microRNA-182抑制人角膜上皮细胞增殖和迁移
曹琼洁,赵晓婷,敬霞,王教,陈晓燕,闫东升,瞿佳
325027 温州医科大学附属眼视光医院 省部共建眼视光学和视觉科学国家重点实验室
MicroRNA-182 Inhibits the Proliferation and Migration of Human Corneal Epithelial Cells
Qiongjie Cao, Xiaoting Zhao, Xia Jing, Jiao Wang, Xiaoyan Chen, Dongsheng Yan, Jia Qu
State Key Laboratory of Ophthalmology, Optometry and Vision Science, Eye Hospital of Wenzhou Medical University, Wenzhou 325027, China
摘要 目的:通过体内动物实验与体外细胞实验来阐明microRNA-182(miR-182)在角膜上皮损伤修复中的功能。方法:实验研究。体内实验选取5 只C57BL/6J野生型小鼠,通过机械刮伤法构建小鼠角膜上皮损伤修复模型作为实验组,对侧眼作为对照组,通过实时定量RT-PCR法检测miR-182 在损伤修复过程( 损伤后48 h)的表达。体外实验采用脂质体介导的方法将miR-182和随机序列寡核苷酸链转染入人角膜上皮细胞(HCECs),通过细胞增殖实验(MTS法)和细胞克隆形成实验检测细胞增殖和生长能力,流式细胞术检测细胞周期,细胞划痕实验检测细胞迁移能力。MiR-182 表达量及细胞实验各参数2 组间比较采用独立样本t检验。结果:体内实验中,与对照组相比,实验组中5 个样本量的miR-182 在角膜上皮损伤修复过程中表达水平显著下调(t=147.6、79.2、136.8、41.3、89.8,均P<0.001)。体外实验中,miR-182组相对细胞数目为(81±5)%,与阴性对照组(100%)相比显著减少(t=6.6,P=0.003),同时miR-182组细胞克隆数明显少于阴性对照组。细胞周期检测结果显示实验组处于G1期细胞数量比例为(49±7)%,明显高于阴性对照组的(35±4)%(t=-3.0,P=0.041)。此外,细胞迁移实验结果显示miR-182组HCECs细胞迁移的距离为(99±12)μm,而阴性对照组的迁移距离为(213±14)μm(t=10.8,P=0.001),迁移速度明显变慢。结论:miR-182通过抑制角膜上皮细胞的增殖与迁移,从而对角膜上皮损伤修复起到负调控的作用。
关键词 :
角膜损伤修复 ,
miR-182 ,
角膜上皮细胞 ,
增殖 ,
迁移
Abstract :Objective: We investigated the in vivo and in vitro effects of microRNA-182 (miR-182) , which modulates post-transcriptional regulation of gene expression, on corneal epithelial cell proliferation, migration, and wound healing. Methods: In this experimental research, mouse corneal epithelial wound healing of five mouse was initiated by a scrape injury. Realtime reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect the expression level of miR-182 in the mouse corneal epithelium during the wound healing process (48 hours post scraping). Lipofectamine RNAiMAX reagent was used to transfect human corneal epithelial cells (HCECs) in vitro with either miR-182 or negative control RNA oligo. Cell proliferation assay (MTS), cell colony-forming assay, and flow cytometry were used to evaluate the effects of miR-182 on cell proliferation and cell cycle. An in vitro wound-healing assay (the HCECs were wounded by placing a scratch along the surface of each culture) was carried out to evaluate the effect of miR-182 on cell migration. Data were analyzed using independent t test. Results: The in vivo expression of miR-182 in experimentally wounded corneal epithelia was significantly lower than in the non-wounded controls (t=147.6, 79.2, 136.8, 41.3, 89.8, all P<0.001). In vitro, the relative number of cells in the experimental group transfected with miR-182 was (81±5)%, which was significantly less than that in the control group transfected with negative control RNA oligo, 100% (t=6.6, P=0.003). Further, the number of clones in the experimental group was remarkably decreased compared to that in the control group. The percentage of transfected cells in G1 arrest was (49±7)%, which was significantly higher than that in the control group, (35±4)% (t=-3.0, P=0.041). In addition, the migration distance of HCECs in the transfected cells, 99±12 μm, was shorter than in the control cells, 213±14 μm (t=10.8, P=0.001). Conclusions: miR-182 negatively regulates the corneal epithelial wound healing process by inhibiting corneal epithelial cell proliferation and migration.
Key words :
corneal wound healing
miR-182
corneal epithelium cell
proliferation
migration
收稿日期: 2018-06-22
基金资助: 温州市科技计划项目(Y20160188)
通讯作者:
瞿佳(ORCID:0000-0003-1678-966X),Email:jqu@wzmc.net
引用本文:
曹琼洁,赵晓婷,敬霞,王教,陈晓燕,闫东升,瞿佳. microRNA-182抑制人角膜上皮细胞增殖和迁移[J]. 中华眼视光学与视觉科学杂志, 2018, 20(8): 503-508.
Qiongjie Cao, Xiaoting Zhao, Xia Jing, Jiao Wang, Xiaoyan Chen, Dongsheng Yan, Jia Qu. MicroRNA-182 Inhibits the Proliferation and Migration of Human Corneal Epithelial Cells. Chinese Journal of Optometry Ophthalmology and Visual science, 2018, 20(8): 503-508. DOI: 10.3760/cma.j.issn.1674-845X.2018.08.009.
链接本文:
https://www.cjoovs.com/CN/10.3760/cma.j.issn.1674-845X.2018.08.009. 或 https://www.cjoovs.com/CN/Y2018/V20/I8/503
及其相关因素. 中华眼视光学和视觉科学杂志, 2015, 17(12):
71
2-716. DOI: 10.3760/cma.j.issn.1674-845X.2015.12.003.
[1]
许琛琛, 吴斯琪, 王勤美. 正常人眼角膜上皮层厚度的分布
[2]
张丽颖, 瞿杨洛娃, 邹笃雷, 等. 组织工程角膜上皮移植重建
及其相关因素. 中华眼视光学和视觉科学杂志, 2015, 17(12):
71
2-716. DOI: 10.3760/cma.j.issn.1674-845X.2015.12.003.
眼表面的临床研究. 中华眼视光学与视觉科学杂志, 2016,
18
(4): 209-214. DOI: 10.3760/cma.j.issn.1674-845X.2016.04.
[2]
张丽颖, 瞿杨洛娃, 邹笃雷, 等. 组织工程角膜上皮移植重建
004.
眼表面的临床研究. 中华眼视光学与视觉科学杂志, 2016,
18
(4): 209-214. DOI: 10.3760/cma.j.issn.1674-845X.2016.04.
[3]
Daniels JT, Notara M, Shortt AJ, et al. Limbal epithelial stem
cell therapy. Expert Opin Biol Ther, 2007, 7(1): 1-3. DOI:
004.
10
1517/14712598.7.1.1.
[3]
Daniels JT, Notara M, Shortt AJ, et al. Limbal epithelial stem
cell therapy. Expert Opin Biol Ther, 2007, 7(1): 1-3. DOI:
[4]
Kenchegowda S, Bazan HE. Significance of lipid mediators in
10
1517/14712598.7.1.1.
corneal injury and repair. J Lipid Res, 2010, 51(5): 879-891.
DOI: 10.1194/jlr.R001347.
[4]
Kenchegowda S, Bazan HE. Significance of lipid mediators in
[5]
He L, Hannon GJ. MicroRNAs: small RNAs with a big role
in gene regulation. Nat Rev Genet, 2004, 5(7): 522-531. DOI:
corneal injury and repair. J Lipid Res, 2010, 51(5): 879-891.
DOI: 10.1194/jlr.R001347.
10
1038/nrg1379.
[5]
He L, Hannon GJ. MicroRNAs: small RNAs with a big role
in gene regulation. Nat Rev Genet, 2004, 5(7): 522-531. DOI:
[6]
Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and
10
1038/nrg1379.
function. Cell, 2004, 116(2): 281-297.
[6]
Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and
[7]
Funari VA, Winkler M, Brown J, et al. Differentially expressed
function. Cell, 2004, 116(2): 281-297.
wound healing-related microRNAs in the human diabetic
cornea. PLoS One, 2013, 8(12): e84425. DOI: 10.1371/journal.
[7]
Funari VA, Winkler M, Brown J, et al. Differentially expressed
pone.0084425.
wound healing-related microRNAs in the human diabetic
cornea. PLoS One, 2013, 8(12): e84425. DOI: 10.1371/journal.
[8]
Lin D, Halilovic A, Yue P, et al. Inhibition of miR-205 impairs
pone.0084425.
the wound-healing process in human corneal epithelial cells by
[8]
Lin D, Halilovic A, Yue P, et al. Inhibition of miR-205 impairs
targeting KIR4.1 (KCNJ10). Invest Ophthalmol Vis Sci, 2013,
the wound-healing process in human corneal epithelial cells by
54
(9): 6167-6178. DOI: 10.1167/iovs.12-11577.
[9]
Winkler MA, Dib C, Ljubimov AV, et al. Targeting miR-146a to
targeting KIR4.1 (KCNJ10). Invest Ophthalmol Vis Sci, 2013,
54
(9): 6167-6178. DOI: 10.1167/iovs.12-11577.
treat delayed wound healing in human diabetic organ-cultured
corneas. PLoS One, 2014, 9(12): e114692. DOI: 10.1371/journal.
[9]
Winkler MA, Dib C, Ljubimov AV, et al. Targeting miR-146a to
pone.0114692.
treat delayed wound healing in human diabetic organ-cultured
corneas. PLoS One, 2014, 9(12): e114692. DOI: 10.1371/journal.
[10]
An J, Chen X, Chen W, et al. MicroRNA expression profile
pone.0114692.
and the role of miR-204 in corneal wound healing. Invest
Ophthalmol Vis Sci, 2015, 56(6): 3673-3683. DOI: 10.1167/
[10]
An J, Chen X, Chen W, et al. MicroRNA expression profile
iovs.15-16467.
and the role of miR-204 in corneal wound healing. Invest
Ophthalmol Vis Sci, 2015, 56(6): 3673-3683. DOI: 10.1167/
[11]
袁一飞, 钱江, 张艳青. 还原型谷胱甘肽滴眼液促进翼状胬
iovs.15-16467.
肉术后角膜上皮修复. 眼视光学杂志, 2009, 11(5): 372-374.
DOI: 10.3760/cma.j.issn.1674-845X.2009.05.015.
[11]
袁一飞, 钱江, 张艳青. 还原型谷胱甘肽滴眼液促进翼状胬
[12]
Solomon A. Corneal complications of vernal keratoconjunctivitis.
Curr Opin Allergy Clin Immunol, 2015, 15(5): 489-494. DOI:
肉术后角膜上皮修复. 眼视光学杂志, 2009, 11(5): 372-374.
DOI: 10.3760/cma.j.issn.1674-845X.2009.05.015.
10
1097/ACI.0000000000000202.
[12]
Solomon A. Corneal complications of vernal keratoconjunctivitis.
Curr Opin Allergy Clin Immunol, 2015, 15(5): 489-494. DOI:
[13]
Alcalde I, Íñigo-Portugués A, Carreño N, et al. Effects of new
10
1097/ACI.0000000000000202.
biomimetic regenerating agents on corneal wound healing in an
[13]
Alcalde I, Íñigo-Portugués A, Carreño N, et al. Effects of new
biomimetic regenerating agents on corneal wound healing in an
experimental model of post-surgical corneal ulcers. Arch Soc
Esp Oftalmol, 2015, 90(10): 467-474. DOI: 10.1016/j.oftal.
experimental model of post-surgical corneal ulcers. Arch Soc
Esp Oftalmol, 2015, 90(10): 467-474. DOI: 10.1016/j.oftal.
20
15.04.006.
20
15.04.006.
[14]
Guo Y, Lu X, Wang H. Downregulation of miR-18a induces
[14]
Guo Y, Lu X, Wang H. Downregulation of miR-18a induces
CTGF and promotes proliferation and migration of sodium
CTGF and promotes proliferation and migration of sodium
hyaluronate treated human corneal epithelial cells. Gene, 2016,
59
1(1): 129-136. DOI: 10.1016/j.gene.2016.07.008.
hyaluronate treated human corneal epithelial cells. Gene, 2016,
59
1(1): 129-136. DOI: 10.1016/j.gene.2016.07.008.
[15]
Robinson PM, Chuang TD, Sriram S, et al. MicroRNA signature
[15]
Robinson PM, Chuang TD, Sriram S, et al. MicroRNA signature
in wound healing following excimer laser ablation: role of miR-
13
3b on TGFβ1, CTGF, SMA, and COL1A1 expression levels
in wound healing following excimer laser ablation: role of miR-
in rabbit corneal fibroblasts. Invest Ophthalmol Vis Sci, 2013,
13
3b on TGFβ1, CTGF, SMA, and COL1A1 expression levels
54
(10): 6944-6951. DOI: 10.1167/iovs.13-12621.
[16]
Muraleedharan CK, McClellan SA, Barrett RP, et al. Inactivation
in rabbit corneal fibroblasts. Invest Ophthalmol Vis Sci, 2013,
54
(10): 6944-6951. DOI: 10.1167/iovs.13-12621.
of the miR-183/96/182 cluster decreases the severity of
[16]
Muraleedharan CK, McClellan SA, Barrett RP, et al. Inactivation
pseudomonas aeruginosa-induced keratitis. Invest Ophthalmol
Vis Sci, 2016, 57(4): 1506-1517. DOI: 10.1167/iovs.16-19134.
of the miR-183/96/182 cluster decreases the severity of
pseudomonas aeruginosa-induced keratitis. Invest Ophthalmol
[17]
Wang Y, Zhao X, Wu X, et al. microRNA-182 mediates sirt1-
Vis Sci, 2016, 57(4): 1506-1517. DOI: 10.1167/iovs.16-19134.
[17]
Wang Y, Zhao X, Wu X, et al. microRNA-182 mediates sirt1-
induced diabetic corneal nerve regeneration. Diabetes, 2016,
65
(7): 2020-2031. DOI: 10.2337/db15-1283.
induced diabetic corneal nerve regeneration. Diabetes, 2016,
65
(7): 2020-2031. DOI: 10.2337/db15-1283.
[18]
Liu CY, Kao WW. Corneal epithelial wound healing. Prog Mol
Biol Transl Sci, 2015, 134: 61-71. DOI: 10.1016/bs.pmbts.2015.
[18]
Liu CY, Kao WW. Corneal epithelial wound healing. Prog Mol
Biol Transl Sci, 2015, 134: 61-71. DOI: 10.1016/bs.pmbts.2015.
05
002.
05
002.
[19]
Ziaei M, Greene C, Green CR. Wound healing in the eye:
[19]
Ziaei M, Greene C, Green CR. Wound healing in the eye:
Therapeutic prospects. Adv Drug Deliv Rev, 2018, 126: 162-
17
6. DOI: 10.1016/j.addr.2018.01.006.
Therapeutic prospects. Adv Drug Deliv Rev, 2018, 126: 162-
17
6. DOI: 10.1016/j.addr.2018.01.006.
[20]
Aomatsu K, Arao T, Abe K, et al. Slug is upregulated during
[20]
Aomatsu K, Arao T, Abe K, et al. Slug is upregulated during
wound healing and regulates cellular phenotypes in corneal
wound healing and regulates cellular phenotypes in corneal
epithelial cells. Invest Ophthalmol Vis Sci, 2012, 53(2): 751-
75
6. DOI: 10.1167/iovs.11-8222.
epithelial cells. Invest Ophthalmol Vis Sci, 2012, 53(2): 751-
75
6. DOI: 10.1167/iovs.11-8222.
[21]
Aomatsu K, Arao T, Sugioka K, et al. TGF-β induces sustained
[21]
Aomatsu K, Arao T, Sugioka K, et al. TGF-β induces sustained
upregulation of SNAI1 and SNAI2 through Smad and non-
upregulation of SNAI1 and SNAI2 through Smad and non-
Smad pathways in a human corneal epithelial cell line. Invest
Ophthalmol Vis Sci, 2011, 52(5): 2437-2443. DOI: 10.1167/
Smad pathways in a human corneal epithelial cell line. Invest
iovs.10-5635.
Ophthalmol Vis Sci, 2011, 52(5): 2437-2443. DOI: 10.1167/
iovs.10-5635.
[22]
Li Y, Zhang H, Li Y, et al. MiR-182 inhibits the epithelial to
[22]
Li Y, Zhang H, Li Y, et al. MiR-182 inhibits the epithelial to
mesenchymal transition and metastasis of lung cancer cells by
targeting the Met gene. Mol Carcinog, 2018, 57(1): 125-136.
mesenchymal transition and metastasis of lung cancer cells by
DOI: 10.1002/mc.22741.
targeting the Met gene. Mol Carcinog, 2018, 57(1): 125-136.
[23]
Yan D, Dong XD, Chen X, et al. Role of microRNA-182 in
DOI: 10.1002/mc.22741.
[23]
Yan D, Dong XD, Chen X, et al. Role of microRNA-182 in
posterior uveal melanoma: regulation of tumor development
posterior uveal melanoma: regulation of tumor development
through MITF, BCL2 and cyclin D2. PLoS One, 2012, 7(7):
e40967. DOI: 10.1371/journal.pone.0040967.
through MITF, BCL2 and cyclin D2. PLoS One, 2012, 7(7):
e40967. DOI: 10.1371/journal.pone.0040967.
[24]
Wang LH, Dong F, Reinach PS, et al. MicroRNA-182 suppresses
[24]
Wang LH, Dong F, Reinach PS, et al. MicroRNA-182 suppresses
HGF/SF-induced increases in retinal pigment epithelial cell
HGF/SF-induced increases in retinal pigment epithelial cell
proliferation and migration through targeting c-Met. PLoS One,
20
16, 11(12): e0167684. DOI: 10.1371/journal.pone.0167684.
proliferation and migration through targeting c-Met. PLoS One,
20
16, 11(12): e0167684. DOI: 10.1371/journal.pone.0167684.
[1]
林永, 杨汝森, 闫东升, 等. CD146在视网膜血管内皮细胞中的功能和机制 [J]. 中华眼视光学与视觉科学杂志, 2023, 25(5): 332-339.
[2]
林永, 杨汝森, 叶菊秀, 等. miR-135a/b靶向SIRT1调控葡萄膜黑色素瘤细胞的迁移 [J]. 中华眼视光学与视觉科学杂志, 2023, 25(2): 88-95.
[3]
林永, 李丽,刘琦, 等.. SIRT1对葡萄膜黑色素瘤细胞增殖的表观遗传调控 [J]. 中华眼视光学与视觉科学杂志, 2022, 24(5): 329-336.
[4]
赵云萍 金珊珊 刘琦 王教 王丽花 闫东升. DZNep靶向EZH2抑制葡萄膜黑色素瘤细胞增殖 [J]. 中华眼视光学与视觉科学杂志, 2020, 22(7): 492-499.
[5]
王丽花 赵云萍 金珊珊 王教 陈晓燕 闫东升. MicroRNA-96对人视网膜色素上皮细胞增殖与迁移的影响 [J]. 中华眼视光学与视觉科学杂志, 2020, 22(3): 185-191.
[6]
王教 王丽花 曹琼洁 赵云萍 陈林华. MicroRNA-199a-3p对脉络膜黑色素瘤细胞增殖的抑制作用 [J]. 中华眼视光学与视觉科学杂志, 2019, 21(8): 569-575.
[7]
曹琼洁,董菁,李丽,陈晓燕,王教,闫东升. microRNA-127抑制葡萄膜黑色素瘤细胞增殖的表观遗传调控研究 [J]. 中华眼视光学与视觉科学杂志, 2018, 20(9): 546-551.
[8]
李创,吕勇,赵宏. 可溶性鸟苷酸环化酶抑制剂对体外培养人胚胎眼巩膜成纤维细胞胶原合成的影响 [J]. 中华眼视光学与视觉科学杂志, 2016, 18(7): 415-419.
[9]
张夏茵,龙尔平,刘臻臻,吴晓航,林浩添. 生长因子与晶状体的正常发育 [J]. 中华眼视光学与视觉科学杂志, 2016, 18(10): 636-640.
[10]
蒋剑,夏晓波,张露. PGC-1α对人血管内皮细胞VEGF表达及管腔形成的调控 [J]. 中华眼视光学与视觉科学杂志, 2015, 17(5): 280-283.
[11]
张筠,程亚辉,闫志鹏,赵平. 纤溶酶对兔后发性白内障的抑制作用 [J]. 中华眼视光学与视觉科学杂志, 2013, 15(1): 41-44.
[12]
王晓琴,陈震,邢怡桥,龚文容,刘剑平. MiR-145对视网膜母细胞瘤Y79细胞增殖和凋亡的影响 [J]. 中华眼视光学与视觉科学杂志, 2012, 14(3): 153-156.
[13]
卢建民,李小磊,王会芳,宋秀君. 小鼠角膜基质细胞的KSFM培养基培养法 [J]. 中华眼视光学与视觉科学杂志, 2012, 14(2): 100-105.
[14]
张潇,董方田,于伟泓,戴荣平. 增殖性糖尿病视网膜病变玻璃体切除术后残留膜的转归分析 [J]. 中华眼视光学与视觉科学杂志, 2012, 14(10): 614-616.
[15]
高妍,李冰,宁晓玲,赵矩伟,马骎. 2型糖尿病患者眼表改变的临床分析 [J]. 中华眼视光学与视觉科学杂志, 2011, 13(4): 304-308.