Investigating the Mechanism of β-Elemene-Mediated LINC00511 Modulation to Suppress Cisplatin Resistance in Lung Cancer

Summary

Cisplatin (DDP) resistance limits chemotherapy efficacy in lung cancer. This study aimed to elucidate how β-Elemene(β-Ele) reverses resistance in A549/DDP cells by downregulating LINC00511. In vitro and RNA-seq analyses showed that this regulation inhibits aerobic glycolysis and Wnt/β-catenin signaling, offering a potential strategy to overcome chemoresistance.

Abstract

Cisplatin resistance significantly limits the efficacy of chemotherapy in non-small cell lung cancer, necessitating the development of new strategies to overcome this barrier. This in vitro study aimed to elucidate the mechanism by which β-Ele reverses cisplatin resistance in lung adenocarcinoma cells via the LINC00511-mediated glycolysis and Wnt/β-catenin signaling pathways. The cisplatin-resistant human lung adenocarcinoma cell line (A549/DDP), with either LINC00511 overexpression or knockdown, was established through plasmid transfection. The transfection efficiency was evaluated using Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) to detect LINC00511 expression levels. Assessment via the 5-ethynyl-2'-deoxyuridine (EdU) assay indicated significant β-Ele-mediated inhibition of A549/DDP cell proliferation. Further RNA-seq analysis revealed that the inhibitory effect of β-Elemene on cisplatin resistance was closely associated with the interference of cellular glycolysis and the suppression of Wnt/β-catenin signaling pathway activation. Western blot analysis of key proteins, including glucose transporter 1 (GLUT1), pyruvate kinase M2 (PKM2), lactate dehydrogenase A (LDHA), β-catenin, and glycogen synthase kinase 3β (GSK-3β), showed that β-Elemene reversed cisplatin resistance by inhibiting aerobic glycolysis and blocking the activation of the Wnt/β-catenin signaling pathway through downregulation of LINC00511. Collectively, these results demonstrate that β-Elemene overcomes cisplatin resistance in lung cancer by targeting LINC00511 and its associated aerobic glycolysis/Wnt/β-catenin pathway. This establishes a theoretical framework for future preclinical and clinical investigations while revealing potential therapeutic targets.

Introduction

Among malignant tumors worldwide, lung cancer exhibits exceptionally high incidence and fatality rates, characterized by the malignant transformation and invasive growth of bronchial epithelial cells^1,2. Based on histopathological features, lung cancer is categorized as either non-small cell carcinoma (NSCLC) or small cell carcinoma (SCLC)³. It is worth mentioning that NSCLC represents roughly 85% of total lung cancer cases^4,5. As a first-line chemotherapeutic standard for NSCLC, cisplatin (DDP) is clinically indispensable

Protocol

1. Solvent preparation

1. Preparation of Medium Solution for A549/DDP cells: Cultivate A549/DDP cells in Ham's F-12K complete medium (see Table of Materials for details), supplemented with 10% fetal bovine serum (FBS, see Table of Materials for details), 1% penicillin-streptomycin solution (P/S, see Table of Materials for details), and 2 μg/mL cisplatin (DDP, see Table of Materials for details).
   1. Preparation of Complete Medium Solution: Prepare a complete medium solution by adding 50 mL of FBS and 5 mL of P/S to 500 mL of Ham's F-12K medium, followed by thorough mi....

Results

β-Ele significantly suppresses the proliferation of A549/DDP cells

In this experiment, the administration concentrations of β-Ele and DDP were determined based on the results of our previous studies^20,21. A549/DDP cells were treated with β-Ele, alone or combined with DDP, to dissect its impact on DDP chemoresistance. EdU analysis (Fi.......

Discussion

This study focused on the critical clinical issue of cisplatin resistance in lung cancer. Lung cancer, responsible for the highest number of cancer fatalities globally, continues to have a five-year survival rate below 20%^29,30. Cisplatin, as the cornerstone of platinum-based chemotherapy drugs, is an efficient, broad-spectrum anticancer agent that inhibits DNA replication to exert its anticancer effects³¹. However, it has been observed th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
A549/DDP cells	Procell	CL-0519	URLs:https://www.procell.com.cn/p/a549-ddp--cl-0519
An exceptionally clean workbench	Suzhou Antai Airtech	SW-CJ-2FD	URLs:http://www.sz-antai.com
BCA Protein concentration Assay kit	Solarbio	PC0020	URLs:https://www.solarbio.com
Benchtop Centrifuge	Thermo	Pico17	URLs:https://www.thermofisher.com
Cell culture chamber	Thermo	3111	URLs:https://www.thermofisher.com
Cell culture dish	BIOFIL	TCD000100	URLs:http://www.biofil.com.cn
Chemiluminescence Analyzer	Qinxiang Instruments	4300	URLs:https://www.clinx.cn/about_us
Cisplatin	Aladdin	C295225-250mg	URLs:https://www.aladdin-e.com/
DNBSEQ-T7 Gene Sequencing Instrument	MGI Tech CO., Ltd	T7	URLs:https://www.mgitech.cn/
EasyPure Micro Genomic DNA Kit	TransGen Biotech	EE181-01	URLs:https://www.transgen.com/
ECL chromogenic solution	Solarbio	PE0010	URLs:https://www.solarbio.com
EDU detection kit	Beyotime	C0071S	URLs:https://www.beyotime.com
Enzyme labeled instrument	Detie Equipment	HBS-1096A	URLs:https://www.detiebio.cn/
Fetal bovine serum FBS)	CLARK	FB15015	URLs:https://zn.clarkbio.com/
Frag/Prime Buffer	Yeasen	11277ES	URLs:https://www.yeasen.com/products/detail/2151
GeneMapper Software 6	Thermo	Version 6	Fragment analysis software. URLs:https://www.thermofisher.cn/
GenReader platform	Beijing Yuewei Gene Technology Co., Ltd.	https://yyigou.com/product/detail/0000037250.html	Automated genetic analyzer used for capillary electrophoresis
Glucose test kit	Jiancheng Bio	A154-1-1	URLs:http://www.njjcbio.com
GLUT1 Polyclonal antibody	Proteintech Group	21829-1-AP	URLs:https://www.ptglab.com
GraphPad Prism	GraphPad Software	Version 8.0.2	URLs:https://www.graphpad.com
GSK-3βAntibody	Affinity	AF5016	URLs:https://www.affbiotech.com
Ham's F-12K	Procell	PM150910	URLs:https://www.procell.com.cn/
Hieff NGS DNA selection Beads	Yeasen	12601ES	URLs:https://www.yeasen.com/products/detail/939
HieffNGS mRNA Isolation Master Kit	Yeasen	12603ZH	URLs:https://www.yeasen.com/products/detail/1119
HRP labeled goat anti-rabbit IgG	Servicebio	GB23303	URLs:https://www.servicebio.cn
Lactate test kit	Jiancheng Bio	A019-2-1	URLs:http://www.njjcbio.com
LDHA-Specific Polyclonal Antibody	Proteintech Group	19987-1-AP	URLs:https://www.ptglab.com
Light Cycler 96	Roche	LC96	URLs:https://lifescience.roche.com
Lipofectamine 2000	Invitrogen	11668-019	URLs:https://www.thermofisher.com/invitrogen
Opti-MEM	Gibco	31985-070	URLs:https://www.thermofisher.com/gibco
Penicillin-streptomycin(P/S)	Biosharp	BL505A	URLs:https://www.biosharp.cn/
PKM2-specific Polyclonal Antibody	Proteintech Group	15822-1-AP	URLs:https://www.ptglab.com
PMSF	Beyotime	ST506	URLs:https://www.beyotime.com
PrimeScript RT reagent Kit (Perfect Real Time)	TaKaRa	RR037A	URLs:https://www.takarabio.com
RIPA lysate	Beyotime	P0013B	URLs:https://www.beyotime.com
Sequencing platform	BGI	DNBSEQ-T7	URLs:https://www.bgi.com/global
TB Green Premix Ex Taq	TaKaRa	RR420A	URLs:https://www.takarabio.com
Trace nucleic acid tester	Aosheng Instrument	Nano-100	URLs:http://www.hzaosheng.com
TRIzon PaI	Canvax Biotech	CW0597S	URLs:https://www.canvaxbiotech.com
Trypsin	Biosharp	BL501A	URLs:https://www.biosharp.cn/
Ultrapure RNA Kit (DNase I)	Canvax Biotech	CW0597S	URLs:https://www.canvaxbiotech.com
β-Actin Antibody	Proteintech Group	60008-1-Ig	URLs:https://www.ptglab.com
β-catenin Antibody	Affinity	DF6794	URLs:https://www.affbiotech.com
β-Elemene	Sigma	63965-25MG	URLs:https://www.sigmaaldrich.com