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miR-23b-3p Modulating Cytoprotective Autophagy and Glutamine Addiction in Sorafenib Resistant HepG2, a Hepatocellular Carcinoma Cell Line
Background: Hepatocellular carcinoma (HCC) is the second most common malignancy with increasing cancer deaths worldwide. HCC is mainly diagnosed at its advanced stage, and treatment with FDA-approved sorafenib, the multikinase inhibitor drug, is advised. Acquired resistance against sorafenib develop...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9407556/ https://www.ncbi.nlm.nih.gov/pubmed/36011286 http://dx.doi.org/10.3390/genes13081375 |
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author | Kaur, Ramanpreet Kanthaje, Shruthi Taneja, Sunil Dhiman, Radha K. Chakraborti, Anuradha |
author_facet | Kaur, Ramanpreet Kanthaje, Shruthi Taneja, Sunil Dhiman, Radha K. Chakraborti, Anuradha |
author_sort | Kaur, Ramanpreet |
collection | PubMed |
description | Background: Hepatocellular carcinoma (HCC) is the second most common malignancy with increasing cancer deaths worldwide. HCC is mainly diagnosed at its advanced stage, and treatment with FDA-approved sorafenib, the multikinase inhibitor drug, is advised. Acquired resistance against sorafenib develops through several pathways involving hypoxia, autophagy, high glycolysis, or glutaminolysis. Small non-coding RNAs, similar to microRNAs (miRNAs), are also known to affect sorafenib resistance in HCC. However, there is a lack of information regarding the significance of differentially expressed miRNA (if any) on autophagy and glutamine regulation in sorafenib-resistant HCC. Methods: The expression of autophagy and glutaminolysis genes was checked in both parental and sorafenib resistant HepG2 cell lines by real-time PCR. MTT and Annexin/PI assays were also performed in the presence of inhibitors such as chloroquine (autophagy inhibitor) and BPTES (glutaminolysis inhibitor). Next generation sequencing and in silico analysis were performed to select autophagy and glutamine addiction-specific microRNA. Selected miRNA were transfected into both HepG2 cells to examine its effect on autophagy and glutamine addiction in regulating sorafenib-resistant HCC. Results: Our in vitro study depicted a higher expression of genes encoding autophagy and glutaminolysis in sorafenib-resistant HepG2 cells. Moreover, inhibitors for autophagy (chloroquine) and glutaminolysis (BPTES) showed a diminished level of cell viability and augmentation in cell apoptosis of sorafenib-resistant HepG2 cells. NGS and real-time PCR demonstrated the downregulated expression of miR-23b-3p in sorafenib-resistant cells compared to parental cells. In silico analysis showed that miR-23b-3p specifically targeted autophagy through ATG12 and glutaminolysis through GLS1. In transfection assays, mimics of miR-23b-3p demonstrated reduced gene expression for both ATG12 and GLS1, decreased cell viability, and increased cell apoptosis of sorafenib-resistant HepG2 cells, whereas the antimiRs of miR-23b-3p demonstrated contrasting results. Conclusion: Our study highlights the cytoprotective role of autophagy and glutamine addiction modulated by miR-23b-3p (tumor suppressor), suggesting new approaches to curb sorafenib resistance in HCC. |
format | Online Article Text |
id | pubmed-9407556 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-94075562022-08-26 miR-23b-3p Modulating Cytoprotective Autophagy and Glutamine Addiction in Sorafenib Resistant HepG2, a Hepatocellular Carcinoma Cell Line Kaur, Ramanpreet Kanthaje, Shruthi Taneja, Sunil Dhiman, Radha K. Chakraborti, Anuradha Genes (Basel) Article Background: Hepatocellular carcinoma (HCC) is the second most common malignancy with increasing cancer deaths worldwide. HCC is mainly diagnosed at its advanced stage, and treatment with FDA-approved sorafenib, the multikinase inhibitor drug, is advised. Acquired resistance against sorafenib develops through several pathways involving hypoxia, autophagy, high glycolysis, or glutaminolysis. Small non-coding RNAs, similar to microRNAs (miRNAs), are also known to affect sorafenib resistance in HCC. However, there is a lack of information regarding the significance of differentially expressed miRNA (if any) on autophagy and glutamine regulation in sorafenib-resistant HCC. Methods: The expression of autophagy and glutaminolysis genes was checked in both parental and sorafenib resistant HepG2 cell lines by real-time PCR. MTT and Annexin/PI assays were also performed in the presence of inhibitors such as chloroquine (autophagy inhibitor) and BPTES (glutaminolysis inhibitor). Next generation sequencing and in silico analysis were performed to select autophagy and glutamine addiction-specific microRNA. Selected miRNA were transfected into both HepG2 cells to examine its effect on autophagy and glutamine addiction in regulating sorafenib-resistant HCC. Results: Our in vitro study depicted a higher expression of genes encoding autophagy and glutaminolysis in sorafenib-resistant HepG2 cells. Moreover, inhibitors for autophagy (chloroquine) and glutaminolysis (BPTES) showed a diminished level of cell viability and augmentation in cell apoptosis of sorafenib-resistant HepG2 cells. NGS and real-time PCR demonstrated the downregulated expression of miR-23b-3p in sorafenib-resistant cells compared to parental cells. In silico analysis showed that miR-23b-3p specifically targeted autophagy through ATG12 and glutaminolysis through GLS1. In transfection assays, mimics of miR-23b-3p demonstrated reduced gene expression for both ATG12 and GLS1, decreased cell viability, and increased cell apoptosis of sorafenib-resistant HepG2 cells, whereas the antimiRs of miR-23b-3p demonstrated contrasting results. Conclusion: Our study highlights the cytoprotective role of autophagy and glutamine addiction modulated by miR-23b-3p (tumor suppressor), suggesting new approaches to curb sorafenib resistance in HCC. MDPI 2022-08-01 /pmc/articles/PMC9407556/ /pubmed/36011286 http://dx.doi.org/10.3390/genes13081375 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Kaur, Ramanpreet Kanthaje, Shruthi Taneja, Sunil Dhiman, Radha K. Chakraborti, Anuradha miR-23b-3p Modulating Cytoprotective Autophagy and Glutamine Addiction in Sorafenib Resistant HepG2, a Hepatocellular Carcinoma Cell Line |
title | miR-23b-3p Modulating Cytoprotective Autophagy and Glutamine Addiction in Sorafenib Resistant HepG2, a Hepatocellular Carcinoma Cell Line |
title_full | miR-23b-3p Modulating Cytoprotective Autophagy and Glutamine Addiction in Sorafenib Resistant HepG2, a Hepatocellular Carcinoma Cell Line |
title_fullStr | miR-23b-3p Modulating Cytoprotective Autophagy and Glutamine Addiction in Sorafenib Resistant HepG2, a Hepatocellular Carcinoma Cell Line |
title_full_unstemmed | miR-23b-3p Modulating Cytoprotective Autophagy and Glutamine Addiction in Sorafenib Resistant HepG2, a Hepatocellular Carcinoma Cell Line |
title_short | miR-23b-3p Modulating Cytoprotective Autophagy and Glutamine Addiction in Sorafenib Resistant HepG2, a Hepatocellular Carcinoma Cell Line |
title_sort | mir-23b-3p modulating cytoprotective autophagy and glutamine addiction in sorafenib resistant hepg2, a hepatocellular carcinoma cell line |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9407556/ https://www.ncbi.nlm.nih.gov/pubmed/36011286 http://dx.doi.org/10.3390/genes13081375 |
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