摘要
目的探讨糖尿病性白内障患者晶状体上皮细胞(LEC)高表达的miR-124在糖尿病性白内障病理反应中的作用及机制。方法收集糖尿病性白内障患者与健康捐献眼的晶状体前囊膜,并通过高通量测序技术对晶状体前囊膜中的miRNA进行检测。使用EdgeR,利用F检验筛选差异表达miRNA。使用RegRNA检测差异表达miRNA和靶基因的结合关系以及结合位点,并进一步使用qRT-PCR验证miR-124和Bcl-2表达量的相关性。采用人LEC细胞分别转染dsControl、miR-124 inhibitor、dsBcl-2-640、miR-124 inhibitor+dsBcl-2-640,使用Western Blot检测Bcl-2表达量的变化;将各组细胞置于35.5 mmol·L^(-1)高糖环境中培养48 h,利用流式细胞术检测细胞凋亡率。结果糖尿病性白内障眼和健康对照眼miR-124丰度值分别为281208和64669(变化倍数为2.26,P<0.05),是上调最为显著的miRNAs之一。生物信息学分析显示miR-124可与Bcl-2启动子序列中相对于转录起始位点的核苷酸-645处序列结合。qRT-PCR检测结果表明miR-124与Bcl-2表达量呈负相关性(r=-0.8729,P=0.0237)。与空白对照组相比,dsBcl-2-640组LEC细胞Bcl-2蛋白表达显著降低[(0.22±0.03)比(0.41±0.04),P<0.05],miR-124 inhibitor组显著升高[(0.91±0.09)比(0.41±0.04),P<0.05],而miR-124 inhibitor+dsBcl-2-640组无明显差异[(0.37±0.03)比(0.41±0.04),P>0.05]。与空白对照组相比,miR-124 inhibitor组LEC细胞凋亡率显著降低[(11.33±1.86)%比(25.81±2.33)%,P<0.05],dsBcl-2-640组显著增加[(32.30±4.54)%比(25.81±2.33)%,P<0.05],而miR-124 inhibitor+dsBcl-2-640组无明显差异[(23.20±4.30)%比(25.81±2.33)%,P>0.05]。结论糖尿病性白内障患者LEC高表达的miR-124可靶向Bcl-2启动子序列诱导LEC细胞凋亡。
Objective To investigate the role of miR-124 in lens epithelial cells(LECs)in the pathogenesis of diabetic cataract and its possible mechanism.Methods The anterior lens capsules were collected from diabetic cataractous patients and healthy donor eyes,and the miRNA expression was detected by high-throughput sequencing technology.The differentially expressed miRNAs were screened by F-test using EdgeR software.The RegRNA was used to predict the target genes of miRNAs and their binding sites.Furthermore,qRT-PCR was performed to verify the relationship between miR-124 and Bcl-2.Human LECs were transfected with dsControl,miR-124 inhibitor,dsBcl-2-640 and miR-124 inhibitor+dsBcl-2-640,respectively.The change in Bcl-2 expression was detected by Western blot.The cells in each group were exposed to 35.5 mmol·L^(-1) of high glucose for 48 hours,and the apoptosis rate was measured by flow cytometry.Results The abundance values of miR-124 were,respectively,281208 and 64669 in diabetic cataractous eyes and healthy control eyes(fold change:2.26,P<0.05).It was one of the most significantly upregulated miRNAs in the anterior lens capsules of diabetic cataractous patients.Bioinformatics analysis showed that miR-124 could bind to the nucleotide-645 relative to the transcription start site in the Bcl-2 promoter sequence.The qRT-PCR showed that miR-124 was negatively correlated with the expression of Bcl-2(r=-0.8729,P=0.0237).Compared with dsControl group,the expression of Bcl-2 protein decreased in dsBcl-2-640 group[(0.22±0.03)vs(0.41±0.04),P<0.05],but increased in miR-124 inhibitor group[(0.91±0.09)vs(0.41±0.04),P<0.05].However,there was no significant difference in Bcl-2 expression between miR-124 inhibitor+dsBcl-2-640 group and dsControl group[(0.37±0.03)vs(0.41±0.04),P>0.05].Compared with dsControl group,the apoptosis rate in miR-124 inhibitor group decreased[(11.33±1.86)%vs(25.81±2.33)%,P<0.05),but increased in dsBcl-2-640 group[(32.30±4.54)%vs(25.81±2.33)%,P<0.05].Nevertheless,no remarkable difference was found in a
作者
陈祎祎
彭建军
CHEN Yi-yi;PENG Jian-jun(Department of Ophthalmology,Puren Hospital Affiliated to Wuhan University of Science and Technology,Wuhan 430081,China)
出处
《南昌大学学报（医学版）》
2022年第1期9-15,共7页
Journal of Nanchang University：Medical Sciences
基金
湖北省卫生和计划生育委员会科研项目(WJ2016F013)。
