Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Dec 27, 2020; 12(12): 1211-1227
Published online Dec 27, 2020. doi: 10.4254/wjh.v12.i12.1211
Pivotal role of long non-coding ribonucleic acid-X-inactive specific transcript in regulating immune checkpoint programmed death ligand 1 through a shared pathway between miR-194-5p and miR-155-5p in hepatocellular carcinoma
Sara M Atwa, Heba Handoussa, Karim M Hosny, Margarete Odenthal, Hend M El Tayebi
Sara M Atwa, Heba Handoussa, Pharmaceutical Biology Department, German University in Cairo, Cairo 11865, Egypt
Karim M Hosny, Department of General Surgery, Faculty of Medicine, Cairo University, Cairo 11562, Egypt
Margarete Odenthal, Institute for Pathology, University Hospital Cologne, Cologne 50924, Germany
Hend M El Tayebi, Molecular Pharmacology Research Group, Department of Pharmacology and Toxicology, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt
Author contributions: El-Tayebi HM and Odenthal M designed and coordinated the study; El-Tayebi HM supported data interpretation and analysis; Handoussa H designed and supervised the natural product study; Atwa SM performed the experiments, acquired and analyzed the data and wrote the manuscript; Hosny KA provided tissue samples and clinical data; all authors approved the final version of the article to be published.
Institutional review board statement: The use of human tissue samples and clinical data was approved by the ethics committee of the German University in Cairo, all patients provided signed informed consent, and the research was carried out in accordance with the Helsinki Declaration.
Conflict-of-interest statement: The authors have no conflicts of interests.
Data sharing statement: No additional data are available.
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: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Hend M El Tayebi, PhD, Assistant Professor, Lecturer, Pharmacist, Senior Postdoctoral Fellow, Molecular Pharmacology Research Group, Department of Pharmacology and Toxicology, Faculty of Pharmacy and Biotechnology, German University in Cairo, New Cairo City - Main Entrance Al Tagamoa Al Khames, Cairo 11835, Egypt. hend.saber@guc.edu.eg
Received: June 30, 2020
Peer-review started: June 30, 2020
First decision: September 14, 2020
Revised: September 30, 2020
Accepted: October 29, 2020
Article in press: October 29, 2020
Published online: December 27, 2020
Processing time: 170 Days and 15.6 Hours
Abstract
BACKGROUND

Anti-programmed death therapy has thrust immunotherapy into the spotlight. However, such therapy has a modest response in hepatocellular carcinoma (HCC). Epigenetic immunomodulation is a suggestive combinatorial therapy with immune checkpoint blockade. Non-coding ribonucleic acid (ncRNA) driven regulation is a major mechanism of epigenetic modulation. Given the wide range of ncRNAs that co-opt in programmed cell-death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) regulation, and based on the literature, we hypothesized that miR-155-5p, miR-194-5p and long non-coding RNAs (lncRNAs) X-inactive specific transcript (XIST) and MALAT-1 are involved in a regulatory upstream pathway for PD-1/PD-L1. Recently, nutraceutical therapeutics in cancers have received increasing attention. Thus, it is interesting to study the impact of oleuropein on the respective study key players.

AIM

To explore potential upstream regulatory ncRNAs for the immune checkpoint PD-1/PD-L1.

METHODS

Bioinformatics tools including microrna.org and lnCeDB software were adopted to detect targeting of miR-155-5p, miR-194-5p and lncRNAs XIST and MALAT-1 to PD-L1 mRNA, respectively. In addition, Diana tool was used to predict targeting of both aforementioned miRNAs to lncRNAs XIST and MALAT-1. HCC and normal tissue samples were collected for scanning of PD-L1, XIST and MALAT-1 expression. To study the interaction among miR-155-5p, miR-194-5p, lncRNAs XIST and MALAT-1, as well as PD-L1 mRNA, a series of transfections of the Huh-7 cell line was carried out.

RESULTS

Bioinformatics software predicted that miR-155-5p and miR-194-5p can target PD-L1, MALAT-1 and XIST. MALAT-1 and XIST were predicted to target PD-L1 mRNA. PD-L1 and XIST were significantly upregulated in 23 HCC biopsies compared to healthy controls; however, MALAT-1 was barely detected. MiR-194 induced expression elevated the expression of PD-L1, XIST and MALAT-1. However, overexpression of miR-155-5p induced the upregulation of PD-L1 and XIST, while it had a negative impact on MALAT-1 expression. Knockdown of XIST did have an impact on PD-L1 expression; however, following knockdown of the negative regulator of X-inactive specific transcript (TSIX), PD-L1 expression was elevated, and abolished MALAT-1 activity. Upon co-transfection of miR-194-5p with siMALAT-1, PD-L1 expression was elevated. Co-transfection of miR-194-5p with siXIST did not have an impact on PD-L1 expression. Upon co-transfection of miR-194 with siTSIX, PD-L1 expression was upregulated. Interestingly, the same PD-L1 expression pattern was observed following miR-155-5p co-transfections. Oleuropein treatment of Huh-7 cells reduced the expression profile of PD-L1, XIST, and miR-155-5p, upregulated the expression of miR-194-5p and had no significant impact on the MALAT-1 expression profile.

CONCLUSION

This study reported a novel finding revealing that opposing acting miRNAs in HCC, have the same impact on PD-1/PD-L1 immune checkpoint by sharing a common signaling pathway.

Keywords: Hepatocellular carcinoma; X-inactive specific transcript; MiR-155-5p; MiR-194-5p; Programmed cell-death protein 1/Programmed death ligand 1; Immune checkpoint

Core Tip: Due to the immune rich milieu of hepatocellular carcinoma (HCC), it is a good candidate for immune-based therapies. In this study, our aim was to identify potential upstream epigenetic regulators of immune checkpoint programmed cell-death protein 1/programmed death ligand 1 in HCC which could be regarded as therapeutic targets. The findings of this study revealed the re-questioning of the role of certain non-coding ribonucleic acids in HCC. Here we deduced a novel shared upstream regulatory signaling pathway for programmed cell-death protein 1/programmed death ligand 1 immune checkpoint between paradoxically acting tumor suppressor miR-194-5p and onco-miR-155-5p, in HCC through X-inactive specific transcript expression modulation.