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Journal of Zhejiang University SCIENCE B

ISSN 1673-1581(Print), 1862-1783(Online), Monthly

Involvement of microRNA-718, a new regulator of EGR3, in regulation of malignant phenotype of HCC cells

Abstract: Objective: Hepatocellular carcinoma (HCC) is still one of the most common death-related malignancies worldwide. Because the way onset and progression are hidden most, HCC diagnoses are made at an advanced stage, when they are unsuitable for surgical resection. MicroRNAs are a class of small non-coding RNAs, participating in many aspects of cancers. In this study, we tried to establish the role of microRNA-718 (miR-718) in the malignant phenotype of HCC cells and its possible role in HCC diagnosis. Methods: Here we first used a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, Transwell migration and invasion assays, and colony formation assay to evaluate the impact of miR-718 on the malignant phenotypes of HCC cells. Then, we used bioinformatic methods to predict the target gene of miR-718 and used green fluorescence protein (GFP) reporter assay, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) to validate the regulation relationship. Finally, we determined the role of the target gene in the HCC phenotype. Results: We found that the expression of miR-718 was significantly reduced in various HCC cell lines and HCC tissues. Re-expression of miR-718 significantly reduced the cellular viability and colony formation ability as well as inhibited the migration and invasion abilities of HCC cell lines. Early growth response protein 3 (EGR3) is a direct target of miR-718 and is negatively regulated by miR-718. EGR3 could increase the viability and proliferation of HCC cells, and promot the migration and invasion of HCC cells. Conclusions: miR-718 acts as a tumor suppressive microRNA in HCC via regulating the expression of EGR3, which may provide a new diagnostic marker and treatment target for HCC.

Key words: miR-718, MicroRNA, Early growth response protein 3 (EGR3), Hepatocellular carcinoma (HCC), Malignant phenotype

Chinese Summary  <21> miR-718和EGR3参与调控肝癌细胞系的恶性表型的研究

目的:研究miR-718和早期生长反应蛋白3(EGR3)在肝癌细胞系HepG2及SMMC-7721中对细胞恶性行为的作用。
创新点:首次在肝癌细胞系HepG2和SMMC-7721中发现miR-718作为抑癌基因及EGR3作为促癌基因起到调控肿瘤恶性行为的作用。
方法:采用聚合酶链反应(PCR)方法构建miR-718和EGR3的过表达及敲降质粒,并采用实时定量PCR(qRT-PCR)方法验证质粒有效性;采用MTT法和克隆形成实验检测肝癌细胞系HepG2和SMMC-7721的生长增殖能力;采用Transwell迁移侵袭实验检测肝癌细胞系HepG2和SMMC-7721的迁移和侵袭能力;采用增强型绿色荧光蛋白(EGFP)荧光报告载体实验验证miR-718的靶基因;采用qRT-PCR和蛋白质印迹(Western blot)检测相关基因表达水平的变化。
结论:miR-718在肝癌细胞系HepG2和SMMC-7721中起到抑癌基因的作用;EGR3在肝癌细胞系 HepG2和SMMC-7721中起到促癌基因的作用;miR-718是通过靶定EGR3 mRNA 3’ UTR下调EGR3的表达起到抑癌基因的作用。

关键词组:miR-718;小非编码RNA;早期生长反应蛋白3;肝癌;表型


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DOI:

10.1631/jzus.B1600205

CLC number:

R735.2

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On-line Access:

2017-01-03

Received:

2016-05-10

Revision Accepted:

2016-08-01

Crosschecked:

2016-12-13

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