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 indispensable⁶. However, clinical observations have revealed that tumor cells can develop drug resistance via multiple mechanisms, primarily involving the activation of immune escape pathways, mutations in drug target genes, and dysregulation of drug metabolism pathways^7,8,9.

Long non-coding RNAs (lncRNA) represent transcripts > 200 nucleotides (nt) that lack protein-coding capacity¹⁰. Accumulating evidence indicates that lncRNA can reverse cisplatin resistance in tumor cells through multiple mechanisms, such as functioning as competing endogenous RNAs (ceRNAs) to sponge miRNAs and release their target genes¹¹, modulating apoptosis signaling pathways¹², participating in DNA damage repair¹³, and regulating cellular autophagy¹⁴. Furthermore, with the advancement of research on tumor metabolic reprogramming, substantial evidence has emerged demonstrating that lncRNA play a critical regulatory role in this process. The Warburg effect, denoting aerobic glycolysis, constitutes a fundamental metabolic characteristic of malignant cells¹⁵. It not only supplies energy for rapid cell proliferation but also generates intermediate metabolites that serve as precursors for biosynthesis, supporting protein and lipid synthesis to meet the demands of malignant tumor growth¹⁶. In lung cancer, it has been confirmed that lncRNA significantly influences tumor progression and chemotherapy resistance by regulating the aerobic glycolysis pathway^17,18.

Among these, LINC00511, a widely studied lncRNA molecule in recent years, has been reported to be closely associated with the initiation and development of various malignant tumors¹⁹. These findings not only elucidate the specific mechanisms of lncRNA involvement in tumor metabolic reprogramming but also provide a theoretical basis for lncRNA-targeted tumor treatment strategies, opening up potential therapeutic avenues.

Derived from the rhizomes of Curcuma zedoaria, β-Ele is a bioactive sesquiterpenoid with established antitumor efficacy. Our previous studies have demonstrated that β-Ele serves as a selective inhibitor of the PI3K/Akt/mTOR signaling pathway and produces a synergistic effect with cisplatin by targeting tumor metabolism. It significantly suppressed tumor growth in the NSCLC mouse xenograft model²⁰. Subsequent investigations demonstrate β-Ele's capacity to attenuate aerobic glycolysis in NSCLC cells via modulation of the miR-301a-3p/AMPKα signaling axis, resulting in oncostatic outcomes²¹. Based on our previous findings, we have confirmed that LINC00511 attenuates cisplatin resistance in lung cancer; nevertheless, the exact molecular mechanism of action remains to be fully elucidated.

Considering the pivotal role of aerobic glycolysis in tumor metabolic reprogramming and the critical role of LINC00511 in tumor development and progression, this study aims to investigate whether β-Ele can reverse cisplatin resistance in lung cancer by modulating the LINC00511-mediated aerobic glycolysis pathway.

Prior evidence indicates β-Ele can reverse cisplatin resistance and enhance its anti-tumor effects by activating apoptotic pathways²², regulating exosome secretion²³, and arresting the cell cycle²⁴. However, these known mechanisms typically exhibit broad effects and lack precise regulation of specific molecular targets, thereby limiting their clinical translatability. Therefore, there is an urgent need to explore novel strategies that are more targeted, have well-defined mechanisms, and demonstrate strong potential for clinical translation to overcome cisplatin resistance. In contrast to prior research emphasizing β-Ele's immediate biological actions, this study characterizes the impact of β-Ele on Warburg metabolism and the Wnt/β-catenin signaling pathway in A549/DDP cells through the regulation of lncRNA-mediated molecular pathways, aiming to elucidate a novel mechanism underlying its reversal of chemosensitization in lung cancer. This article not only uncovers a new approach for β-Ele to reverse chemoresistance by targeting lncRNA but also provides a fresh theoretical foundation and potential intervention targets for the clinical management of lung cancer. These findings, through their detailed elucidation of a specific molecular target (LINC00511) and key signaling pathways (Warburg and Wnt/β-catenin), demonstrate significantly enhanced clinical translational potential. They are expected to provide a solid scientific basis for improving chemotherapy sensitivity in lung cancer patients and enhancing their quality of life, as well as to advance the development of precision treatment strategies for lung cancer.

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 mixing.
   2. Preparation of Complete Medium Solution Containing Cisplatin: Weigh 1 mg of cisplatin powder (DDP, see Table of Materials for details) and dissolve it in 1 mL of double-distilled water (ddH₂O) to prepare the stock solution. Add 200 μL of the stock solution to 100 mL of complete medium (prepared according to step 1.1.1) and mix thoroughly to prepare a working solution of complete medium containing 2 μg/mL of cisplatin.
      NOTE: Since cisplatin is a cytotoxic substance, perform all procedures in a fume hood or biosafety cabinet and wear protective equipment such as gloves, lab coats, and goggles.
2. β-Ele administration solution: The drug concentration used in this study was based on the results of previous experiments conducted by our research team^20,21. Dissolve 1 mg of β-Ele (see Table of Materials for details) in 20 μL of dimethyl sulfoxide (DMSO, see Table of Materials for details) to prepare the stock solution. Dilute the stock solution to 25 mL using complete medium to obtain a 40 μg/mL β-Ele working solution.
   NOTE: Since DMSO can enhance the absorption of other substances through the skin, perform all operation steps in a fume hood or biosafety cabinet and wear protective equipment such as gloves, laboratory clothing, and goggles.

2. Cell culture 

1. Use the human lung adenocarcinoma cisplatin-resistant cell line (A549/DDP, Procell, see Table of Materials for details) in this study. Confirm that this cell line has been identified by short tandem repeat (STR) analysis and successfully matched to standard lineages in ATCC and other databases.
   1. For the STR analysis method, collect approximately 1 x 10⁶ cells, and extract genomic DNA using a commercial DNA extraction kit (see Table of Materials for details).
   2. Employ a validated multiplex STR system suitable for DNA typing to amplify 20 STR loci along with the gender-determining locus. Perform PCR amplification under the following thermal cycling conditions: initial denaturation at 95 °C for 2 min, followed by 29 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 45 s, and extension at 72 °C for 45 s, with a final extension step at 72 °C for 10 min.
   3. Analyze the resulting PCR products via capillary electrophoresis using an automated genetic analyzer according to manufacturer instructions. Here, allele typing was conducted using a fragment analysis software (see Table of Materials for details), with a peak detection threshold set at 150 Relative Fluorescence Units (RFU).
      NOTE: The generated 20-locus STR profile was compared against reference profiles of A549/DDP cell lines available in public databases. A match rate exceeding 80% was considered indicative of homology. The absence of extraneous allele peaks further confirmed the absence of cross-contamination.
2. Retrieve the cells from liquid nitrogen and place them in a 37 °C water bath for rapid thawing until the liquid is completely melted. Transfer the cell suspension to a 5 mL centrifuge tube, add two volumes of complete medium solution, and gently resuspend the mixture. Following centrifugation (358 × g, 5 min, room temperature), discard the supernatant carefully.
   NOTE: When using cell cryopreservation tubes from liquid nitrogen, wear anti-freezing gloves and either goggles or a face mask, and do not face the tube mouth. 
3. Resuspend the cell pellet in 1 mL of complete medium with gentle pipetting, then dilute the suspension to 10 mL by adding 9 mL of complete medium. Seed the cells into 100 mm cell culture dishes and culture them in an incubator (see Table of Materials for details) at 37 °C and 5% CO₂. The cell passage ratio was maintained between 1:2 and 1:3.
   NOTE: Cisplatin is a platinum chemotherapeutic drug with cytotoxicity. In this method, A549/DDP cells are cultured with medium containing cisplatin. Therefore, collect the waste culture medium containing cisplatin into a special waste liquid barrel and regularly send it to the hazardous waste treatment company for professional disposal.

3. Plasmid transfection 

1. Employ a plasmid construct for LINC00511 overexpression and a gene-specific siRNA targeting LINC00511 (gene ID: 400619; transcript ID: NR_033876.1) for cell transfection. The utilized siRNA sequences are provided in Table 1.

siRNA sequences
Name	Sense 5'-3'	Antisense 5'-3'
LINC00511-Homo-1625	CAGCUGUGAACUUCCUUUATT	UAAAGGAAGUUCACAGCUGTT

Table 1: siRNA sequence list

2. Randomly select A549/DDP cells that have been passaged to the third generation after resuscitation, and uniformly inoculate them into 6-well plates at a density of 1 x 10⁵ cells/well. Each well was randomly numbered, and the wells were assigned to experimental groups based on these random numbers.
3. Transfect the cells using a transfection reagent (see Table of Materials for details)  when their density reaches approximately 70%.
   1. Prepare the siRNA transfection solution.
      1. Mix 250 μL of Optimized Minimum Essential Medium (Opti-MEM, see Table of Materials for details) with 5 μL of transfection reagent, gently vortex 3–5x to ensure homogeneity, and incubate at room temperature for 5 min.
      2. Dilute 5 μL of siRNA (50 nM) in 250 μL of Opti-MEM, vortex gently 3–5x to mix thoroughly. Combine the diluted transfection reagent with the siRNA solution and incubate at room temperature for 20 min to form the siRNA transfection complex.
   2. Prepare the plasmid transfection solution.
      1. Mix 250 μL of Opti-MEM with 5 μL of transfection reagent, gently vortex 3–5x to ensure homogeneity, and incubate at room temperature for 5 min.
      2. Dilute 2 μg of plasmid in 250 μL of Opti-MEM, gently vortex 3–5x to ensure thorough mixing. Combine the diluted transfection reagent with the plasmid solution and incubate at room temperature for 20 min to prepare the plasmid transfection solution.
      3. Add the siRNA transfection solution and plasmid transfection solution to the 6-well plates, respectively. Add 1,500 μL of complete medium to each well. Gently mix the cell plates before and after the addition to ensure even distribution. Incubate the plates at 37 °C, 5% CO₂ for 24 h post-transfection prior to analysis.

4. Detecting the transfection efficiency by Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR)

NOTE: Take the cells after transfection for 24 h in step 3.3 for qRT-PCR detection according to the following procedure. All experiments were performed in three independent biological replicates (n = 3).

1. RNA extraction 
   1. Use an ultrapure RNA extraction kit (see Table of Materials for details). Discard the medium in the 6-well plate according to the kit instructions and wash the cells with PBS. Subsequently, add 500 μL of phenol–guanidine-based lysis reagent solution to each well, gently blow the cells with the tip of the gun to fully suspend them, and transfer the mixture to a centrifuge tube. Leave it for 5 min to ensure complete lysis of the cell sample.
   2.  Add 100 μL of phenol-guanidine isothiocyanate lysis reagent, shake sharply for 15 s, and leave it for 2 min at room temperature. Finally, carefully aspirate the upper aqueous phase, transfer it to the adsorption column, and centrifuge at 13,400 x g for 10 min at 4 °C.
   3. Bind RNA to the silica gel membrane. Wash it with Buffer RW1 and Buffer RW2 to remove proteins. Subsequently, add DNase I to digest residual DNA and perform a secondary wash with Buffer RW2 to ensure complete removal of impurities. Finally, collect RNA samples by eluting RNA with RNase-free water and centrifuging at 13,400 x g for 1 min.
2. Measure the RNA concentration.
   1. Place each 1.5 μL of sample RNA solution in a micro nucleic acid detector (see Table of Materials for details) and read the A260/A280 values.
      NOTE: If the measured A260/A280 values are within the range of 1.8 to 2.0 and the RNA concentration is between 20 ng/μL and 200 ng/μL, this indicates that the RNA quality is acceptable and can be used for subsequent experiments.
   2. Based on the measurements, dilute the RNA solution with Diethyl Pyrocarbonate (DEPC) water to ensure consistent RNA concentrations for all samples, using the measured minimum RNA concentration as a benchmark, followed by PCR experiments.
3. Reverse transcription of RNA
   1. Use the extracted total RNA as a template and synthesize cDNA according to the reverse transcription kit instructions (see Table of Materials for details). Prepare the reaction system according to the kit requirements.
   2. Add the extracted RNA solution and the reaction system to an eight-row tube, and set two replicate wells in each group. Initiate the reaction with sequential conditions: 15 min incubation at 37 °C, 5 s heat denaturation at 85 °C, 5 min cooling at 4 °C.
4. PCR reaction
   1. Conduct PCR amplification on a real-time fluorescence quantitative PCR system (see Table of Materials for details) using the SYBR Green-based premixed PCR master mix (see Table of Materials for details). Use GADPH as the reference gene, with primer sequences listed in Table 2, and prepare the reaction mixture as follows: sequentially add 0.4 μL of the corresponding primer, 2 μL of cDNA template, and the reaction mixture to an eight-well strip tube to achieve the final reaction volume.
   2. Run PCR amplification: 95 °C 30 s (1 cycle), 95 °C 5 s (40 cycles), 60 °C 20 s (40 cycles). Calculate the relative expression levels of target genes using the 2 ^-ΔΔCt method²⁵. Independently repeat all experiments three times to ensure the reliability of the data. 
      NOTE: Chemical waste generated during experimental procedures must be collected in designated hazardous waste containers and disposed of by certified hazardous waste management services in accordance with institutional policies.

Primer sequences
Primer	Sequence（5'-3'）
human-LINC00511-F	TTTCCCAGCACAGCTCAATC
human-LINC00511-R	TCCCTTCTCCCTCGGTCA
GAPDH-F	AATCCCATCACCATCTTCCA
GAPDH-R	AAATGAGCCCCAGCCTTCT

Table 2: Primer sequences for PCR assays

5. EdU detection

1. Evaluate the effect of altered LINC00511 expression on the proliferation of A549/DDP cells using an EdU assay kit (see Table of Materials for details).
   1. Randomly select A549/DDP cells that have been passaged to the third generation after resuscitation, and uniformly inoculate them into 24-well plates at a density of 2 x 10⁴ cells/well.
      NOTE: Here, all experiments were performed in three independent biological replicates (n = 3).
   2. Randomly number each well, and assign the wells to one of four experimental groups based on these random numbers: Control group, β-Ele group, β-Ele + LINC00511 overexpression (β-Ele + OV-LINC00511) group, and β-Ele + LINC00511 knockdown (β-Ele + si-LINC00511) group.
2. When the cell density reaches approximately 70%, perform plasmid transfection and interference treatment according to step 3.3. After 24 h of transfection, discard the original medium and add 1 mL of medium solution containing β-Ele (40 μg/mL) to each well of the drug treatment group.
3. Incubate the cells with EdU for labeling after treating them with 40 μg/mL of β-Ele for 24 h. Then fix the cells with 4% paraformaldehyde for 10 min and permeabilize them with 0.5% Triton X-100.
4. Add the click reaction solution (prepared by dissolving one vial of the kit-provided Click Additive reagent in 1.3 mL of deionized water with thorough mixing) in the dark and maintain the reaction in the dark for 30 min. Wash the cells with PBS after the reaction is complete, followed by Hoechst staining for 5 min. Finally, observe the results using fluorescence microscopy (Figure 1 and Figure 2).
   NOTE: DMSO is highly permeable and a flammable liquid. In this scheme, the β-Ele administration solution contains DMSO. Therefore, collect the β-Ele administration solution into a special waste liquid barrel after use and hand it to the hazardous waste treatment company for professional disposal on a regular basis.

6. Glucose content detection 

NOTE: All experiments were performed in three independent biological replicates (n = 3).

1. Randomly select A549/DDP cells that have been passaged to the third generation after resuscitation, and uniformly inoculate them into 6-well plates at a density of 1 x 10⁵ cells/well. Randomly number each well, and assign the wells to experimental groups based on these random numbers.
2. When the cell density reaches approximately 70%, proceed with plasmid transfection and interference as described in step 3.3. After 24 h of transfection, treat the cells in the treated groups with 40 μg/mL of β-Ele.
3. After 24 h of drug treatment, collect the upper culture medium from the cells and centrifuge it at 1000 × g for 5 min. Collect the supernatant. According to the instructions in the glucose test kit (see Table of Materials for details), prepare the reaction solution and mix it thoroughly with the supernatant.
4. Place the samples into 96-well plates with five replicate wells per group. Gently shake the plates and incubate them at 37 °C for 10 min. Measure the absorbance of each well using a microplate reader at a wavelength of 505 nm, and calculate the glucose content using the provided formula in the test kit:
   
   Here, C_standard = 5.55 mmol/L, N = Dilution factor.
   NOTE: Chemical waste generated during experimental procedures must be collected in designated hazardous waste containers and disposed of by certified hazardous waste management services in accordance with institutional policies.

7. Lactic acid content detection 

NOTE: All experiments were performed in three independent biological replicates (n = 3).

1. Randomly select A549/DDP cells that have been passaged to the third generation after resuscitation, and uniformly inoculate them into 6-well plates (1 × 10⁵ cells/well). Randomly number each well, and assign the wells to experimental groups based on these random numbers. When the cell density reaches approximately 70%, proceed with plasmid transfection and interference as described in step 3.3. 24 h after transfection, treat the cells in the treated groups with 40 μg/mL of β-Ele.
2. After 24 h of drug treatment, collect the upper culture medium from the cells and centrifuge it at 1000 x g for 5 min. Collect the supernatant. According to the instructions in the lactic acid test kit (see Table of Materials for details), prepare the reaction solution and mix it thoroughly with the supernatant.
3. Add a total of 250 μL of sample to each well of a 96-well plate, setting five replicate wells for each group. Gently shake the plate and incubate it at 37 °C for 10 min. Measure the absorbance of each well at a wavelength of 530 nm using a microplate reader (see Table of Materials for details), and calculate the lactate content using the provided formula in the test kit:
   
   Here, C_standard =3 mmol/L, N = Dilution factor.
   NOTE: Chemical waste generated during experimental procedures must be collected in designated hazardous waste containers and disposed of by certified hazardous waste management services in accordance with institutional policies. 

8. Bulk RNA-seq

1. Establish a sample database
   1. Select A549/DDP cells for the experiment and divide them into the following groups: Control group, DDP group, β-Ele (40 μg/mL) + DDP (48 μM) group, β-Ele+ DDP + OV-LINC00511 group and β-Ele + DDP +si-LINC00511 group. Include 3 samples in each group.
   2. Extract total RNA from each cell group. Use a magnetic bead-based mRNA isolation kit (see Table of Materials for details) to enrich eukaryotic mRNA, in which Oligo(dT)-coated beads selectively bind to the poly(A) tails of mRNA via A-T base pairing. Add fragmentation buffer (1 μL/μg of RNA, see Table of Materials for details) and incubate at 94 °C for 7 min to fragment the mRNA into short segments.
   3. Proceed with first-strand cDNA synthesis using random hexamer primers according to the manufacturer's instructions. For second-strand cDNA synthesis, add appropriate amounts of reaction buffer, RNase H, and DNA polymerase I to complete the process.
   4. Perform end repair and A-tailing on double-stranded cDNA by incubation at 25 °C for 30 min (end repair), immediately followed by 72 °C for 30 min (A-tailing). Ligate sequencing adapters to the end-repaired fragments.
   5. Purify and size-select ligated products using carboxyl-modified magnetic silica beads (see Table of Materials for details). Finally, construct the sequencing library by PCR amplification.
   6. Following sample preparation, analyze the sequencing library on the DNBSEQ-T7 platform (see Table of Materials for details). Load the sample onto a Patterned Array microarray chip for sequencing.
      NOTE: The instrument collected, read, and identified optical signals using a high-resolution imaging system to determine the sequence information of individual bases. Subsequently, perform additional sequencing cycles to obtain the sequence of the following bases. After multiple cycles, the raw sequencing data were generated.
2. Bioinformatics analysis 
   1. Sequence the samples on the machine to obtain the raw data, also known as the off-machine data. Further, process the sequencing data using fastp (v 0.21.0). Remove reads containing 3' adapter sequences and eliminate low-quality reads with an average quality score below Q20 as part of the primary filtering criteria. Conduct all downstream analyses based on the cleaned, high-quality data.
      NOTE: Conduct initial filtering of sequencing data using fastp. Refer to the official documentation for transcriptomic datasets (https://github.com/OpenGene/fastp#rna-seq-processing) for the specific command-line parameters.
   2. Use FastQC (v 0.12.0) to assess the quality of the cleaned sequencing data. The quality requirements are as follows: at least 90% of bases must have a quality score higher than Q20, and the percentage of remaining adapter sequences should not exceed 1%. Data that meet these criteria are considered qualified and can be used for downstream analysis.
      NOTE: Assess the quality of the cleaned sequencing data using FastQC, following the official guidelines (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
3. Gene expression analysis 
   1. Use DESeq2 software to perform differential expression analysis between the two comparison groups.
      1. First, input the data into the software to construct a DESeq DataSet. Then, filter out genes with a total count of fewer than 10 across all samples.
      2. Finally, conduct the differential gene analysis and extract the results. The conditions for screening differentially expressed genes are as follows: the fold change in expression |log2FoldChange| > 1, and the significance padj ≤ 0.05.
         NOTE: For detailed operation steps of DESeq2 software, please refer to the detailed steps on the official website: https://www.bioconductor.org/packages/release/bioc/html/DESeq2.html.

9. Detection of protein expression level changes by Western blot (WB) 

NOTE: All experiments were performed in three independent biological replicates (n = 3).

1. Perform Western blotting to quantify cellular protein expression levels.
   1. Randomly select A549/DDP cells that have been passaged to the third generation after resuscitation, and uniformly inoculate them into 6-well plates at a density of 1 x 10⁵ cells/well.
   2. Randomly number each well, and assign the wells to one of the six experimental groups based on these random numbers: Control, β-Ele, OV-LINC00511, si-LINC00511, β-Ele + OV-LINC00511, and β-Ele + si-LINC00511.
   3. When the cell density reaches approximately 70%, perform plasmid transfection and interference as described in step 3.3. After 24 h of transfection, expose the treatment group to β-Ele (40 μg/mL), while providing fresh medium to the control group. Culture both groups for an additional 24 h.
   4. Subsequently, collect cell pellets by centrifugation at 1675 × g for 5 min. Lyse each group of cells with 500 μL of RIPA lysis buffer containing 1 mM PMSF on ice for 30 min (see Table of Materials for details).
2. Centrifuge the cell lysates at 13,400 × g for 10 min at 4 °C and collect the supernatant. Measure protein concentrations using the BCA Protein Concentration Assay Kit (see Table of Materials for details). Separate equal amounts of protein samples by 10% SDS-PAGE and transfer them to polyvinylidene fluoride (PVDF) membranes via electrophoresis.
3. Immerse membranes in blocking solution (5% skim milk/Tris-buffered saline with 0.1% Tween-20 (TBST)) for 2 h at room temperature and incubate them overnight at 4 °C with primary antibodies against GLUT1, PKM2, LDHA, β-catenin, GSK-3β (1:1000), and β-Actin (1:2000, see Table of Materials for details).
   1. The next day, wash the membranes three times with TBST, 5 min each, at room temperature with gentle shaking.
4. The next day, incubate the membranes with secondary antibody solutions (HRP-labeled goat anti-rabbit IgG, 1:2000) and shake gently at room temperature for 1 h.
5. Finally, add 0.1 mL/cm² of commercially available ECL chromogenic solution to fully cover the PVDF membranes. Incubate for 1 min at room temperature before exposure. Visualize and analyze the bands using an enhanced chemiluminescence system (see Table of Materials for details). Please refer to Figure 4C and Figure 5B.
   NOTE: Chemical waste generated during experimental procedures must be collected in designated hazardous waste containers and disposed of by certified hazardous waste management services in accordance with institutional policies.

10. Statistical anaysis 

NOTE: All experiments were performed in three independent biological replicates (n = 3).

1. Organize the experimental data by group and input them into GraphPad Prism software (Version 8.0.2; see Table of Materials for details). Perform data visualization and statistical significance analysis in accordance with the software guidelines. Measurement data were expressed as mean ± standard deviation. Statistical differences between groups were assessed using One-way ANOVA with η² (eta-squared) effect size calculation, followed by Tukey's multiple comparisons test²⁶. Consider **P < 0.01 as statistically significant.

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