上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

miRNA-449对SH-SY5Y细胞增殖活性的抑制作用

王丹   

  1. 第二军医大学 基础部神经生物学教研室， 上海 200433
  • 出版日期:2016-04-28 发布日期:2016-05-26
  • 作者简介:王丹（1980—）， 女， 助理实验师, 学士; 电子信箱: dannay08@163.com。

Inhibitory effect of miRNA-449 on proliferation activity of SH-SY5Y cells

WANG Dan   

  1. Departments of Neurobiology， The Second Military Medical University, Shanghai 200433, China
  • Online:2016-04-28 Published:2016-05-26

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摘要：

目的 寻找并验证miRNA-449的靶基因，探讨miRNA-449调控SH-SY5Y细胞增殖活性的分子机制。方法 检测神经母细胞瘤及其癌旁组织中的miRNA-449及NOTCH1蛋白的表达并分析其相关性；生物信息学预测miRNA-449与NOTCH1基因3′UTR区的结合靶位，并通过荧光素酶报告基因法进行验证；化学合成miRNA-449模拟物、 miRNA-449抑制剂及miRNA-449错义序列,经脂质体转染至SH-SY5Y细胞，转染后48 h，real-time PCR检测细胞中miRNA-449表达，Western blotting检测细胞中NOTCH1蛋白表达；转染后24、48和72 h，CCK-8法检测肿瘤细胞增殖活性。结果 与癌旁组织比较，肿瘤组织中miRNA-449表达明显升高，NOTCH1蛋白表达则明显降低，差异均有统计学意义（P=0.002和P=0.021）。miRNA-449在NOTCH1基因3′UTR区存在结合位点5′-CACTGCC-3′，并且通过结合位点对NOTCH1基因表达进行负调控。miRNA-449 模拟物转染SH-SY5Y细胞，明显抑制细胞内NOTCH1蛋白表达（P=0.006）；而miRNA-449 抑制剂转染细胞后可明显增强胞内NOTCH1蛋白表达（P=0.018）。在48和72 h时间点，miRNA-449 模拟物可明显抑制SH-SY5Y细胞增殖活性，而miRNA-449抑制剂则使细胞增殖活性进一步增强。结论 miRNA-449可以通过抑制其靶基因NOTCH1表达抑制SH-SY5Y细胞增殖活性。

关键词: miRNA-449, SH-SY5Y细胞, 荧光素酶, NOTCH1, 细胞活性

Abstract:

Objective To identify and verify the target gene of miRNA-449 and explore the molecular mechanism of regulating the proliferation activity of neuroblastoma cell SH-SY5Y by miRNA-449. Methods Expressions of miRNA-449 and NOTCH1 protein in neuroblastoma tissues and adjacent tissues were detected and their correlation was analyzed. The binding site between miRNA-449 and 3′UTR region of NOTCH1 gene was predicted with bioinformatic software and verified with luciferase reporter gene assay. The mimic, inhibitor, and scrambled sequence of miRNA-449 were chemically synthesized and transfected to SH-SY5Y cells via liposome. Expressions of miRNA-449 and NOTCH1 protein were detected by real-time PCR and Western blotting 48 h after transfection, respectively. The proliferation activity of neuroblastoma cells was measured by CCK-8 method 24, 48, and 72 h after transfection. Results Compared with adjacent tissues, the miRNA-449 expression in neuroblastoma tissues was significantly higher and the NOTCH1 protein expression was significantly lower. The differences were statistically significant (P=0.002; P=0.021). There was a binding site 5′-CACTGCC-3′ in 3′UTR region of NOTCH1 gene. Gene expression was negatively regulated via the binding site. Transfection of SH-SY5Y cells with miRNA-449 mimic significantly inhibited the NOTCH1 protein expression (P=0.006), whereas transfection with miRNA-449 inhibitor significantly increased the NOTCH1 protein expression (P=0.018).miRNA-449 mimic significantly inhibited the proliferation activity of SH-SY5Y cells and miRNA-449 inhibitor significantly enhanced the proliferation activity at time points of 48 and 72 h. Conclusion miRNA-449 can inhibit the proliferation activity of SH-SY5Y cells via inhibiting the expression of its target gene NOTCH1.

Key words: miRNA-449, SH-SY5Y cell, luciferase, NOTCH1, cell viability