MicroRNA-1 inhibits proliferation of hepatocarcinoma cells by targeting endothelin-1
MicroRNA-1 inhibits proliferation of hepatocarcinoma cells by targeting endothelin-1
复制标题
MicroRNA-1通过靶向内皮素-1抑制肝癌细胞增殖
DOI：
10.1016/j.lfs.2012.08.015
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发表时间：
2012-10-05
期刊：
影响因子：
6.1
通讯作者：
Zhang, Tao
中科院分区：
文献类型：
--
作者：
Li, Dong;Yang, Pengyuan;Zhang, Tao
目的：MicroRNA-1 (miR-1) 已被证明是一种肿瘤抑制 miRNA，在包括肝细胞癌 (HCC) 在内的多种人类恶性肿瘤中显示出下调模式。然而，miR-1在HCC肿瘤发生中的病理生理作用及其机制尚未完全阐明。主要方法：将Pre-miR-1克隆到pSuper质粒中，在肝癌细胞中过表达miR-1。实时荧光定量PCR和Western blot分别检测miR-1、ET-1 mRNA和蛋白水平。进行双荧光素酶报告基因测定以研究 ET-1 mRNA 3'UTR 上 miR-1 的结合位点。通过 MTT 测定评估肝癌细胞的增殖。 主要发现：我们观察到，在 HepG2 和 Hep3B 细胞中通过 miRNA 表达质粒转染过度表达 miR-1 显着降低了这些细胞的增殖。为了探究其机制，我们通过生物信息学研究了miR-1的潜在靶基因。一种有效的有丝分裂原，内皮素-1 (ET-1)，引起了我们的注意。使用 Western Blot 和 miRNA 实时 PCR 分别检测到人肝癌组织和肝癌细胞系中 ET-1 表达升高，但 miR-1 水平降低。通过miR-1在HepG2和Hep3B中的过表达和抑制，我们证实miR-1负向调节肝癌细胞中ET-1的表达。荧光素酶报告基因检测表明，miR-1 调节是通过与 ET-1 3'UTR 内的互补结合位点配对而建立的。最后，外源性ET-1治疗可以部分恢复因miR-1过度表达而减弱的肝癌细胞增殖。意义：我们的研究结果表明，miR-1可以抑制ET-1表达，从而减弱肝癌细胞的增殖。 (c) 2012 Elsevier Inc. 保留所有权利。
Aims: MicroRNA-1 (miR-1) has been demonstrated as a tumor-suppressive miRNA, which shows a down-regulated pattern in several human malignancies including hepatocellular carcinoma (HCC). However, the pathophysiologic roles of miR-1 and their mechanisms in HCC tumorigenesis are still not totally elucidated.Main methods: Pre-miR-1 was cloned into pSuper plasmid to overexpress the miR-1 in hepatoma cells. Real-time PCR and Western blot were applied to detect miR-1, ET-1 mRNA and protein levels respectively. Dual luciferase reporter assay was conducted to investigate the binding site of miR-1 on 3'UTR of ET-1 mRNA. Proliferation of hepatoma cells was evaluated by MTT assay.Key findings: We observed that over-expression of miR-1 by miRNA-expressing plasmid transfection in HepG2 and Hep3B cells significantly reduced the proliferation of these cells. To explore the mechanism, we examined the potential target genes of miR-1 by bioinformatics. A potent mitogen, Endothelin-1 (ET-1), attracted our attention. Elevated expression of ET-1 but reduced miR-1 level was detected both in human liver cancer tissues and in hepatoma cell lines using Western Blot and miRNA real-time PCR respectively. By the over-expression and inhibition of miR-1 in HepG2 and Hep3B, we confirmed that miR-1 negatively regulated ET-1 expression in hepatoma cells. A luciferase reporter assay showed that miR-1 regulation was established by pairing to a complementary binding site within the ET-1 3'UTR. Finally, attenuated proliferation of hepatoma cells by over-expression of miR-1 could be partially restored by exogenous ET-1 treatment.Significance: Our findings demonstrate that miR-1 could inhibit ET-1 expression to attenuate the proliferation of hepatoma cells. (c) 2012 Elsevier Inc. All rights reserved.