Basic Study
Copyright ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Mar 27, 2021; 13(3): 328-342
Published online Mar 27, 2021. doi: 10.4254/wjh.v13.i3.328
Promotive action of 2-acetylaminofluorene on hepatic precancerous lesions initiated by diethylnitrosamine in rats: Molecular study
Amany Helmy Hasanin, Eman K Habib, Nesreen El Gayar, Marwa Matboli
Amany Helmy Hasanin, Nesreen El Gayar, Clinical Pharmacology Department, Faculty of Medicine, Ain Shams University, Cairo 11318, Egypt
Eman K Habib, Anatomy and Embryology Department, Faculty of Medicine, Ain Shams University, Cairo 11318, Egypt
Marwa Matboli, Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Ain Shams University, Cairo 11381, Egypt
Author contributions: Hasanin AH and El Gayar N conceived the idea and designed the animal model and statistical analysis; Habib EK performed histochemical examinations; Matboli M conducted bioinformatics analysis, biochemical and molecular assay; all authors drafted the manuscript and critically reviewed the manuscript and approved the final version of the manuscript for publication.
Institutional animal care and use committee statement: All animal procedures were carried out in accordance with the National Institute of Health guide for the care and use of laboratory animals (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Animal Ethics Committee for Ain Shams University, Faculty of Medicine (approval No. 17585).
Conflict-of-interest statement: The authors declare no competing interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE Guidelines, and the manuscript was prepared and revised according to the ARRIVE Guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (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: Marwa Matboli, MD, Adjunct Professor, Associate Professor, Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Ain Shams University, Abbassia, P.O. box 11381, Cairo 11381, Egypt.
Received: November 23, 2020
Peer-review started: November 23, 2020
First decision: December 12, 2020
Revised: December 14, 2020
Accepted: March 2, 2021
Article in press: March 2, 2021
Published online: March 27, 2021
Research background

2-Acetylaminofluorene (2-AAF) dose dependently promoted hepatic precancerous lesion. Over diethylnitrosamine (DEN), 2-AAF decreased autophagy. Over DEN, 2-AAF decreased apoptosis and tumor suppression gene. Over DEN, 2-AAF increased hepatic cell proliferation. 2-AAF epigenetically regulated long-noncoding RNA (lncRNA) RP11-513I15.6/miRNA-1262/miR-1298 (microRNA = miRNA = miR).

Research motivation

Urgent need for hepatocellular carcinoma (HCC) rat model for preclinical trials.

Research objectives

The present study aimed to develop a model of chemically-induced pre precancerous nodules in rat liver using DEN + 2-AAF and explore the putative molecular mechanism at the genetic and epigenetic levels.

Research methods

Bioinformatics-based selection of molecular parameters to investigate the oncogenic mechanism of the chemicals used in the HCC model followed by induction of animal model by. intraperitoneal injection of DEN for three weeks consecutively, followed by one intraperitoneal injection of 2-AAF at three different doses (100, 200 and 300 mg/kg. Rats were sacrificed after 10 wk and 16 wk. Liver functions, level of alpha-fetoprotein, glutathione S-transferase-P and proliferating cell nuclear antigen staining of liver tissues were performed. The mRNA level of RAB11A, BAX, p53, and Cyclin E and epigenetic regulation by lncRNA RP11-513I15.6, miR-1262, and miR-1298 were assessed in the sera and liver tissues of the rats.

Research results

2-AAF administration significantly increased the percent area of the precancerous foci and cell proliferation along with a significant decrease in RAB11A, BAX, and p53 mRNA, and the increase in Cyclin E mRNA was associated with a marked decrease in lncRNA RP11-513I15.6 expression with a significant increase in both miR-1262 and miR-1298.

Research conclusions

Administration of DEN to rats produced changes in hepatocytes with increased glutathione S-transferase-P and proliferating cell nuclear antigen expression and development of precancerous hepatic foci. The transformed cells proliferated when challenged with another carcinogen (2-AAF) as a promoter. These changes increased with the elevated dose of 2-AFF and duration of the experiment. DEN and 2-AAF affected the mRNA-biomarkers, including RAB11A, BAX, p53, and Cyclin E. Thus, the oncogenic properties of DEN and 2-AAF were observed in induced HCC model, which might be attributed to the suppression of p53, autophagy, and apoptosis along with the activation of the cell cycle. Moreover, it significantly increased the level of miR-1262 and miR-1298 with a concomitant decrease in the expression of lncRNA-RP11-513I15.6. This phenomenon led to the hypothesis that lncRNA-RP11-513I15.6 is a part of competing endogenous RNA, decreasing the level of miR-1262 and miR-1298, which, in turn, regulates the selected target mRNAs.

Research perspectives

More in vitro functional studies are urgently need to explore the competing endogenur role of lncRNA in HCC pathogenesis.