[1]夏侯梨,胡军华,吴碧,等.miR-17-5p对小梁网细胞外基质蛋白表达的调控作用及其机制[J].眼科新进展,2021,41(1):037-41.[doi:10.13389/j.cnki.rao.2021.0008]
　XIA Houli,HU Junhua,WU Bi,et al.miR-17-5p targets Smad3 to regulate extracellular matrix protein expression in trabecular meshwork cells[J].Recent Advances in Ophthalmology,2021,41(1):037-41.[doi:10.13389/j.cnki.rao.2021.0008]
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miR-17-5p对小梁网细胞外基质蛋白表达的调控作用及其机制/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
41卷
期数:
2021年1期
页码:
037-41
栏目:
实验研究
出版日期:
2021-01-05

文章信息/Info

Title:
miR-17-5p targets Smad3 to regulate extracellular matrix protein expression in trabecular meshwork cells
作者:
夏侯梨; 胡军华; 吴碧; 廖莹琳
338025　江西省新余市，新余市人民医院眼科（夏侯梨，胡军华，廖莹琳）；430014　湖北省武汉市，武汉市中心医院眼科（吴碧）
Author(s):
XIA Houli1; HU Junhua1; WU Bi2; LIAO Yinglin1
1.Department of Ophthalmology,Xinyu People’s Hospital,Xinyu 338025,Jiangxi Province,China
2.Department of Ophthalmology,Wuhan Central Hospital,Wuhan 430014， Hubei Province,China
关键词:
人眼小梁网细胞; miR-17-5p; 细胞外基质; Smad3
Keywords:
human trabecular meshwork cells;  miR-17-5p;  extracellular matrix;  Smad3
分类号:
R775
DOI:
10.13389/j.cnki.rao.2021.0008
文献标志码:
A
摘要:
目的　探讨miR-17-5p对小梁网细胞外基质（ECM）蛋白表达的调控作用及其机制。方法　收集原发性开角型青光眼（POAG）患者和正常小梁网组织，利用RT-PCR和Western blot分别检测miR-17-5p和Smad3表达；将人小梁网细胞（HTMCs）分为miR-17-5p mimics和miR-17-5p NC组，分别转染载有miR-17-5p mimics组和miR-17-5p NC的载体，Western blot检测HTMCs中ECM蛋白纤连蛋白（FN）、胶原蛋白I（CoL-I）、α-平滑肌肌动蛋白（α-SMA）的表达；靶基因预测库预测miR-17-5p靶基因，荧光素酶报告载体联合Western blot鉴定靶基因。HTMCs中共转染miR-17-5p mimics和Smad3过表达载体，Western blot检测各转染组细胞中Smad3、FN、CoL-I、α-SMA的蛋白表达。结果　正常小梁网组织中miR-17-5p表达量为1.16±0.11，高于POAG小梁网组织的0.19±0.04，差异有统计学意义（t=10.641,P<0.001）；正常小梁网组织中Smad3蛋白表达量为0.31±0.06，低于POAG小梁网组织的0.86±0.07，差异有统计学意义（t=8.105,P<0.001）。miR-17-5p mimics组HTMCs中FN、CoL-I和α-SMA蛋白表达均低于miR-17-5p NC组，差异均有统计学意义（均为P<0.001）；靶基因预测库预测及荧光素酶报告载体联合Western blot鉴定证实Smad3为miR-17-5p下游靶基因；miR-17-5p+Smad3转染组HTMCs中Smad3、FN、CoL-I和α-SMA蛋白表达均高于miR-17-5p+vector转染组，差异均有统计学意义(均为P<0.001)。结论　miR-17-5p 能抑制HTMCs的ECM蛋白表达，这一过程可能是通过靶向抑制Smad3表达而实现的。
Abstract:
Objective　To investigate the regulatory effect of miR-17-5p on the expression of extracellular matrix protein in trabecular meshwork and its possible mechanism.Methods　Trabecular meshwork tissues were collected from patients with primary open angle glaucoma (POAG) and those with no history of eye disease. RT-PCR and Western Blot were used to detect miR-17-5p and Smad3 expression. Human trabecular meshwork cells (HTMCs) were divided into miR-17-5p mimics group and miR-17-5p NC group, and transfected with plasmids carrying miR-17-5p mimics group and miR-17-5p NC. Western blot were used to detect HTMCs extracellular matrix (ECM) related proteins such as fibronectin (FN), collagen I (CoL-I), Laminin. The target gene prediction library predicted the miR-17-5p target gene, and the luciferase reporter vector combined with Western blot identified the target gene; HTMCs were co-transfected with miR-17-5p mimics and Smad3 overexpression vectors. Western blot detected the expression of Smad3, FN, CoL-I, α-SMA in each transfection group.Results　The expression level of miR-17-5p in normal trabecular meshwork tissue was 1.16±0.11, which was higher than that in POAG trabecular meshwork tissue (0.19±0.04), and the difference was statistically significant (t=10.641, P<0.001). The expression of Smad3 protein in normal trabecular meshwork tissue was 0.31±0.06, which was lower than that in POAG trabecular meshwork tissue (0.86±0.07), and the difference was statistically significant (t=8.105, P<0.001). The protein expression of FN, CoL-I and α-SMA in HTMCs of miR-17-5p mimics group was lower than that of miR-17-5p NC group, and the difference was statistically significant (all P<0.001). Target gene prediction library prediction and luciferase reporter vector combined with Western blot identification confirmed that Smad3 was the downstream target gene of miR-17-5p.The expression of Smad3, FN, CoL-I and α-SMA protein in HTMCs of miR-17-5p + Smad3 transfection group was higher than that of miR-17-5p + vector transfection group, and the difference was statistically significant (all P<0.001).Conclusion　miR-17-5p can inhibit the expression of HTMCs extracellular matrix, and this process may be achieved by targeted inhibition of Smad3 expression.

参考文献/References:

［1］　De VIENCE E,MCMILLAN B D,GROSS R L.Screening for primary open-angle glaucoma(POAG)［J］.Int Ophthalmol Clin，2018,58(3):1-9.
［2］　孙瑞竹,张绍丹,梁远波.氧化应激对小梁网的损伤［J］.中华实验眼科杂志,2016,34(4):375-379.
SUN R Z，ZHANG S D，LIANG Y B.Oxidative injury on trabecular meshwork ［J］.Chin J Exp Ophthalmol,2016,34(4):375-379.
［3］　WANG K,READ A T,SULCHEK T,ETHIER C R.Trabecular meshwork stiffness in glaucoma［J］.Exp Eye Res,2017,158:3-12.
［4］　HERRERA J,HENKE C A,BITTERMAN P B.Extracellular matrix as a driver of progressive fibrosis［J］.J Clin Invest,2018,128(1):45-53.
［5］　O’CALLAGHAN,JEFFRE Y,CASSIDY P S,HUMPHRIES P.Open-angle glaucoma:Therapeutically targeting the extracellular matrix of the conventional outflow pathway［J］.Expert Opin Ther Targets,2017,21(11):1037-1050.
［6］　ZHAO J,WANG S,ZHONG W,YANG B,SUN L X,SUN Y J,et al.Oxidative stress in the trabecular meshwork［J］.Int J Mol Med,2016,38(4):995-1002.
［7］　XU L Q,ZHANG Y,GUO R R,SHEN W C,YAN Q,WANG Q S,et al.HES1 promotes extracellular matrix protein expression and inhibits proliferation and migration in human trabecular meshwork cells under oxidative stress［J］.Oncotarget,2017,8(13):21818-21833.
［8］　WANG X Y,LI Z J,BAI J,SONG W Q,ZHANG F M.miR-17-5p regulates the proliferation and apoptosis of human trabecular meshwork cells by targeting phosphatase and tensin homolog［J］.Mol Med Rep,2019,19(4):3132-3138.
［9］　WANG W,DONG R L,GUO Y,HE J A,SHAO C P,YI P,et al.CircMTO1 inhibits liver fibrosis via regulation of miR and Smad7［J］.J Cell Mol Med,2019,23(8):5486-5496.
［10］　HASAN H F,ABDEL-RAFEI M K,GALAL S M.Diosmin attenuates radiation-induced hepatic fibrosis via boosting PPAR-γ expression and hampering miR-17-5p activated canonical Wnt/ β-catenin signaling［J］.Biochem Cell Biol,2017,95(3):400-414.
［11］　刘爱华,高美子,黄亮瑜,刘勋,苏睿虹,赵今稚,等.叉头框转录因子F2小发夹RNA对人眼小梁网细胞外基质蛋白表达的抑制作用［J］.中华实验眼科杂志,2019,37(6):405-410.
LIU A H,GAO M Z,HUANG L Y,LIU X,SU R H,ZHAO J Z,et al.Inhibitory effect of forkhead box transcription factor F2 small hairpin RNA on the expression of extracellular matrix proteins of human trabecular meshwork［J］.Chin J Exp Ophthalmol,2019,37(6):405-410.
［12］　KENNEDY S M,SHERIDAN C,KEARNS V R,BILIR E K,CHOUDHARY A.Thrombospondin-2 is up-regulated by TGFβ2 and increases fibronectin expression in human trabecular meshwork cells［J］.Exp Eye Res,2019,189:107820.
［13］　FARALLI J A,FILLA M S,PETERS D M.Role of fibronectin in primary open angle glaucoma［J］.Cells,2019,8(12):E1518.
［14］　SHEN W C,HUANG B Q,HE Y,SHI L K,YANG J.Long non-coding RNA RP11-820 promotes extracellular matrix production via regulating miR-3178/MYOD1 in human trabecular meshwork cells［J］.FEBS J,2020,287(5):978-990.
［15］　党晓洁,任梅,朱江,许治国,杨新光.氧化应激条件下miR-21对人眼小梁网细胞胞外基质蛋白表达的影响［J］.眼科新进展,2017,37(1):30-34.
DANG X J,REN M,ZHU J,XU Z G,YANG X G.Effects of miR-21 on the expression of extracellular matrix proteins in human trabecular meshwork cells under oxidative stress［J］.Rec Adv Ophthalmol,2017,37(1):30-34.
［16］　WANG Y Z,XU W J,WANG Y N,XU X P,LV S M,DONG X J.miR-17-5p promotes migration and invasion in breast cancer cells by repressing netrin 4［J］.Int J Clin Exp Pathol,2019,12(5):1649-1657.
［17］　YU F J,LU Z Q,HUANG K,WANG X D,XU Z Q,CHEN B C,et al.MicroRNA-17-5p-activated Wnt/β-catenin pathway contributes to the progression of liver fibrosis［J］.Oncotarget,2016,7(1):81-93.
［18］　PERVAN C L.Smad-independent TGF-β2 signaling pathways in human trabecular meshwork cells ［J］.Exp Eye Res,2017,158:137-145.
［19］　GOETSCH K P,NIESLER C U.The extracellular matrix regulates the effect of decorin and transforming growth factor beta-2 (TGF-β2) on myoblast migration［J］.Biochem Biophys Res Commun,2016,479(2):351-357.
［20］　李华.TGF-β/Smad信号转导通路与白内障发生的研究进展［J］.中华实验眼科杂志,2015,33(4):377-379.
LI H.Research progress of TGF-β/Smad signal transduction pathway and cataract occurrence［J］.Chin J Exp Ophthalmol,2015,33(4):377-379.
［21］　LU Z F,LI X H,XU Y F,CHEN M M,CHEN W,CHEN T,et al.microRNA-17 functions as an oncogene by downregulating Smad3 expression in hepatocellular carcinoma［J］.Cell Death Dis,2019,10(10):723.

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备注/Memo

备注/Memo:
2014年江西省科技支撑计划项目（编号：20142BBG70040）
更新日期/Last Update: 2021-01-05