Basic Study
Copyright ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 21, 2023; 29(27): 4317-4333
Published online Jul 21, 2023. doi: 10.3748/wjg.v29.i27.4317
18β-glycyrrhetinic acid promotes gastric cancer cell autophagy and inhibits proliferation by regulating miR-328-3p/signal transducer and activator of transcription 3
Yi Yang, Yi Nan, Yu-Hua Du, Shi-Cong Huang, Dou-Dou Lu, Jun-Fei Zhang, Xia Li, Yan Chen, Lei Zhang, Ling Yuan
Yi Yang, Yu-Hua Du, Shi-Cong Huang, Dou-Dou Lu, Xia Li, Ling Yuan, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
Yi Nan, Jun-Fei Zhang, Yan Chen, Lei Zhang, Key Laboratory of Ningxia Minority Medicine Modernization Ministry of Education, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
Author contributions: Yang Y carried out most of the studies, analyzed the data and wrote the manuscript; Nan Y designed the study and revised the manuscript; Nan Y, Du YH and Huang SC wrote the manuscript and carried out the chart-making work; Lu DD and Li X were responsible for the total transcriptomic and bioinformatics analyses; Zhang JF performed parts of the in vivo and in vitro experiments and conducted statistical analyses of the data; Yuan L supervised the process of research and provided clinical guidance; Yuan L, Zhang L and Chen Y provided the conceptual and technical guidance as well as revised the manuscript critically for important intellectual content; All authors have read and approved the manuscript.
Supported by Ningxia Medical University Project, No. XZ2021005; Ningxia Natural Science Foundation, Nos. 2022AAC03144 and 2022AAC02039; and National Natural Science Foundation of China, No. 82260879.
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board of Ningxia Medical University (No. 2021-X003, No. 2021-N0063, Nos. 2021-N001 and 2022-G089).
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Ningxia Medical University (IACUC-NYLAC-2022-108).
Conflict-of-interest statement: All the authors report having no relevant conflicts of interest for this article.
Data sharing statement: All data generated or analyzed during this study are included in this paper, and further inquiries can be directed to the corresponding author 20080017@nxmu.edu.cn.
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: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Ling Yuan, MD, PhD, Professor, Ningxia Medical University, No. 1160 Shengli Street, Yinchuan 750004, Ningxia Hui Autonomous Region, China. nxykdx@qq.com
Received: March 28, 2023
Peer-review started: March 28, 2023
First decision: April 27, 2023
Revised: May 10, 2023
Accepted: June 2, 2023
Article in press: June 2, 2023
Published online: July 21, 2023
Abstract
BACKGROUND

Gastric cancer (GC) is one of the most common cancer types worldwide, and its prevention and treatment methods have garnered much attention. As the active ingredient of licorice, 18β-glycyrrhetinic acid (18β-GRA) has a variety of pharmacological effects. The aim of this study was to explore the effective target of 18β-GRA in the treatment of GC, in order to provide effective ideas for the clinical prevention and treatment of GC.

AIM

To investigate the mechanism of 18β-GRA in inhibiting cell proliferation and promoting autophagy flux in GC cells.

METHODS

Whole transcriptomic analyses were used to analyze and screen differentially expressed microRNAs (miRNAs) in GC cells after 18β-GRA intervention. Lentivirus-transfected GC cells and the Cell Counting Kit-8 were used to detect cell proliferation ability, cell colony formation ability was detected by the clone formation assay, and flow cytometry was used to detect the cell cycle and apoptosis. A nude mouse transplantation tumor model of GC cells was constructed to verify the effect of miR-328-3p overexpression on the tumorigenicity of GC cells. Tumor tissue morphology was observed by hematoxylin and eosin staining, and microtubule-associated protein light chain 3 (LC3) expression was detected by immunohistochemistry. TransmiR, STRING, and miRWalk databases were used to predict the relationship between miR-328-3p and signal transducer and activator of transcription 3 (STAT3)-related information. Expression of STAT3 mRNA and miR-328-3p was detected by quantitative polymerase chain reaction (qPCR) and the expression levels of STAT3, phosphorylated STAT3 (p-STAT3), and LC3 were detected by western blot analysis. The targeted relationship between miR-328-3p and STAT3 was detected using the dual-luciferase reporter gene system. AGS cells were infected with monomeric red fluorescent protein-green fluorescent protein-LC3 adenovirus double label. LC3 was labeled and autophagy flow was observed under a confocal laser microscope.

RESULTS

The expression of miR-328-3p was significantly upregulated after 18β-GRA intervention in AGS cells (P = 4.51E-06). Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation ability, arrested the cell cycle in the G0/G1 phase, promoted cell apoptosis, and inhibited the growth of subcutaneous tumors in BALB/c nude mice (P < 0.01). No obvious necrosis was observed in the tumor tissue in the negative control group (no drug intervention or lentivirus transfection) and vector group (the blank vector for lentivirus transfection), and more cells were loose and necrotic in the miR-328-3p group. Bioinformatics tools predicted that miR-328-3p has a targeting relationship with STAT3, and STAT3 was closely related to autophagy markers such as p62. After overexpressing miR-328-3p, the expression level of STAT3 mRNA was significantly decreased (P < 0.01) and p-STAT3 was downregulated (P < 0.05). The dual-luciferase reporter gene assay showed that the luciferase activity of miR-328-3p and STAT3 3’ untranslated regions of the wild-type reporter vector group was significantly decreased (P < 0.001). Overexpressed miR-328-3p combined with bafilomycin A1 (Baf A1) was used to detect the expression of LC3 II. Compared with the vector group, the expression level of LC3 II in the overexpressed miR-328-3p group was downregulated (P < 0.05), and compared with the Baf A1 group, the expression level of LC3 II in the overexpressed miR-328-3p + Baf A1 group was upregulated (P < 0.01). The expression of LC3 II was detected after intervention of 18β-GRA in GC cells, and the results were consistent with the results of miR-328-3p overexpression (P < 0.05). Additional studies showed that 18β-GRA promoted autophagy flow by promoting autophagosome synthesis (P < 0.001). qPCR showed that the expression of STAT3 mRNA was downregulated after drug intervention (P < 0.05). Western blot analysis showed that the expression levels of STAT3 and p-STAT3 were significantly downregulated after drug intervention (P < 0.05).

CONCLUSION

18β-GRA promotes the synthesis of autophagosomes and inhibits GC cell proliferation by regulating the miR-328-3p/STAT3 signaling pathway.

Keywords: 18β-glycyrrhetinic acid, miR-328-3p, Signal transducer and activator of transcription 3, Cell proliferation, Autophagy flow

Core Tip: 18β-glycyrrhetinic acid (18β-GRA) is an important bioactive component of glycyrrhiza liquorice. Our results showed that 18β-GRA upregulated the expression of miR-328-3p in gastric cancer (GC) cells. Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation, arrested the cell cycle, promoted apoptosis, inhibited subcutaneous tumor formation, and inhibited signal transducer and activator of transcription 3 (STAT3) expression. Dual-luciferase reporter assay showed that miR-328-3p targeted the regulation of STAT3. Confocal microscopy and western blotting indicated that 18β-GRA promotes autophagy flow by promoting autophagosome synthesis. Thus, 18β-GRA appears to promote cell autophagy and inhibit GC cell proliferation by regulating miR-328-3p/STAT3.