MicroRNA-96对人视网膜色素上皮细胞增殖与迁移的影响
王丽花 赵云萍 金珊珊 王教 陈晓燕 闫东升
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 325027
MicroRNA-96 Affects the Proliferation and Migration of Human Retinal Pigment Epithelial Cells
Lihua Wang, Yunping Zhao, Shanshan Jin, Jiao Wang, Xiaoyan Chen, Dongsheng Yan
State Key Laboratory of Ophthalmology, Optometry and Vision Science, Eye Hospital, Wenzhou Medical
University, Wenzhou 325027, China
摘要 目的:探讨MicroRNA-96(miR-96)对人视网膜色素上皮(RPE)细胞增殖和迁移的影响。方法:实验研究。通过RNA原位杂交检测人胚胎眼(20周)石蜡切片中RPE层miR-96的表达情况。将miR-96和阴性对照(NC)通过阳离子脂质体介导转染人眼RPE细胞,采用细胞增殖实验(MTS)、流式细胞术和Transwell实验分别检测细胞增殖、细胞周期以及细胞迁移能力。通过生物信息学及Western blot法确定miR-96作用的靶基因。应用Western blot检测miR-96对细胞增殖迁移相关信号通路蛋白(Akt、ERK)及细胞周期相关蛋白(p-Cdc2、CyclinD2、p-Rb)表达的影响。组间数据比较采用独立样本t检
验。结果:MiR-96在人眼RPE细胞中有表达。MTS结果显示,转染NC、miR-96后,人眼RPE细胞的相对增殖速率分别为100%、74%±2%,差异有统计学意义(t=42.174,P=0.002)。流式细胞术检测结果显示,转染miR-96后阻滞在G1期的RPE细胞显著多于转染NC后,且差异有统计学意义(t= -18.444,P=0.003)。Transwell实验结果显示,与转染NC相比,转染miR-96能显著抑制RPE细胞的迁移,差异具有统计学意义(t=6.754,P=0.002)。进而,明确了MITF是miR-96作用的靶基因。Western blot检测结果显示,转染miR-96后细胞中细胞周期相关蛋白p-Rb(t=11.211,P=0.002)、p-Cdc2(t=9.133, P=0.003)、CyclinD2(t=7.542,P=0.005)以及迁移相关信号通路蛋白p-ERK(t=16.699,P<0.001),p-Akt
(t=23.552,P<0.001)的表达水平均降低。结论:miR-96通过作用于靶基因MITF,并调控细胞周期和迁移相关蛋白的表达从而抑制人RPE细胞的增殖和迁移。
关键词 :
视网膜色素上皮 ,
microRNA-96 ,
细胞增殖 ,
细胞迁移 ,
MITF
Abstract :Objective: To investigate the effects of microRNA-96 (miR-96) on the proliferation and migration of
human retinal pigment epithelial (RPE) cells. Methods: In this experimental research, the expression of miR-96 was detected in the RPE layer in paraffin sections of the human embryonic eye (20 weeks) by RNA in situ hybridization. MiR-96 or a negative control (NC) was transfected into RPE cells by a lipofectamineRNAiMAX reagent. MTS, flow cytometry, and transwell assays were used to evaluate the effects of miR-96 on cell proliferation, cell cycle, and cell migration. The target gene of miR-96 was predicted by bioinformatics and verified by Western blot. The expression of cell cycle-related proteins in RPE cells was detected by Western blot analysis. Data were analyzed by an independent t test. Results: MiR-96 was highly expressed in human RPE cells. MTS assay showed that the proliferation of RPE cells was inhibited by miR-96 (t=42.174, P=0.002). Flow cytometry revealed that miR-96 resulted in a G1 phase arrest in RPE cells (t=-18.444, P=0.003). Transwell assay also revealed that miR-96 inhibited the migration of RPE cells (t=6.754, P=0.002). Furthermore, MITF was identified as the target gene of miR-96. In addition, the expression of cell cycle-related proteins p-Cdc2, CyclinD2, p-Rb, as well as p-Akt and p-ERK proteins, had significantly decreased in RPE cells transfected with miR-96 (p-Rb: t=11.211, P=0.002; p-Cdc2: t=9.133, P=0.003; CyclinD2: t=7.542, P=0.005; p-ERK: t=16.699, P<0.001; p-Akt: t=23.552, P<0.001). Conclusions: MiR-96 inhibits human RPE cell proliferation and migration by targeting MITF and regulating the expression of cell cycle and migration-related proteins.
Key words :
retinal pigment epithelium
microRNA-96
cell proliferation
cell migration
MITF
收稿日期: 2019-05-17
基金资助: 浙江省自然科学基金(LQ17H120009,LQ15H160014)
通讯作者:
闫东升(ORCID:0000-0003-0551-2219),Email:dnaprotein@hotmail.com
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