摘要背景 作为人角膜上皮组织的主要细胞成分,角膜上皮细胞通过迁移、增生以及分化等生物学行为在角膜上皮组织的损伤修复中发挥重要作用.微小RNA(miRNA)是内源性表达的单链非编码RNA,参与多种生物活动的调控过程,研究报道miR-204在正常角膜上皮组织内呈高表达,但其生物学功能仍不清楚.目的 研究miR-204对角膜上皮细胞增生的调控及其分子机制.方法 收集角膜屈光手术过程中患者脱落的角膜上皮组织,同时体外培养人角膜上皮细胞株(HCECs).采用实时荧光定量PCR技术分别检测角膜上皮组织和HCECs中miR-204的表达情况.将培养的HCECs分为3个组,分别用含miR-204 mimic的脂质体或空脂质体转染到HCECs内作为miR-204 mimic转染组和阳性对照组,正常培养的细胞作为正常对照组.用平板克隆形成试验检测各组培养细胞的克隆数以评价细胞的增生能力;采用流式细胞仪检测不同细胞周期的细胞百分比;采用Western blot技术检测各组细胞中磷酸化细胞内细胞周期相关蛋白p-RB、转录因子E2F1、p27、细胞周期蛋白CyclinA、细胞周期蛋白依赖性激酶CDC2、磷酸化CDC2 (p-CDC2)、细胞周期蛋白依赖性激酶(CDK2)的表达情况.结果 miR-204 mRNA在正常人角膜上皮组织中的相对表达量为1.077 ±0.268,明显高于HCECs中的0.041±0.018,差异有统计学意义(t=7.700,P＜0.001).平板克隆形成试验显示miR-204mimic转染组细胞克隆数明显少于阳性对照组和空白对照组.流式细胞仪检测结果显示,miR-204 mimic转染组G1期细胞百分比为47.75％,明显高于阳性对照组的37.23％和空白对照组的40.72％.Western blot检测表明,miR-204 mimic转染组细胞中CDK2和p-CDC2蛋白的相对表达量明显低于阳性对照组,E2F1和P27蛋白的相对表达量明显高于阳性对照组,差异均有统计学意义(t=5.39、10.65、14.87、25.11,均P＜0.01).结论 正常的角膜上皮组织中miR-204的高表达可能参与角膜上皮细胞的非增生状态的维持.miR-204可上调E2F1和p27蛋白在HCECs中的表达,抑制复合物CDK2/CyclinA和p-CDC2/CyclinA的形成,使角膜上皮细胞停滞在G1期,从而抑制角膜上皮细胞的增生.

abstractsBackground As the main cellular constituent of corneal epithelium,corneal epithelial cells play critical roles in regulating and controlling the migration, proliferation and differentiation of cells during the repair of damage.MicroRNA (miRNA) is endogenously expressed small non-coding RNAs, which participates in a variety of biological processes.Previous studies demonstrated that miR-204 is highly expressed in normal corneal epithelium,but its function is still unclear.Objective This study was to investigate the function and mechanism of miR-204 in corneal epithelial cell proliferation.Methods Corneal epithelial tissue was collected during the corneal refractive surgery from the patients with refractive error under the informed consent, and human corneal epithelial cells(HCECs) were cultured and passaged.The relative expressing levels of miR-204 mRNA in the normal corneal epithelium and HCECs were detected by real time quantitative PCR.Cultured HCECs were evenly divided into three groups.The liposome with miR-204 mimic was transfected into the cells of the miR-204 mimic group, and blank liposome was transfected in the cells of the positive control group,and regularly cultured cells served as the normal control group.Cell proliferation capability was evaluated by colony-forming assay, and the percentage of the cells in different cell cycles was analyzed by flow cytometry.Western blot assay was employed to detect the expression levels of p-RB, E2F1 ,p27,CyclinA, CDC2, p-CDC2 and CDK2 proteins in the cells.Results The relative expression levels of miR-204 mRNA were 1.077 ±0.268 in the normal corneal epithelium and 0.041 ±0.018 in the HCECs, showing a significant difference between them (t =7.700,P＜0.001).The cloning cell number was evidently decreased in the miR-204 mimic group in comparison with the positive control group and normal control group.The percentage of cells in the G1 phase was 47.75％ in the miR-204 mimic group, which was significantly higher than 37.23％ in the positive control group and 40.72％ in the normal control group.The expression levels of E2F1 and p27 proteins in the cells were elevated (t=14.87,25.11;both at P＜0.01) and those of CDK2 and p-CDC2 proteins were decreased (t=5.39,10.65;both at P＜0.01) in the miR-204 mimic group in comparison with the positive control group.Conclusions The overexpression of miR-204 in the normal corneas probably is associated with non-proliferation status of corneal epithelial cells.Transfection of miR-204 into corneal epithelial cells can inhibit the proliferation of corneal epithelial cells probably by up-regulating the expression of E2F1 and p27 and suppressing the expression of CDK2/CyclinA and p-CDC2/CyclinA,which lead to cell arrest in G1 phase.