Basic Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Mar 14, 2019; 25(10): 1210-1223
Published online Mar 14, 2019. doi: 10.3748/wjg.v25.i10.1210
Short hairpin RNA-mediated knockdown of nuclear factor erythroid 2-like 3 exhibits tumor-suppressing effects in hepatocellular carcinoma cells
Miao-Mei Yu, Yue-Hua Feng, Lu Zheng, Jun Zhang, Guang-Hua Luo
Miao-Mei Yu, Yue-Hua Feng, Lu Zheng, Jun Zhang, Guang-Hua Luo, Comprehensive Laboratory, the Third Affiliated Hospital of Soochow University, Changzhou 213003, Jiangsu Province, China
Author contributions: Yu MM and Luo GH designed this study; Feng YH and Zhang J performed the experiments; Yu MM, Feng YH, and Zheng L wrote the manuscript; Yu MM and Luo GH performed the bioinformatic prediction; Yu MM and Luo GH conducted statistical analysis; all authors read and approved the final manuscript.
Supported by the Changzhou High-Level Medical Talents Training Project, No. 2016ZCLJ002.
Institutional review board statement: The clinicopathological features involved in this study are all from the TCGA database, thus, no institutional review board document is provided.
Conflict-of-interest statement: None declared.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines and prepared the manuscript accordingly.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Guang-Hua Luo, MD, PhD, Professor, Senior Researcher, Comprehensive Laboratory, the Third Affiliated Hospital of Soochow University, No. 185, Juqian Street, Tianning District, Changzhou 213003, Jiangsu Province, China.
Telephone: +86-519-68870619 Fax: +86-519-86621235
Received: January 4, 2019
Peer-review started: January 4, 2019
First decision: January 23, 2019
Revised: February 13, 2019
Accepted: February 15, 2019
Article in press: February 16, 2019
Published online: March 14, 2019
Research background

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. Many factors can induce HCC, such as viral infection, alcoholic cirrhosis, and aflatoxin, while the underlying mechanism remains unclear. Nuclear factor erythroid 2-like 3 (NFE2L3) is a member of the cap ‘n’ collar basic-region leucine zipper family of transcription factors. Studies have shown that NFE2L3 acts as a crucial regulator in a variety of cancer progression involving proliferation, migration, and invasion of tumor cells.

Research motivation

The roles of NFE2L3 in HCC and their underlying molecular mechanisms have yet to be elucidated.

Research objectives

Our study aimed to examine NFE2L3 expression in HCC and to analyze its association with clinicopathological features, and to systematically investigate the biological effects of NFE2L3 in HCC cell lines.

Research methods

Based on The Cancer Genome Atlas data portal, we analyzed NFE2L3 expression in 344 HCC patients and its correlation with clinicopathological features. Short hairpin RNA (shRNA) interference technology was utilized to knock down NFE2L3 in SMMC-7721 and BEL-7404 cells. NFE2L3 mRNA levels were quantified using qPCR. Flow cytometry, clone-forming, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and transwell assays were performed to evaluate the apoptosis, clone formation, proliferation, migration, and invasion of HCC cells. The protein levels of NFE2L3 and epithelial-mesenchymal transition (EMT) markers were examined by Western blot.

Research results

Our results revealed that NFE2L3 expression in G3/4 HCC patients was significantly higher than that in G1/2 grade patients, and its expression gradually increased with the advancement of T stage and pathologic stage. The Spearman rank correlation analysis showed that NFE2L3 expression was significantly correlated with tumor grade, T stage, and pathologic stage. The qPCR and Western blot results indicated that both mRNA and protein levels of NFE2L3 were markedly decreased after shRNA-mediated knockdown in BEL-7404 and SMMC-7721 cells. Flow cytometry results demonstrated that shRNA-mediated knockdown of NFE2L3 could promote the apoptosis of HCC cells. Meanwhile, NFE2L3 knockdown significantly suppressed the clone formation, cell proliferation, migration, and invasion of HCC cells. Additionally, our results showed that NFE2L3 knockdown dramatically decreased the levels of mesenchymal markers (N-cadherin and Vimentin) and EMT transcription regulators (Snail1 and Snail2) in HCC cells.

Research conclusions

The present study identified that NFE2L3 was closely associated with the grade and stage of HCC patients, and shRNA-mediated knockdown of NFE2L3 exhibited tumor-suppressing effects in HCC cells.

Research perspectives

Our study preliminarily explored the possible role of NFE2L3 in HCC. Future studies should focus on the following aspects. First, we will expand the sample size, strengthen follow-up, and conduct survival analyses. Second, animal experiments will be performed to further validate the role of NFE2L3 in vivo. Finally, we need to further explore the molecular mechanisms underlying its roles in HCC.