed that the rat corneal tissues of 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). ConclusionOverexpression 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.

参考文献/References:

[1]LEE P, WANG C C, ADAMIS A P. Ocular neovascula-rization: an epidemiologic review[J]. Surv Ophthalmol,1998, 43(3): 245-269. DOI:10.1016/s0039-6257(98)00035-6.
[2]LI Z R, LI Y P, LIN M L, et al. Activated macrophages induce neovascularization through upregulation of MMP-9 and VEGF in rat corneas[J]. Cornea, 2012, 31(9): 1028-1035. DOI:10.1097/ico.0b013e31823f8b40.
[3]EBRAHEM Q, CHAURASIA S S, VASANJI A, et al.Cross-talk between vascular endothelial growth factor and matrix metalloproteinases in the induction of neovascularization in vivo[J]. Am J Pathol, 2010, 176(1): 496-503. DOI:10.2353/ajpath.2010.080642.
[4]MATSUYAMA H, SUZUKI H I. Systems and synthetic microRNA biology: from biogenesis to disease pathogenesis[J]. Int J Mol Sci, 2019, 21(1): 132. DOI:10.3390/ijms21010132.
[5]LIU C H, HUANG S, BRITTON W R, et al. MicroRNAs in vascular eye diseases[J]. Int J Mol Sci, 2020, 21(2):649. DOI:10.3390/ijms21020649. 
[6]MUKWAYA A, JENSEN L, PEEBO B, et al. MicroRNAs in the cornea: Role and implications for treatment of corneal neovascularization[J]. Ocul Surf, 2019, 17(3): 400-411. DOI:10.1016/j.jtos.2019.04.002. 
[7]SUN P C, WANG L, LU Y H, et al. MicroRNA-195 targets VEGFR2 and has a tumor suppressive role in ACHN cells via PI3K/Akt and Raf/MEK/ERK signaling pathways[J]. Int J Oncol, 2016, 49(3): 1155-1163. DOI:10.3892/ijo.2016.3608.
[8]毛旖旎, 胡雁, 侯胜平, 等. 大鼠角膜缘新生血管微小RNA与血管内皮生长因子相关性分析[J]. 重庆医科大学学报, 2014, 39(8): 1090-1094. DOI:10.13406/j.cnki.cyxb.000324. 
MAO Y N, HU Y, HOU S P, et al. Correlation between microRNA expression profiles and VEGF of corneal neovascularization in rats[J]. J Chongqing Med Univ, 2014, 39(8): 1090-1094. DOI:10.13406/j.cnki.cyxb.000324. 
[9]SHARIF Z, SHARIF W. Corneal neovascularization: updates on pathophysiology, investigations & management[J]. Rom J Ophthalmol, 2019, 63(1): 15-22. 
[10]BAI Y, BAI X, WANG Z, et al. MicroRNA-126 inhibits ischemia-induced retinal neovascularization via regulating angiogenic growth factors[J]. Exp Mol Pathol, 2011, 91(1): 471-477. DOI:10.1016/j.yexmp.2011.04.016. 
[11]ZHANG L X, TAO L J. miR-132 promotes retinal neovascularization under anoxia and reoxygenation conditions through up-regulating Egr1, ERK2, MMP2, VEGFA and VEGFC expression[J]. Int J Clin Exp Pathol, 2017, 10(8): 8845-8857. 
[12]PARK J K, PENG H, YANG W D, et al. miR-184 exhibits angiostatic properties via regulation of Akt and VEGF signaling pathways[J]. FASEB J, 2017, 31(1): 256-265. DOI:10.1096/fj.201600746R.
[13]GHOSH G, SUBRAMANIAN I V, ADHIKARI N, et al. Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis[J]. J Clin Invest, 2010, 120(11): 4141-4154. DOI:10.1172/jci42980.
[14]SOUFI-ZOMORROD M, HAJIFATHALI A, KOUHKAN F, et al. MicroRNAs modulating angiogenesis: miR-129-1 and miR-133 act as angio-miR in HUVECs[J]. Tumour Biol, 2016, 37(7): 9527-9534. DOI:10.1007/s13277-016-4845-0.
[15]李雪, 曹浪, 甘敏, 等. MiR-200b通过调控PIK3CA/AKT通路抑制大鼠角膜血管新生[J]. 第三军医大学学报, 2020, 42(4): 407-413. DOI:10.16016/j.1000-5404.201909141.
LI X, CAO L, GAN M, et al. MiR-200b inhibits corneal angiogenesis in rats by regulating PIK3CA/AKT pathway[J]. J Third Mil Med Univ, 2020, 42(4): 407-413. DOI:10.16016/j.1000-5404.201909141.
[16]APTE R S, CHEN D S, FERRARA N. VEGF in signaling and disease: beyond discovery and development[J]. Cell, 2019, 176(6): 1248-1264. DOI:10.1016/j.cell.2019.01.021.
[17]POTENTE M, CARMELIET P. The link between angiogenesis and endothelial metabolism[J]. Annu Rev Physiol, 2017, 79: 43-66. DOI:10.1146/annurev-physiol-021115-105134. 
[18]ZHU S, WANG H, ZHANG Z, et al. IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway[J]. Mol Med Rep, 2020, 22(6): 4837-4847. DOI:10.3892/mmr.2020.11578.
[19]GUO C M, ZHAO D T, ZHANG Q L, et al. MiR-429 suppresses tumor migration and invasion by targeting CRKL in hepatocellular carcinoma via inhibiting Raf/MEK/ERK pathway and epithelial-mesenchymal transition[J]. Sci Rep, 2018, 8(1): 2375. DOI:10.1038/s41598-018-20258-8. 
[20]ZHANG F, CAO H. MicroRNA-143-3p suppresses cell growth and invasion in laryngeal squamous cell carcinoma via targeting the k-Ras/Raf/MEK/ERK signaling pathway[J]. Int J Oncol, 2019, 54(2): 689-701. DOI:10.3892/ijo.2018.4655. 
[21]WU R S, QIU E H, ZHU J J, et al. MiR-101 promotes nasopharyngeal carcinoma cell apoptosis through inhibiting Ras/Raf/MEK/ERK signaling pathway[J]. Eur Rev Med Pharmacol Sci, 2020, 24(16): 8240. DOI:10.26355/eurrev_202008_22580. 

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更新日期/Last Update: 2021-09-03