MicroRNA-96对人视网膜色素上皮细胞增殖与迁移的影响

MicroRNA-96 Affects the Proliferation and Migration of Human Retinal Pigment Epithelial Cells

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和随机序列寡核苷酸链[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细胞明显多于转染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,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。

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 lipofectamine RNAiMAX 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.