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.

概要

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。