miR-182通过靶向调节细胞周期蛋白依赖性激酶1促进由氧化应激诱导的HLE-B3细胞凋亡  

miR-182 inhibited oxidative stress induce apoptosis in HLEB-3 cells by targeting cyclin dependent kinase 1

作　　者：马宁 朱琳[2] 杨沚涵 盖佳鑫 房春来 Ma Ning;Zhu Lin;Yang Zhihan;Gai Jiaxin;Fang Chunlai(Department of Ophthalmology,Harbin Ophthalmology Hospital,Harbin 150081,China)

机构地区：[1]哈尔滨市眼科医院眼科,150080 [2]哈尔滨医科大学,150081 [3]哈尔滨医科大学附属第四医院眼科房,150001

出　　处：《国际遗传学杂志》2018年第6期459-464,共6页International Journal of Genetics

基　　金：哈尔滨医科大学创新科学研究(2017lczx101);黑龙江卫生计生委科研课题(2017-140);黑龙江省自然科学基金(H2017009).

摘　　要：目的探讨miR-182在氧化应激诱导下的人晶状体上皮细胞(human lens epithelium cells,HLE-B3)凋亡引起白内障发生时的保护作用。方法通过实时定量PCR(realtime quantitative polymerase chain reaction,qRT-PCR)检测由H2O2(100μmol/L,12h)刺激的HLE-B3细胞中miR-182的表达。通过qRT-PCR和Western印迹检测由H2O2刺激HLE-B3细胞中细胞周期蛋白依赖性激酶1(cyclin dependent kinase1,CDK1)的表达。同时通过qRT-PCR检测白内障患者和正常健康的患者晶状体前囊膜中miR-182和CDK1表达。通过生物信息学方法(targetscan和miRanda),预测miR-182潜在的靶基因,Luciferase报告方法确认miR-182的靶基因。通过Western印迹检测凋亡相关蛋白(Bax、Bad、caspase-3)及靶基因CDK1蛋白的变化。结果研究发现,miR-182在人白内障晶状体前囊膜样品和由H2O2诱导的HLE-B3细胞中相对于正常对照组表达上调,CDK1表达相对于正常对照组降低。过表达miR-182能够逆转由H2O2诱导的HLE-B3细胞凋亡。转染miR-182-inhibitor后CDK1在mRNA和蛋白水平表达上调。Luciferase报告检测结果显示CDK1是miR-182的直接靶基因。转染miR-182mimic12h后,氧化应激刺激12h,能够下调CDK1的表达,转染miR-182-inhibitor12h后,氧化应激刺激12h,能够上调CDK1的表达。而转染过表达miR-182后能够逆转由H2O2诱导的HLE-B3细胞中凋亡相关蛋白Bax、Bad和caspase-3下调。结论研究证明,miR-182是白内障的功能基因,能够逆转由氧化应激诱导的HLE-B3细胞凋亡,而这些功能是通过调控CDK1表达实现的。这些结果表明,miR-182/CDK1/Rb/P16INK4a的信号通路是一种新型的影响白内障的发生、发展的因素。Objective To investigate the protective mechanism of miRNA-182 on human lens epithelial cells (HLE-B3) apoptosis induced by oxidative stress during cataract formation. Methods Human lens epithelium cells (HLE-B3) were stimulated by H2O2(100 μmol/L, 12 h) and the expression of miR-182 was evaluated using qRT-PCR. The expression of CDK1 was determined by qRT-PCR and western blot. To confirm our hypothesis, we analyzed miR-182 and CDK1 gene expression in the lens anterior capsules of patients with and without cataracts by qRT-PCR. Luciferase report assay was used to confirm the target gene of miR-182. The changes in apoptosis related protein (Bax, Bad, caspase-3) and target gene CDK1 protein were detected by Western bloting. Results While CDK1 expression was suppressed, a significant increase was observed in the expression of miR-182 in both human cataract lens anterior capsular samples and HLE-B3 cell lines stimulated by H2O2 compared with normal control group. Over-expression of miR-182 significantly suppressed H2O2-induced HLE-B3 cell apoptosis in vitro, a critical step in cataract formation. The expression of miR-182 was also decreased in cataract lens anterior capsular tissues and H2O2-induced HLE-B3 cell lines. nockdown of miR-182 significantly up-regulated the expression of CDK1 at the level of mRNA and protein. Luciferase report assay showed CDK1 was a direct target gene of miR-182. After 12 hours of pre-transfection of miR-182 followed by another 12 hours of oxidative stress stimulation, the expression of CDK1 was down-regulated. In contrast, the expression of CDK1 was promoted by miR-182-inhibitor in the same condition. The transfection of miR-182 could reverse apoptosis process in H2O2 induced HLE-B3 cells through down-regulated of expressions of apoptosis-related proteins Bax, Bad and caspase-3. Conclusion We demonstrated for the first time that miR-182 is a functional gene of cataract and knockdown of miR-182 reduced the expression of CDK1. These results suggest that miR-182/CDK1/Rb/P16INK4a signaling

关 键 词：白内障 氧化应激 MIR-1 82 CDK1 细胞凋亡 

分 类 号：R776.1[医药卫生—眼科]