Paeonol inhibits the malignancy of Apatinib-resistant gastric cancer cells via LINC00665/miR-665/MAPK1 axis

Abstract

Background: Paeonol is the extractive of Paeonia suffruticosa Andr and is reported to reverse the chemotherapy resistance of cancer cells. The present study explores the role of paeonol in inhibiting the malignant biological behaviors of Apatinib-resistant gastric cancer (GC) cells.

Methods: The Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was adopted to screen the target genes of paeonol, and the STRING database was employed to construct a protein-protein interaction (PPI) network. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the target genes was performed employing DAVID online database. The expressions of these target genes in GC tissues and para-cancerous tissues were analyzed with GEPIA database, and GEO datasets (GSE109476 and GSE93415) were utilized to analyze differentially expressed lncRNAs and miRNAs in GC tissues and para-cancerous tissues. The expressions of LINC00665, miR-665 and MAPK1 mRNA in Apatinib-resistant GC cells were detected through quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8) assay was conducted to detect cell proliferation; Transwell assays were employed to detect cell migration and invasion, and TdT-mediated dUTP nick end labeling (TUNEL) assay was utilized to detect cell apoptosis. Dual-luciferase reporter gene assay was performed to detect the binding relationships between miR-665 and LINC00665, as well as between miR-665 and MAPK1 mRNA. The expressions of MAPK1 protein and glycolysis-associated proteins (GLUT1, LDHB and HK2) were detected by Western blot. Additionally, a tumor xenograft mice model was constructed to evaluate the effects of paeonol on lung metastasis.

Results: Paeonol could inhibit the proliferation, migration, invasion and glycolysis, and promote the apoptosis of Apatinib-resistant GC cells. TCMSP database suggested that Paeonol had 17 target genes, and 17 target genes were mainly enriched in signaling pathways related to apoptosis, glucose and lipid metabolism, etc.; GEPIA database suggests that MAPK1, among the 17 target genes, was markedly elevated in GC tissues. Paeonol could decrease LINC00665 and MAPK1 expressions in GC cells but increase the expression of miR-665. LINC00665 overexpression, MAPK1 overexpression or inhibition of miR-665 could abolish the inhibitive effects of paeonol on the malignant phenotypes of Apatinib-resistant GC cells. miR-665 is verified as an upstream regulator of MAPK1 and a target of LINC00665. Additionally, paeonol could significantly inhibit the lung metastasis in the tumor xenograft mice model.

Conclusions: Paeonol can inhibit the malignancy of Apatinib-resistant GC cells through LINC00665/miR-665/MAPK1 axis. For the first time, our study imply that paeonol may be a potential drug to reverse Apatinib-resistant of GC cells.

Keywords: Apatinib; Gastric cancer; LINC00665; MAPK1; Paeonol; miR-665.