miR-23b-3p Modulating Cytoprotective Autophagy and Glutamine Addiction in Sorafenib Resistant HepG2, a Hepatocellular Carcinoma Cell Line

Abstract

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.

Targeting and regulation of autophagy in hepatocellular carcinoma: revisiting the molecular interactions and mechanisms for new therapy approaches

“…Beclin-1 KU-0063794 Silencing of Beclin-1 suppresses autophagy and increases drug sensitivity [243] CD13/p38/Hsp27/CREB/ATG7 5-flourouracil CD13 increases ATG7 expression via the p38/Hsp27/CREB axis, which induces chemoresistance [244] miR-23b-3p/ATG12 Sorafenib miR-23b-3p reduces ATG12 expression, impairing autophagy and reversing sorafenib resistance [245] p57/autophagy EGFR inhibitors p57 induces autophagy and enhances the potential of EGFR inhibitors in combating HCC [246] VRK2 JNK1/MAPK8/Bcl-2/Beclin-1 VRK2 induces Beclin-1 expression via activation of the JNK1/MAPK8 axis to mediate dissociation of Bcl-2 from Beclin-1, triggering autophagy and mediating drug resistance [247] Akt Sorafenib Suppression of Akt signaling reverses autophagy that promotes survival to autophagy that promotes death, thereby reversing drug resistance [248] miR-101-3p/Beclin-1 oxaliplatin miR-101-3p reduces the expression of Beclin-1 in inhibiting autophagy and reversing drug resistance [249] miR-651-3p/ATG3 Cisplatin miR-651-3p reduces ATG3 expression in suppressing autophagy and chemoresistance increase the formation of ROS and mediates oxidative stress [187]. Thus, when the levels of ROS decrease, the sensitivity of HCC cells to radiotherapy decreases.…”

An Insight into the Arising Role of MicroRNAs in Hepatocellular Carcinoma: Future Diagnostic and Therapeutic Approaches

“…Similarly, it is also demonstrated that miR-23b-3p modulates the autophagy-induced drug resistance of sorafenib in the HCC HepG2 cell line, while it not only targets ATG12 but also GLS1 (glutaminase), which is related to the high exogenous glutamine that is associated with progressed disease [ 171 ].…”

Little things with significant impact: miRNAs in hepatocellular carcinoma

“…Therefore, we conclude that miRNAs in HCC inhibit cellular autophagy and reverse HCC chemoresistance by altering the expression of autophagy-related genes or affecting key links in the autophagic process, providing new therapeutic targets for HCC. In Figure 4, we illustrate the mechanism by which some miRNAs enhance the chemosensitivity of HCC by mediating HCC cellular autophagy (91,96,(98)(99)(100).…”