miR-15b-5p靶向CDK4抑制脉络膜黑色素瘤细胞增殖  

miR-15b-5p Inhibits Choroid Melanoma Cell Proliferation by Targeting CDK4

作　　者：张晓楠 冯浩[1] 白雪[1] 应曼曼 孙胜男 宁宏[1] ZHANG Xiaonan;FENG Hao;BAI Xue;YING Manman;SUN Shengnan;NING Hong(Department of Ophthalmology,The First Hospital,China Medical University,Shenyang 110001,China)

机构地区：[1]中国医科大学附属第一医院眼科,沈阳110001

出　　处：《中国医科大学学报》2019年第3期230-235,共6页Journal of China Medical University

基　　金：辽宁省自然科学基金(20180551171)

摘　　要：目的探讨miR-15b-5p通过靶向抑制细胞周期素依赖性激酶4 (CDK4)的表达进而发挥负向调节脉络膜黑色素瘤细胞增殖的作用。方法使用荧光素酶报告分析检测miR-15b-5p与CDK4结合情况。体外培养人眼侵袭性脉络膜黑色素瘤细胞系MUM-2B,分别转染negative control RNA、miR-15b-5p mimics、inhibitor nc RNA和miR-15b-5p inhibitor;采用实时定量PCR检测转染后miR-15b-5p的表达水平;采用Western blotting检测4组细胞中CDK4蛋白的表达量;采用CCK8检测4组细胞的增殖能力;采用流式细胞术检测4组细胞周期情况。结果荧光素酶报告基因分析验证miR-15b-5p能够与CDK4 m RNA 3’UTR结合。与阴性对照组相比,mimics组miR-15b-5p表达显著增加(t=25.01,P <0.000 1),CDK4的蛋白表达水平降低,细胞增殖能力降低,细胞G1期比例增高。而与inhibitor nc组相比,inhibitor组miR-15b-5p表达减少,CDK4表达水平升高,细胞增殖能力增强,细胞G1期比例降低。结论miR-15b-5p能够靶向抑制CDK4的表达,造成肿瘤细胞发生G1期阻滞,从而有效抑制脉络膜黑色素瘤细胞增殖。Objective To explore the inhibitory effects of miR-15b-5p on choroid melanoma cell line proliferation by targeting CDK4. Methods Dual-luciferase assay was used to verify the direct binding site between miR-15b-5p and CDK4 3’-UTR. MUM-2B cells were cultured in vitro and transfected with negative control RNA,miR-15b-5p mimics,inhibitor normal control( nc) RNA,and miR-15b-5p inhibitor. qRT-PCR was used to detect miR-15b-5p expression,Western blotting was used to measure the expression levels of CDK4 in the cells,CCK-8 assay was used to detect proliferation capacity,and flow cytometry was used to detect cell cycle. Results Dual-luciferase assay verified that miR-15b-5p could bind to CDK4 mRNA 3’-UTR successfully. Compared to the negative control group,the mimics group showed significantly increased miR-15b-5p expression,decreased CDK4 levels,decreased cell proliferation rate,and increased proportion of G1-phase cells. Compared to the inhibitor nc group,the inhibitor group showed significantly decreased miR-15b-5p expression( t = 25.01,P < 0.000 1),increased CDK4 protein level,increased cell proliferation rate,and decreased proportion of G1-phase cells. Conclusion miR-15b-5p can target CDK4,induce G1 phase arrest in cells,and thus,reduce the proliferation rate of choroid melanoma cells.

关 键 词：脉络膜黑色素瘤 细胞周期素依赖性激酶4 miR-15b-5p 

分 类 号：R773.4[医药卫生—眼科]