[1]李静,李迪,李雪颖,等.上调microRNA-27b对血小板衍生因子诱导下的人视网膜色素上皮细胞增殖的抑制作用[J].眼科新进展,2020,40(9):822-825.[doi:10.13389/j.cnki.rao.2020.0187]
　LI Jing,LI Di,LI Xueying,et al.Up-regulation of microRNA-27b inhibits PDGF-induced cell proliferation in ARPE-19 cells[J].Recent Advances in Ophthalmology,2020,40(9):822-825.[doi:10.13389/j.cnki.rao.2020.0187]
点击复制

上调microRNA-27b对血小板衍生因子诱导下的人视网膜色素上皮细胞增殖的抑制作用/HTML
分享到：

《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
40卷
期数:
2020年9期
页码:
822-825
栏目:
实验研究
出版日期:
2020-09-05

文章信息/Info

Title:
Up-regulation of microRNA-27b inhibits PDGF-induced cell proliferation in ARPE-19 cells
作者:
李静; 李迪; 李雪颖; 寇列玲; 车选义
710068　陕西省西安市，陕西省人民医院眼科（李静，李迪，李雪颖，车选义）；725000　陕西省安康市，安康市中医院（寇列玲）
Author(s):
LI Jing1; LI Di1; LI Xueying1; KOU Lieling2; CHE Xuanyi1
1.Department of Ophthalmology,Shaanxi Provincial People’s Hospital,Xi’an 710068,Shaanxi Province,China
2.Department of Ophthalmology,Ankang Hospital of Traditional Chinese Medicine,Ankang 725000,Shaanxi Province,China
关键词:
miR-27b; 人视网膜色素上皮细胞; 血小板衍生因子; 细胞周期; 细胞增殖
Keywords:
miR-27b;  human retinal pigment epithelium cell;  platelet-derived growth factor;  cell cycle;  cell proliferation
分类号:
R774.5
DOI:
10.13389/j.cnki.rao.2020.0187
文献标志码:
A
摘要:
目的　观察过表达的microRNA-27b(miR-27b)对血小板衍生因子（platelet-derived growth factor,PDGF）诱导下的人视网膜色素上皮细胞（ARPE-19）增殖的抑制作用，探讨miR-27b在ARPE细胞生物学行为中的作用及其调控机制。方法　将miR-27b过表达质粒或空载体转入ARPE-19细胞36 h后，用终浓度为20 μg·L-1的PDGF预处理ARPE-19细胞5 h。根据转染物不同，将实验分为空白对照组（control组）、miR-27b阴性对照组（miR-27b NC组）、miR-27b模拟物转染组（PDGF+mimics组）和空载体转染组（PDGF+NC组）。实时荧光定量PCR(qRT-PCR)检测转染后各组ARPE-19细胞中miR-27b的表达水平；MTT法测定各组细胞活性；通过流式细胞术评估各组细胞周期的分布变化；Western blot检测各组细胞周期的正向调控因子cyclinD1、CDK4和负向调控因子p21CIP1和p27KIP1的表达水平。结果　qRT-PCR检测结果显示：经PDGF处理5 h，与miR-27b NC组相比，PDGF+NC组miR-27b的表达显著降低（P<0.01）；与PDGF+NC组相比，PDGF+mimics组miR-27b的表达增加（P<0.01）。MTT检测结果显示：与miR-27b NC组相比，PDGF+NC组ARPE-19细胞的光密度（D）值增加(P<0.01)；而与PDGF+NC组相比，PDGF+mimics组可明显抑制ARPE-19细胞的D值(P<0.01)。流式细胞术检测结果显示：与空白对照组和miR-27b NC组相比，PDGF+NC组G0/G1期细胞的百分比显著降低，S期细胞的百分比增加（P<0.05）；而与PDGF+NC组相比，PDGF+mimics组miR-27b的过表达显著增加了G0/G1期细胞的百分比，并抑制了细胞增殖（均为P<0.05）。Western blot检测结果显示：与PDGF-NC组相比，PDGF+mimics组miR-27b的过表达可显著降低cyclinD1蛋白和CDK4蛋白的表达，同时增强了p21CIP1蛋白和p27KIP1蛋白的表达（均为P<0.05）。结论　上调miR-27b表达可抑制PDGF诱导的ARPE细胞增殖。
Abstract:
Objective　To observe the inhibitory effect of overexpressed miR-27b on the proliferation of human retinal pigment epithelial cells (ARPE-19) induced by platelet-derived growth factor (PDGF), and to explore the role and regulatory mechanism of miR-27b in the biological behavior of human RPE cells.Methods　After transferring miR-27b over-expression plasmid or empty body into ARPE-19 cells for 36 hours, ARPE-19 cells were pretreated with PDGF with final concentration of 20 μg·L-1 for 5 hours. According to the different transfection materials, the experiment was divided into control group, miR-27b negative control group (miR-27b NC group), miR-27b analog transfection group (PDGF+mimics group) and empty vector transfection group (PDGF+NC group). The expression level of miR-27b in ARPE-19 cells was detected by real-time quantitative PCR (qRT-PCR). The cell activity was measured by MTT and the distribution of cell cycle was evaluated by flow cytometry. Western blot was used to detect the expression levels of CyclinD1, CDK4, p21CIP1 and p27KIP1.Results　qRT-PCR showed that, 5 hours after PDGF pretreat, the expression of miR-27b in PDGF+NC group was significantly lower than that in miR-27b NC group (P<0.01), and PDGF+mimics group was significantly higher than PDGF+NC group (P<0.01). The results of MTT showed that the optical density (D) of ARPE-19 cells in PDGF + NC group was higher than that in miR-27b NC group (P<0.01), while PDGF+mimics group significantly inhibited the D value when compared with PDGF+NC group (P<0.01). The results of flow cytometry showed that compared with the control group and miR-27b NC group, the percentage of G0/G1 phase cells in PDGF+NC group decreased significantly, while the percentage of S phase cells increased (P<0.05). Compared with PDGF+NC group, over expression of miR-27b in PDGF+mimics group significantly increased the proportion of cells in G0/G1 phase and inhibited cell proliferation (all P<0.05). Western blot showed that the overexpression of miR-27b in PDGF+mimics group significantly decreased the expression of cyclin D1 and CKD4, and enhanced the expression of p21CIP1 and p27KIP1 compared with PDGF+NC group (all P<0.05).Conclusion　Up-regulation of miR-27b expression can inhibit PDGF induced proliferation of ARPE cells.

参考文献/References:

［1］SIMO R,VILLARREL M,CORRALIZA L,HERNANDEZ C,GARCIA-RAMIREZ M.The retinal pigment epithelium:something more than a constituent of the blood-retinal barrier—implications for the pathogenesis of diabetic retinopathy［J］.J Biomed Biotechnol,2010,17(2):1-15.
［2］　FUHRMANN S,ZOU C,LEVINE E M.Retinal pigment epithelium development,plasticity,and tissue homeostasis［J］.Exp Eye Res,2014,123(6):141-150.
［3］　CHARTERIS D G,SETHI C S,LEWIS G P,FISHER S K.Proliferative vitreoretinopathy-developments in adjunctive treatment and retinal pathology［J］.Eye(Lond),2002,16(4):369-374.
［4］　HE S,KUMAR S R,ZHOU P,KRASNOPEROV V,RYAN S J,GILL P S,et al.Soluble EphB4 inhibition of PDGF-induced RPE migration in vitro［J］.Invest Ophthalmol Vis Sci,2010,51(1):543-552.
［5］　HAMMOND S M.An overview of microRNAs［J］.Adv Drug Deliv Rev,2015,87(1):3-14.
［6］　WAHID F,KHAN T,KIM Y Y.MicroRNA and diseases:therapeutic potential as new generation of drugs［J］.Biochimie,2014,104:12-26.
［7］　AYAZ L,DINC E.Evaluation of microRNA responses in ARPE-19 cells against the oxidative stress［J］.Cutan Ocul Toxicol,2018,37(2):121-126.
［8］　CHANG J H,KANG S W,HAM D I.Sensitivity of CD95-induced apoptosis in different proliferative status of human retinal pigment epithelial cells［J］.Korean J Ophthalmol,2001,15(2):74-80.
［9］　KUZNETSOVA A V,KURINOV A M,ALEKSANDROVA M A.Cell models to study regulation of cell transformation in pathologies of retinal pigment epithelium［J］.J Ophthalmol,2014,1：1-18.
［10］　ROBBINS S G,MIXON R N,WILSON D J,HART C E,ROBERTON J E,WESTRA I,et al.Platelet-derived growth factor ligands and receptors immunolocalized in proliferative retinal diseases［J］.Invest Ophthalmol Vis Sci,1994,35(10):3649-3463.
［11］　LI R,MAMINISHKIS A,WANG F E,MILLER S S.PDGF-C and -D induced proliferation/migration of human RPE is abolished by inflammatory cytokines［J］.Invest Ophthalmol Vis Sci,2007,48(12):5722-5732.
［12］　CHAKRABORTY C,DOSS C G,BANDYOPADHYAY S,AGORAMOORTHY G.Influence of miRNA in insulin signaling pathway and insulin resistance:micro-molecules with a major role in type-2 diabetes［J］.Wiley Interdiscip Rev RNA,2014,5(5):697-712.
［13］　WAN L,ZHANG L,FAN K,WANG J.MiR-27b targets LIMK1 to inhibit growth and invasion of NSCLC cells［J］.Mol Cell Biochem,2014,390(1-2):85-91.
［14］　JIN L,WESSELY O,MARCUSSON E G,IVAN C,CALIN G A,ALAHARI S K.Prooncogenic factors miR-23b and miR-27b are regulated by Her2/Neu,EGF,and TNF-α in breast cancer［J］.Cancer Res,2013,73(9):2884-2896.
［15］　OHANA R,WEIMAN-KELMAN B,RAVIV S,TAMM E R,PASMANIK-CHOR M,RINON A,et al.MicroRNAs are essential for differentiation of the retinal pigmented epithelium and maturation of adjacent photoreceptors［J］.Development,2015,142(14)：2487-2498.
［16］　SUNDERMEIER T R,PALCZEWSKI K.The impact of microRNA gene regulation on the survival and function of mature cell types in the eye［J］.FASEB J,2016,30(1):23-33.
［17］　SANTAMARIA D,ORTEGA S.Cyclins and CDKS in development and cancer:lessons from genetically modified mice［J］.Front Biosci,2006,11(10):1164-1188.
［18］　TANE S,IKENISHI A,OKAYAMA H,IWAMOTO N,NAKAYAMA K I,TAKEUCHI T.CDK inhibitors,p21(Cip1)and p27(Kip1),participate in cell cycle exit of mammalian cardiomyocytes［J］.Biochem Biophys Res Commun,2014,443(3):1105-1109.
［19］　VELICEASA D,BIYASHEV D,QIN G,MISENER S,MACKIE AR,KISHORE R,et al.Therapeutic manipulation of angiogenesis with miR-27b［J］.Vasc Cell,2015,7(1):6.
［20］　LIU H T,XING A Y,CHEN X,MA R R,WANG Y W,SHI D B,et al.MicroRNA-27b,microRNA-101 and microRNA-128 inhibit angiogenesis by down-regulating vascular endothelial growth factor C expression in gastric cancers［J］.Oncotarget,2015,6(35):37458-37470.
［21］　LUDWIG N,LEIDINGER P,BECKER K,BACKES C,FEHLMANN T,PALLASCH C,et al.Distribution of miRNA expression across human tissues［J］.Nucleic Acids Res,2016,44(8):3865-3877.

相似文献/References:

[1]司艳芳 王君 关娟 韩泉洪 惠延年.α-平滑肌肌动蛋白在PVR增生膜中的表达以及PDGF对其在人RPE细胞中表达的影响[J].眼科新进展,2012,32(11):000.
[2]李姣 王兴荣 崔彦 刘正峰 王影 李洋.雷珠单抗选择性体外抑制睫状体恶性黑色素瘤[J].眼科新进展,2013,33(11):000.
[3]俞永珍,徐哲,邹秀兰,等. 蓝光诱导氧化应激反应参与视网膜色素上皮细胞凋亡机制研究[J].眼科新进展,2015,35(6):520.[doi:10.13389/j.cnki.rao.2015.0141]
　YU Yong-Zhen,XU Zhe,ZOU Xiu-Lan,et al. Role of oxidative stress induced by blue light in human retinal pigment epithelium cells apoptosis[J].Recent Advances in Ophthalmology,2015,35(9):520.[doi:10.13389/j.cnki.rao.2015.0141]
[4]彭素芬,韩泉洪,李丽华,等.LED光源对人视网膜色素上皮细胞分泌单核细胞趋化因子-1和白细胞介素-8的影响[J].眼科新进展,2016,36(3):201.[doi:10.13389/j.cnki.rao.2016.0055]
　PENG Su-Fen,HAN Quan-Hong,LI Li-Hua,et al.Effects of light emitting diode on expression of MCP-1 and IL-8 in cultured human retinal pigment epithelial cells[J].Recent Advances in Ophthalmology,2016,36(9):201.[doi:10.13389/j.cnki.rao.2016.0055]
[5]张照华,杨玲玲,张盼盼,等.贝伐单抗对视网膜色素上皮细胞抗氧化功能的影响[J].眼科新进展,2017,37(4):305.[doi:10.13389/j.cnki.rao.2017.0077]
　ZHANG Zhao-Hua,YANG Ling-Ling,ZHANG Pan-Pan,et al.Effects of bevacizumab on antioxidative function in human retinal pigment epithelial cells[J].Recent Advances in Ophthalmology,2017,37(9):305.[doi:10.13389/j.cnki.rao.2017.0077]
[6]赵洁文,李明新.康柏西普联合PDGF受体抑制剂对缺氧条件下视网膜色素上皮细胞增殖、迁移及VEGF表达的影响[J].眼科新进展,2017,37(12):1127.[doi:10.13389/j.cnki.rao.2017.0284]
　ZHAO Jie-Wen,LI Ming-Xin.Effects of conbercept combined with PDGF receptor inhibitor on the proliferation，migration of RPE cells and VEGF expression in hypoxic conditions[J].Recent Advances in Ophthalmology,2017,37(9):1127.[doi:10.13389/j.cnki.rao.2017.0284]
[7]张盼盼,董琪,华英彬,等.贝伐单抗对人视网膜色素上皮细胞形态、凋亡率及凋亡相关因子表达的影响[J].眼科新进展,2018,38(4):314.[doi:10.13389/j.cnki.rao.2018.0074]
　ZHANG Pan-Pan,DONG Qi,HUA Ying-Bin,et al.Effects of bevacizumab on morphology and apoptosis of human retinal pigment epithelial cells as well as the expression of apoptosis-related factors[J].Recent Advances in Ophthalmology,2018,38(9):314.[doi:10.13389/j.cnki.rao.2018.0074]
[8]邱梅园,丁芝祥,廖妙云,等.表没食子儿茶素没食子酸酯对视网膜色素上皮细胞增殖及相关因子表达的影响[J].眼科新进展,2018,38(7):638.[doi:10.13389/j.cnki.rao.2018.0150]
　QIU Mei-Yuan,DING Zhi-Xiang,LIAO Miao-Yun,et al.Effects of epigallocatechin gallate on proliferation of retinal pigment epithelium cells and the expression of P21，P27，PDGFR，NF-κB and IκB[J].Recent Advances in Ophthalmology,2018,38(9):638.[doi:10.13389/j.cnki.rao.2018.0150]
[9]孙凯,刘丽娟.雷公藤甲素对人视网膜色素上皮细胞氧化损伤的抑制作用[J].眼科新进展,2018,38(12):1133.[doi:10.13389/j.cnki.rao.2018.0267]
　SUN Kai,LIU Li-Juan.Inhibition effects of triptolide on human retinal pigment epithelium cells against oxidative damage[J].Recent Advances in Ophthalmology,2018,38(9):1133.[doi:10.13389/j.cnki.rao.2018.0267]
[10]崔铮,靖鹏举,朱冬梅,等.地塞米松对脂多糖诱导人视网膜色素上皮细胞炎症损伤及NLRP3/Caspase-1通路的影响[J].眼科新进展,2020,40(9):826.[doi:10.13389/j.cnki.rao.2020.0188]
　CUI Zheng,JING Pengju,ZHU Dongmei,et al.Effects of dexamethasone on lipopolysaccharide-induced inflammatory damage of human retinal pigment epithelial cells and NLRP3/Caspase-1 pathway[J].Recent Advances in Ophthalmology,2020,40(9):826.[doi:10.13389/j.cnki.rao.2020.0188]

备注/Memo

备注/Memo:
陕西省重点研发计划项目（编号：2018SF-155）
更新日期/Last Update: 2020-09-05