中国医科大学学报

中国医科大学学报
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中国医科大学学报 ›› 2019, Vol. 48 ›› Issue (3): 230-235.doi: 10.12007/j.issn.0258-4646.2019.03.008

• 论著 • 上一篇    下一篇

miR-15b-5p靶向CDK4抑制脉络膜黑色素瘤细胞增殖

张晓楠, 冯浩, 白雪, 应曼曼, 孙胜男, 宁宏   

  1. 中国医科大学附属第一医院眼科, 沈阳 110001
  • 收稿日期:2018-11-15 出版日期:2019-03-30 发布日期:2019-03-06
  • 通讯作者: 宁宏 E-mail:xiaoxiao1998@21.cn.com
  • 作者简介:张晓楠(1992-),女,医师,硕士.
  • 基金资助:
    辽宁省自然科学基金(20180551171)

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

ZHANG Xiaonan, FENG Hao, BAI Xue, YING Manman, SUN Shengnan, NING Hong   

  1. Department of Ophthalmology, The First Hospital, China Medical University, Shenyang 110001, China
  • Received:2018-11-15 Online:2019-03-30 Published:2019-03-06

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 mRNA 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。

关键词: 脉络膜黑色素瘤, 细胞周期素依赖性激酶4, miR-15b-5p

Abstract: 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.

Key words: choroidal melanoma, CDK4, miR-15b-5p

中图分类号: 

  • R773.4
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