IGF2BP1-mediated methylation of ABCA1 facilitates tumor progression by affecting cholesterol metabolism in lung adenocarcinoma

Abstract

ABCA1 is a key protein in maintaining cholesterol homeostasis, and its abnormal expression is associated with the progression of many cancers. Nonetheless, the specific molecular mechanisms by which ABCA1 facilitates the development of LUAD remain largely unexplored, necessitating further in-depth investigation. The TCGA-LUAD database was used to analyze the expression of ABCA1 in LUAD tissues. Subsequently, a cell model with overexpressed ABCA1 was constructed for verification through cell experiments. Cell function was evaluated using the Transwell assay and the colony formation assay. Intracellular cholesterol levels were detected using a kit. At the same time, the online database RM2 Target was employed to predict upstream factors that may have a methylation regulatory relationship with ABCA1. On this basis, Dot blot and MeRIP-qPCR techniques were employed to determine the degree of m6A modification. To clarify the mechanism of IGF2BP1 regulating ABCA1 through the m6A pathway, RNA pull-down binding experiments were carried out, and changes in mRNA stability were assessed using actinomycin D treatment. Finally, the biological function of the IGF2BP1/ABCA1 signaling axis during the growth and metastasis of LUAD in vivo was evaluated by establishing a xenograft animal model. Bioinformatics analysis and cell experimental results confirmed the low expression of ABCA1 in LUAD tissues and cells. ABCA1 significantly inhibited cell proliferation, migration, and invasion capabilities, promoted apoptosis, and reduced intracellular cholesterol levels. From a molecular perspective, IGF2BP1 recognized and bound to methylation sites on ABCA1 mRNA, thereby accelerating its degradation process, resulting in a substantial decrease in the stability of ABCA1 mRNA. Moreover, in vivo and in vitro experiments further confirmed that IGF2BP1 affected cholesterol metabolism by regulating the expression of ABCA1, thereby facilitating the malignant progression of LUAD. Overall, our research revealed that IGF2BP1 affects cholesterol metabolism by reducing the stability of ABCA1 mRNA through m6A modification, thereby boosting the malignant progression of LUAD and formulating a theoretical basis for subsequent LUAD treatment.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00726-025-03474-1.

Introduction

According to global cancer statistics, lung cancer accounted for 12.4% of all new cases of malignant tumors in 2022, making it the top cause of death among various types of cancers (Bray et al. 2024). Among them, lung adenocarcinoma (LUAD) is the most common histological subtype, and its global prevalence remains high. Age-standardised incidence rates for adenocarcinoma were estimated to be 12.4 per 100,000 person-years in males and 8.3 per 100,000 person-years in females worldwide (Zhang et al. 2023a, b). In terms of clinical treatment, surgery remains the preferred treatment option for LUAD (Bian et al. 2023), accompanied by chemotherapy (Wang et al. 2021a, b, c), specific molecule-targeted drugs (Wang et al. 2023), and other comprehensive treatment strategies. Although these treatments have elevated the survival rates of LUAD patients to some extent, the emergence of drug resistance and adverse effects has considerably impeded clinical treatment efficacy. Consequently, delving deeper into the biological mechanisms of LUAD progression to identify novel biomarkers and provide theoretical foundations for the development of more effective therapeutic strategies is imperative.

As we all know, metabolism is a crucial indicator of cancer progress (Yang et al. 2023). Cholesterol metabolism serves as an important metabolic adaptation strategy and exerts a pivotal influence in pancreatic cancer (PC) (Zheng et al. 2022), colorectal cancer (CRC) (Ren et al. 2024), and liver cancer (Wang et al. 2021a, b, c). A study has shown that gene alpha-endosulfine (ENSA) amplification increases cholesterol biosynthesis by up-regulating sterol regulatory element-binding transcription factor (SREBP) and reinforces the progression of triple-negative breast cancer (TNBC) (Chen et al. 2022a, b, c). Hydroxymethylglutaryl-CoA Synthase 1 (HMGCS1) facilitates tumor growth by reinforcing cholesterol synthesis in glioblastomas (Zhao et al. 2024). Based on the aforementioned evidence, disturbed cholesterol metabolism can exert great influence on the occurrence and development of malignant tumors, which offers an important theoretical foundation for the development of novel anti-cancer strategies targeting cholesterol metabolic pathways. ATP-binding cassette transporter A1 (ABCA1), as one of the proteins involved in cholesterol homeostasis, is mainly responsible for transporting excess cholesterol in cells to lipoproteins to strengthen its efflux (Chen et al. 2022a, b, c). In various types of tumor cells, such as small cell lung cancer (Kashiwagi et al. 2022), and renal clear cell carcinoma (Liang et al. 2022), upregulating ABCA1 can inhibit tumor progression by regulating cholesterol metabolism. This suggests that targeting ABCA1 to restore cellular cholesterol metabolic balance is expected to become a potential LUAD treatment strategy. At the same time, in-depth research on its upstream regulatory genes can lay the foundation for us to reveal the molecular mechanism of LUAD progression more comprehensively.

N6-methyladenosine (m6A), one of the most prevalent modifications of eukaryotic mRNA, plays a pivotal role in tumor progression (Ma et al. 2019). The dynamic regulation process of m6A modification is mainly mediated by three types of functional molecules: the methyltransferase (writer) responsible for adding methyl groups, the demethylase (eraser) that performs demethylation, and the RNA binding protein (reader) that recognizes methylation sites. These regulatory factors cooperate to participate in mRNA splicing processing, nucleoplasmic transport, translation regulation, and degradation metabolism (An and Duan 2022; Wang et al. 2020). Among the many m6A reading proteins, the IGF2 mRNA binding protein family (IGF2BP1/2/3) has garnered much attention for its ability to specifically recognize m6A modification sites (Duan et al. 2024). This protein family plays an important part in the occurrence and development of various malignant tumors by regulating the stability of target mRNAs, such as CRC (Liu et al. 2022), head and neck squamous cell carcinoma (Paramasivam et al. 2021), and breast cancer (BC) (Jiang et al. 2022). Collectively, Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is essential in driving tumor progression, but its function in LUAD is not fully understood and further exploration is still required.

In the current study, we identified that ABCA1 was considerably down-regulated in LUAD tissues and cells, and its overexpression repressed cell proliferation, migration, and invasion, enhanced apoptosis, and reduced intracellular cholesterol accumulation. We also revealed that the RNA-binding protein IGF2BP1 mediated the m6A methylation of ABCA1, which degraded ABCA1 by reducing its mRNA stability, which in turn leads to the accumulation of cholesterol in LUAD cells, and ultimately promotes the malignant progression of tumors. To sum up, this work revealed the molecular regulatory network of ABCA1 in the occurrence and development of LUAD. The discovery not only deepens the understanding of the pathological mechanism of LUAD but also offers a crucial theoretical basis for developing treatment strategies based on ABCA1 targets.

Materials and methods

Data collection and analysis

We utilized the Gene Expression Profiling Interactive Analysis (GEPIA) online website (http://gepia.cancer-pku.cn/detail.php) Tang et al. 2017, combined with the TCGA database (https://portal.gdc.cancer.gov/) Cancer Genome Atlas Research, N 2014 and the Prototype Tissue Expression (GTEx) databases, to obtain the transcriptome sequencing data of LUAD. Bioinformatics analysis was performed using the R language to compare the expression profiles of ABCA1 between 483 tumor samples and 347 normal tissues adjacent to cancer. The screening criteria were set to ensure that differentially expressed genes met both the statistical thresholds of P < 0.05 and | Log2FC |>0.585. The GEPIA website was utilized to analyze the relationship between ABCA1 expression and patient survival. Furthermore, by leveraging the online database RM2 Target, genes related to ABCA1 m6A methylation were predicted.

Cell cultivation

The cell lines used here included human normal lung epithelial cells HBE (FH1014), LUAD cells A549 (BNCC337696), and Calu-3 (BNCC359757), all purchased from Shanghai FuHeng Biology and BeNa Culture Collection (BNCC) (China). The cell culture system is as follows: HBE cells were kept in the KM medium (BNCC, China). A549 cells were cultivated in the F-12 K medium (BNCC, China), and Calu-3 cells were in the EMEM medium (BNCC, China). All media were supplemented with 1% streptomycin-penicillin solution (Penocillin, China) and 10% fetal bovine serum (FBS). Cells were housed in an incubator (Thermo Fisher Scientific, USA) at 37 ℃ with 5% CO₂.

Cell transfection

To achieve overexpression of IGF2BP1 and ABCA1, specific plasmids pcDNA3.1 were provided and synthesized by RIBOBIO (China). The oe-ABCA1 and oe-IGF2BP1 overexpression plasmids and their corresponding control vectors were introduced into the A549 cell line using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, USA). After 48 h of transfection treatment, the transfection efficiency was tested.

Quantitative real-time polymerase chain reaction (qRT-PCR)

The transfected cells were collected after 48 h of transfection. TRIzol reagent (Invitrogen, USA) was employed to extract total RNA from A549 cells. We strictly followed the experimental protocol provided by the manufacturer to ensure the integrity and purity of RNA. Subsequently, a reverse transcription reaction was conducted using the PrimeScript RT kit (Takara, Japan) to obtain a cDNA template. The qPCR testing was completed on an ABI7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific, USA). The reaction conditions were pre-denaturation at 95 ℃ for 30 s, with 40 times of cycling. The denaturation was at 95 ℃ for 5 s. Annealing and extension were at 60 ℃ for 30 s. With three technical replicates set up for each sample. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous reference, the relative expression of the target gene was calculated by the 2^−ΔΔCt method. Results were expressed as the expression fold of the experimental group relative to the control group. All targeting primer sequences are shown in Table 1. To ensure the reproducibility of the experiments, we repeated the experiments in each group three times.

Table 1.

Information on primer sequence

Gene	Primer sequence
ABCA1	Forward primer: 5’-ATTCCTCAAGGTGGCCGAAG-3’; Reverse primer: 5’-AAGACAGCTCTGCTTGTCCC-3’
IGF2BP1	Forward primer: 5’-GTGACACACCAGCCCTCTC-3’; Reverse primer: 5’-CCCCACCCCAGAAGTTGTC-3’
GAPDH	Forward primer: 5’-AATGGGCAGCCGTTAGGAAA-3’; Reverse primer: 5’-GCGCCCAATACGACCAAATC-3’

Western blot (WB)

After the experimental samples were processed, they were rinsed three times with pre-cooled PBS buffer. After lysing cells with RIPA lysis buffer (containing protease and phosphatase inhibitors), the protein was quantified using the BCA protein quantification kit (Thermo Fisher Scientific, USA). An equal amount of protein was loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The blocking step was treated with 5% skimmed milk for 45 min, followed by incubation with the primary antibody at 4 ℃ for 12–16 h. The next day, the horseradish peroxidase (HRP)-conjugated secondary antibody (ab205718, Abcam, UK) was applied to incubate for 60 min at room temperature. After rinsing with phosphate-buffered saline with Tween-20 (PBST) buffer, the color reaction was carried out using an ECL chemiluminescent kit (Biorad, USA). The image data was finally collected and analyzed utilizing an iBright imaging system (Thermo Fisher Scientific, USA). Antibodies are shown in Table 2. To ensure the reproducibility of the experiments, we conducted three independent replicates for each group of experiments.

Table 2.

Information on antibodies

Antibody	Catalog Number	Company
ABCA1 (Primary antibody)	ab307534	Abcam (UK)
IGF2BP1 (Primary antibody)	ab184305	Abcam (UK)
GAPDH (Primary antibody)	ab181602	Abcam (UK)
IgG (Secondary antibody)	ab238531	Abcam (UK)

Transwell

Cells were treated under different conditions for 24 h, then digested with trypsin (Beyotime, China) and collected. For migration experiments, 2 × 10⁴ cells were resuspended in 200 µL serum-free medium and seeded into the upper chamber of a Transwell (Corning, USA). A complete medium containing 10% FBS was added to the lower chamber as a chemical attractant. In the invasion experiment, the Transwell membrane was pre-coated with 100 µL of Matrigel (BD Biosciences, USA) and incubated at 37 ℃ for 2 h to fully solidify it. Subsequently, the concentration of the cell suspension was adjusted to 2 × 10⁴ cells/mL, 200 µL was added to the upper chamber of Transwell, and 500 µL of DMEM medium containing 10% fetal bovine serum (FBS) (BNCC, China) was added to the lower chamber, following 24 h of cultivation at 37 ℃. After the experiment, non-migrating cells on the membrane surface were removed with a moist cotton swab. After fixation with 75% ethanol, they were stained with 0.5% crystal violet (Beyotime, China) for 10 min. Finally, three fields of view were randomly selected under a microscope (Carl Zeiss, Germany) for photography and cell counting analysis.

Colony formation assay

Treated cells were inoculated into a 12-well plate, with 150 cells per well, and the medium was changed regularly to maintain normal cell growth. Following the cultivation for 10–14 days, upon observing clear cell clumps under the microscope, two washes with phosphate buffer saline (PBS) solution were performed. Subsequently, the samples were fixed in 4% paraformaldehyde solution (Beyotime, China) at room temperature for 15 min. After fixation was completed, 0.5% crystal violet solution (Beyotime, China) was added for dyeing for 30 min. Unbound cells were washed away with PBS. The cell colonies in each well plate were counted. To ensure the reproducibility of the experiments, we conducted three independent replicates for each group.

Apoptosis detection

The apoptosis rate was detected using the Annexin V-FITC/PI double staining method, and the kit was purchased from Qcbio Science & Technologies (China). Specifically, the cells were collected and rinsed twice with PBS buffer, then the cells were resuspended in staining buffer containing 50 µg/mL RNase A (Sigma, USA). Annexin V-FITC and PI staining solutions were added. After incubation at room temperature without light for 120 min, an Agilent flow cytometer (Agilent, USA) was used for detection. The apoptotic cell population was quantitatively analyzed through analysis software. To ensure the reproducibility of the experiments, we conducted three independent replicates for each group.

Quantification of cholesterol

The total cholesterol detection kit from Solarbio Company (China) was applied to measure the total cholesterol content in cell samples and tumor tissue samples, respectively. In short, 0.1 g of tissue was collected, and 1 mL of extract was added for ice bath homogenization. Centrifugation was conducted at 10,000 g, 4 ℃ for 10 min. The supernatant was removed and placed on ice for testing. 5 million cells were added to the 1 mL of extraction solution, and disrupted with ice bath ultrasound (power 300 w, ultrasound 2 s, interval 3 s, total time 3 min), then centrifuged at 10,000 g at 4 ℃ for 10 min. The supernatant was placed on ice for testing. Each reaction reagent was added and mixed well, being undisturbed at 37 ℃ for 15 min. The absorbance at 500 nm was examined after the reaction was completed. The extracellular cholesterol content detection kit (Applegen, E1005) was employed to detect the cholesterol content in the culture medium. To ensure the reproducibility of the experiments, we conducted three independent replicates for each group.

FNA pull-down

The Pierce™ Magnetic Beads RNA-Protein Interaction Enrichment Kit (Thermo Fisher Scientific, USA) was used to conduct RNA pull-down experiments. The experimental steps are as follows: First, RNA was synthesized by in vitro transcription, and ABCA1-WT and ABCA1–306MUT were labeled with biotin, respectively. Among them, ABCA1-306MUT was constructed as a mutant by replacing adenosine (A) at position 306 with thymine (T), using Poly (A) 25RNA as a negative control. Subsequently, the biotin-labeled RNA was mixed with pre-washed magnetic beads and incubated at 4 ℃ for 30–60 min. Subsequently, the cell lysate was prepared and mixed with the RNA-magnetic bead complex, and incubated at 4 ℃ for 45 min. After incubation, the magnetic beads were washed three times with Wash Buffer to remove non-specifically bound proteins, and then the RNA-protein complex was eluted using Biotin Elution Buffer. Samples from each group were collected. Total protein was extracted, and the concentration was kept at ≥ 2 mg/mL. Finally, the expression level of the target protein was detected by WB. We conducted three independent replicates for each group to ensure the reproducibility of the experiment.

The m6A dot blot assay

First, total RNA was extracted from A549 cells using a Trizol reagent (Beyotime, China). Next, the ratio of m6A modification level to total mRNA was determined using the EpiQuik m6A RNA Methylation Quantitative Kit (AmyJet Scientific, China). The experimental steps for RNA methylation detection are as follows: First, a 4 µg total RNA sample was taken and purified using the RNeasy Mini Kit (Qiagen, China). The purified RNA samples were separated by 1.2% formaldehyde-sepharose electrophoresis and then transferred to a Hybond N + nylon membrane (GE Healthcare, USA). Three UV cross-linked fixation was performed using an automatic cross-linking instrument. The crosslinked membrane was stained in 0.02% methylene blue (MB, Sigma, USA)/0.3 M sodium acetate staining solution for 5 min to confirm RNA transfer efficiency. The stained membranes were washed three times with 1× PBST buffer and then blocked in 5% skimmed milk/PBST blocking solution at room temperature for 60 min. The blocked membranes were incubated with m6A-specific primary antibodies (202003, Synaptic Systems, Germany) at 4 ℃ for 12–16 h. The next day, the membrane was incubated with HRP-labeled goat anti-rabbit IgG secondary antibodies (ab238531, Abcam, UK) for 60 min at room temperature. Finally, chemiluminescent development was performed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, USA), and signals were collected using a chemiluminescent imaging system. To ensure the reproducibility of the experiments, we conducted three independent replicates for each group.

MeRIP-qPCR

The N6-Methylated RNA Immunoprecipitation Kit (C11051-1) was purchased from RIBOBIO (China), and the experimental operations were performed according to the instructions. Firstly, 50–100 µg of total RNA sample was taken, with RNA lysis buffer added for lysis. 10% of the lysis product was retained as the Input control. Next, 25 µL Protein A/G magnetic beads were utilized to incubate and bind with 5 µg m6A-specific antibody and IgG control antibody provided by the kit. After the addition of MeRIP reaction buffer, the magnetic beads mixture was incubated at 4 ℃ for 2 h and washed three times. Unbound protein was then removed by digestion with Proteinase K (Magen, China), and the m6A-modified RNA fragment was purified using 6.7 mM N6-methyladenosine 5-monophosphate sodium salt (Sigma, USA). Finally, it was reverse transcribed into cDNA using the FastKing cDNA First Strand Synthesis Kit (TIANGEN, China), and the expression of ABCA1 was detected by qRT-PCR. The reverse transcription and qRT-PCR steps were the same as described previously. Based on the prediction results of the m6A modification site prediction platform SRAMP (http://www.cuilab.cn/sramp), ABCA1 gene-specific MeRIP-qPCR amplification primers were designed and synthesized (5’-CTGTTTTCTCCCCTTCTCCG-3’; 5’-ATAAATAGAGGCCAGGCCAC-3’). To ensure the reproducibility of the experiments, we conducted three independent replicates for each group.

RNA immunoprecipitation (RIP) and qPCR (RIP-qPCR)

The RIP-qPCR was based on methods in a previous experiment (Zhang et al. 2023a, b). Specifically, YTHDF2 labeled with Flag was stably transfected into A549 cells until the cell density reached around 80%. The cells were washed once with PBS and lysed with RIP buffer containing 100 U/mL RNAase inhibitor and protease inhibitor. Next, we incubated the whole cell extract with protein A/G magnetic beads conjugated with YTHDF2 antibody and negative control IgG antibody at 4 ℃ for 6 h. Afterwards, the magnetic beads were rinsed three times with RIP buffer, and then incubated sequentially with DNase I without RNase and proteinase K (Magen, China) at 37 ℃ for 15 min to remove unbound proteins. Finally, RNA was extracted from the co-precipitate and subjected to qRT-PCR analysis.

RNA stability determination

When the cell fusion rate reached 70–80%, treatment with 5 mg/mL puromycin D (MedChemExpress, MCE, USA) for 0, 2, 4, and 6 h was conducted to repress RNA transcription. At each time point, total RNA from cells was extracted using TRIzol reagent (Takara, Japan). Subsequently, the cDNA template was obtained by reverse transcription using PrimeScript RT reagent (Takara, Japan) and amplified on a QLightCycler480 real-time fluorescent quantitative PCR instrument (Roche, Switzerland). The experimental data was calculated by calculating the average of Ct values at each time point, comparing it with the 0 h control group to obtain the ΔCt value, and finally using the 2^−ΔCt method to calculate the relative mRNA expression level. To ensure the reproducibility of the experiments, we conducted three independent replicates for each group.

Animal experiment

The 4-6-week-old BALB/c nude mice used were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. (China). All experimental animals were kept in a specific pathogen-free (SPF) barrier environment for one week to ensure their physiological stability. Subsequently, 15 nude mice were randomly assigned to 3 experimental groups (5 mice per group). The control group was inoculated with A549 cells stably expressing oe-NC, and the experimental group was inoculated with A549 cell suspensions stably transfected with oe-IGF2BP1 or oe-IGF2BP1 + oe-ABCA1. During the modeling period, tumor growth was monitored and recorded regularly every week. At the experimental endpoint (4 weeks), the experimental animals were euthanized humanely, the tumors were completely peeled off and weighed, and the tumor tissues were then split and stored for subsequent analysis. This study protocol was approved by the Laboratory Animal Ethics Review Committee of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Approval Number SRRSH2025-0014.

Immunohistochemistry (IHC)

Subcutaneous transplanted tumor tissue derived from A549 cells was embedded in paraffin, and 4 μm sections were prepared. Sections were first dewaxed with xylene, then hydrated with a series of decreasing concentrations of ethanol (100%, 95%, 80%, 70%), and rinsed 3 times in PBS buffer. Next, an EDTA antigen retrieval solution was employed to perform epitope retrieval under high temperatures. The repaired sections were incubated with 5% BSA blocking solution for 30 min at 37 ℃ to block non-specific binding sites. Sections were incubated at 4 ℃ with the following primary antibodies for 12–16 h: rabbit anti-IGF2BP1 (ab290736, Abcam, UK), rabbit anti-ABCA1 (ab7360, Abcam, UK), and rabbit anti-Ki67 (ab15580, Abcam, UK). The next day, sections were incubated with HRP-labeled secondary antibody (ab6721, Abcam, UK) at 37 ℃ for 30 min. Subsequently, the color reaction was carried out using a DAB color reagent (ZLI-9018, ZSGB-Bio, China) for 5 min, and finally counterstained with hematoxylin stain solution (GT100540, Gene Tech, China) for 60 s. Following dehydration and embedding procedures, positive expression of IGF2BP1, ABCA1, and Ki67 in tissue was observed under an optical microscope. The positively stained cells appeared brown. Each group of experiments consisted of 3 mice, and each mouse’s tumor tissue was independently sliced and stained once.

Data processing

All experiments were independently repeated three times, and data were expressed as mean ± SEM (standard error of the mean). Analysis was performed using SPSS v21.0 statistical software. Student’s t-test was applied in the comparison between the two groups. Charts were drawn using GraphPadPrism8 software. The difference was statistically significant when *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Results

ABCA1 is down-expressed in LUAD and boosts the malignant progression of tumors

To explore the biological function of ABCA1 in LUAD, we analyzed the expression of ABCA1 in LUAD using the GEPIA online website combined with the TCGA and GTEX databases. By comparative analysis of cancer tissue and normal lung tissue samples, the results demonstrated that the transcription level of ABCA1 in LUAD tissue was significantly down-regulated compared with normal tissue (P<0.05), and this difference was statistically significant (Fig. 1A). The survival analysis also indicated that high expression of ABCA1 was associated with poor prognosis in LUAD patients (Fig. 1B). Based on this, we screened LUAD cell lines expressing ABCA1 and explored their potential impact on the development of LUAD in vitro. The expression of ABCA1 in different cell lines was compared. In LUAD cell lines, ABCA1 expression was downregulated compared to normal human lung epithelial cells HBE. (Fig. 1C, D). Based on this expression characteristic, we selected A549 cells with low ABCA1 expression for follow-up analysis. To clarify the biological function of ABCA1, an oe-ABCA1 overexpression vector was constructed, and oe-NC was established as a control. The level of ABCA1 after transfection was considerably elevated by qRT-PCR and WB (Fig. 1E). Functional experimental results revealed that after upregulating ABCA1 expression, the migration and invasion ability of A549 cells were dramatically repressed (Fig. 1F). At the same time, cloning experiments further confirmed that overexpression of ABCA1 greatly reduced the proliferation ability of tumor cells (Fig. 1G). Flow cytometry analysis further revealed that overexpression of ABCA1 enhanced apoptosis of A549 cells (Fig. 1H). Taken together, ABCA1 is significantly downregulated in LUAD, and its high expression can effectively inhibit the malignant progression of tumor cells.

Fig. 1.

ABCA1 is down-expressed in LUAD and facilitates its malignant progression. A The analysis and comparison of the differences in ABCA1 transcription levels between LUAD tissue and normal lung tissue based on the GEPIA website; B The analysis of the relationship between ABCA1 expression and patient survival based on the GEPIA website; C The expression of ABCA1 mRNA in three cell lines: HBE, A549, and Calu-3 was detected by qRT-PCR; D The expression of ABCA1 protein in three cell lines: HBE, A549, and Calu-3 was measured by WB; E qRT-PCR and WB were used to verify the overexpression efficiency of ABCA1 after plasmid transfection; F Transwell assay was used to evaluate the migration and invasion characteristics of cells in each group. Scale bar was 200 μm; G Changes in cell proliferation ability were analyzed using clone formation experiments; H The apoptosis level of cells in each group was detected by flow cytometry. Data are presented as the mean ± SEM. * means P < 0.05, ** means P < 0.01, *** means P < 0.001 and **** means P < 0.0001 were determined by t-test

ABCA1 affects LUAD progress by reinforcing cholesterol efflux

ABCA1 is proven to regulate cellular metabolism by boosting cholesterol efflux, thereby affecting the progression of endometrial cancer (Wen et al. 2023). To further investigate the effect of ABCA1 on cholesterol metabolism during the malignant progression of LUAD, we conducted fluorescence probe assay, Transwell, and flow cytometry. First, cholesterol levels were quantitatively detected using the Amplex^® Red fluorescent probe method. Experimental data demonstrated that ABCA1 overexpression greatly reduced intracellular cholesterol accumulation compared to the control group transfected with an empty vector and increased the accumulation of cholesterol in extracellular culture medium (Fig. 2A). To further verify whether ABCA1 affects the biological function of LUAD cells through cholesterol metabolism, we added Ethanol (dissolve cholesterol) (Beyotime, China) or Cholesterol (CHOL, MCE, USA) to oe-NC or oe-ABCA1 LUAD cells, respectively, to regulate intracellular cholesterol levels, and constructed oe-NC + Ethanol, oe-NC + CHOL, oe-ABCA1 + CHOL groups. The addition of CHOL promoted the uptake of cholesterol by cells and increased the cholesterol content in extracellular culture medium, while overexpression of ABCA1 inhibited CHOL-induced cholesterol accumulation (Fig. 2B). According to the Transwell and colony formation assays, elevated cholesterol uptake boosted cell migration, invasion, and proliferation, and overexpressing ABCA1 partially reversed this effect (Fig. 2C, D). Furthermore, in flow cytometry, the addition of CHOL reduced apoptosis, while overexpression of ABCA1 simultaneously partially elevated cell apoptosis rates (Fig. 2E). Given the above results, ABCA1 affects the progression of LUAD by enhancing the mechanism of cellular cholesterol efflux.

Fig. 2.

ABCA1 affects LUAD progression by reinforcing cholesterol efflux. Cell grouping: oe-NC, oe-ABCA1. A Intracellular and extracellular cholesterol levels were measured by the Amplex^® Red cholesterol assay; cell groups: oe-NC + Ethanol, oe-NC + CHOL, and oe-ABCA1 + CHOL; B Intracellular cholesterol levels in each group were measured by the Amplex^® Red cholesterol assay; C Transwell assay was used to detect the migration and invasion abilities of cells in each group. Scale bar was 200 μm; D Clone formation assay was used to detect the proliferation ability of cells in each group; E Apoptosis was detected by flow cytometry in each group. Data are presented as the mean ± SEM. * means P < 0.05, ** means P < 0.01, *** means P < 0.001 and **** means P < 0.0001 were determined by t-test

IGF2BP1 regulates ABCA1 stability through m6A methylation

The aforementioned assays demonstrated that the down-regulation of ABCA1 expression was closely related to the malignant progression of LUAD, but the mechanism of its abnormally low expression in LUAD has not yet been clarified. Therefore, we further probed into the mechanism underlying this phenomenon. According to a former work, m6A, a type of RNA epigenetic modification, plays a crucial regulatory role in gene expression post-transcriptionally (He et al. 2019). Based on this, we predicted genes potentially associated with the ABCA1 methylation utilizing online tools. IGF2BP1 was observed to be potentially implicated in the regulation of m6A modification at position 306 of ABCA1 (Fig. 3A). To clarify the regulatory mechanism of IGF2BP1 on ABCA1, we employed qRT-PCR and WB to detect the expression of IGF2BP1 in HBE, A549, and Calu-3 cell lines. Compared with normal lung epithelial cells, IGF2BP1 was greatly over-expressed in LUAD cells, which down-regulated the expression of ABCA1 at the transcriptional and translational levels (Fig. 3B–E). To further confirm whether IGF2BP1 binds to ABCA1 mRNA, we used biotin-labeled ABCA1-WT and ABCA1-306 MUT probes to conduct RNA pull-down experiments. The results demonstrated that IGF2BP1 could be specifically captured by biotin-labeled ABCA1-WT probes, but ABCA1-306 MUT probes had no such effect (Fig. 3F). Through Dot blot experiments, it was found that overexpression of IGF2BP1 greatly elevated the overall level of intracellular m6A modification (Fig. 3G). MeRIP-qPCR experiments further confirmed that IGF2BP1 specifically enhanced the m6A modification of ABCA1 mRNA (Fig. 3H). To evaluate the effect of IGF2BP1 on the transcriptional stability of ABCA1, cells were treated with the RNA synthesis inhibitor actinomycin D (5 µg/mL, MCE, USA). Combined with qRT-PCR, overexpression of IGF2BP1 was found to greatly shorten the half-life of ABCA1 mRNA (Fig. 3I). YT521-B homologous domain family member 2 (YTHDF2) is an N6 methyladenosine (m6A) binding protein initially discovered for regulating mRNA stability (Chen et al. 2022a, b, c). To further confirm whether the degradation pathway of ABCA1 was mediated by YTHDF2, we performed RIP-qPCR detection. The test results showed that compared with the control group, IgG, the YTHDF2 treatment group significantly enriched ABCA1 mRNA, which also suggested that IGF2BP1 degradation of ABCA1 may be achieved by regulating YTHDF2 (Fig. 3J). Collectively, IGF2BP1 inhibits the expression of ABCA1 by mediating m6A methylation modification of ABCA1.

Fig. 3.

IGF2BP1 reinforces ABCA1 degradation through m6A methylation modification. A Genes associated with ABCA1 methylation modifications were predicted through the RM2 Target database; B The mRNA expression level of IGF2BP1 in HBE, A549, and Calu-3 cells was measured by qRT-PCR; C WB was used to detect the expression of IGF2BP1 protein in HBE, A549, and Calu-3 cells; cell groups included oe-NC and oe-IGF2BP1; D The expression level of ABCA1 mRNA in each group of cells was analyzed by qRT-PCR; E WB was used to detect the expression level of ABCA1 protein in cells of each group; F The interaction between ABCA1 and IGF2BP1 was verified using RNA pull-down experiments; G The level of m6A methylation in cells in each group was detected by Dot blot; H MeRIP-qPCR was used to quantitatively analyze the m6A methylation level of ABCA1 in cells in each group; I The expression level of ABCA1 mRNA in cells in each group at different time points (0, 2, 4, 6 h) was detected by qRT-PCR; J The expression level of ABCA1 mRNA was detected by RIP-q-PCR. Data are presented as the mean ± SEM. * means P < 0.05, ** means P < 0.01, *** means P < 0.001 and **** means P < 0.0001 were determined by t-test

IGF2BP1/ABCA1 axis facilitates migration and invasion of LUAD cells by affecting cholesterol metabolism

To further dig out how the IGF2BP1/ABCA1 axis affected LUAD progression, we constructed three groups: oe-NC control group, oe-IGF2BP1 experimental group, and oe-IGF2BP1 + oe-ABCA1 rescue group for analysis. Detection by Amplex^® Red fluorescence assay revealed that overexpression of IGF2BP1 resulted in considerable accumulation of intracellular cholesterol, while simultaneous overexpression of ABCA1 restored intracellular cholesterol homeostasis. The extracellular cholesterol level exhibited the opposite results (Fig. 4A). Further functional experiments demonstrated that overexpression of IGF2BP1 enhanced the migration, invasion, and proliferation of A549 cells, but these pro-cancer effects could be greatly suppressed by overexpression of ABCA1 (Fig. 4B–D). At the same time, overexpression of IGF2BP1 repressed apoptosis in LUAD cells, and this inhibition was also reversed by overexpression of ABCA1 (Fig. 4E). In summary, the IGF2BP1/ABCA1 axis boosts the migration and invasion of LUAD cells by regulating cholesterol metabolism.

Fig. 4.

The IGF2BP1/ABCA1 axis reinforces migration and invasion of LUAD cells by affecting cholesterol metabolism. Cell grouping: oe-NC, oe-IGF2BP1, oe-IGF2BP1 + oe-ABCA1. A The intracellular and extracellular cholesterol content of each group was measured using Amplex ^® Red cholesterol assay; B, C The migration and invasion abilities of cells in each group were evaluated by Transwell assay. Scale bar was 200 μm; D The proliferation ability of cells in each group was detected by colony formation assay; E The apoptosis of cells in each group was analyzed by flow cytometry. Data are presented as the mean ± SEM. ns indicates no statistical significance. * means P < 0.05, ** means P < 0.01, *** means P < 0.001 and **** means P < 0.0001 were determined by t-test

The in vivo verification of IGF2BP1/ABCA1 axis affecting LUAD progression through cholesterol metabolism

To validate the biological functions of the IGF2BP1/ABCA1 regulatory axis in vivo, a subcutaneous xenograft tumor model was established in nude mice (Fig. 5A). By dynamically monitoring the tumor growth curve and the final tumor mass, we discovered that overexpression of IGF2BP1 considerably boosted tumorigenesis compared to the control group. However, co-expression of ABCA1 effectively suppressed this pro-oncogenic effect (Fig. 5B, C). Moreover, cholesterol content in tumor tissue was assessed using the Amplex ^® Red method, revealing a remarkable consistency between in vitro cell experiments and in vivo results (Fig. 5D). In addition, IHC analysis demonstrated that in the IGFBP1-overexpressed group, Ki67 levels were greatly elevated, while ABCA1 protein expression was reduced. Conversely, the co-expression of both IGFBP1 and ABCA1 resulted in a decrease in Ki-67 expression (Fig. 5E, F). In conclusion, the IGF2BP1/ABCA1 axis regulates the malignant progression of LUAD via cholesterol metabolism within the body.

Fig. 5.

In vivo experiments verify that the IGF2BP1/ABCA1 axis affects LUAD progression through cholesterol metabolism. Animal groups: oe-NC, oe-IGF2BP1, oe-IGF2BP1 + oe-ABCA1. A Images of isolated xenografts in each group; B Dynamic line chart of tumor volume changes in mice in each group; C Statistical histogram of tumor mass weight in mice in each group; D Cholesterol content determined by the Amplex^® Red method; E, F The expression levels of IGF2BP1, ABCA1, and Ki67 proteins were analyzed by IHC. Scale bar was 50 μm. Data are presented as the mean ± SEM. ns indicates no statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 were determined by t-test

Discussion

In this work, we revealed the molecular mechanism by which IGF2BP1 affects cholesterol metabolism by regulating m6A methylation of ABCA1, thereby boosting the malignant progression of LUAD. These findings highlighted the important function of IGF2BP1, ABCA1, and cholesterol metabolism in LUAD, offering new theoretical basis and research ideas for the treatment of LUAD.

In recent years, ABCA1 has become the focus due to its key role in tumor biology. Its biological functions in a variety of malignant tumors have been extensively studied and discussed. ABCA1 is associated with the malignant phenotype of cholangiocarcinoma (Qian et al. 2024), ovarian cancer (Wang et al. 2021a, b, c), and BC (Pan et al. 2019). According to a study, when ABCA1 is up-regulated, the growth rate of LUAD cells is considerably slowed down, and their migratory and invasive abilities are greatly repressed (Liu et al. 2019). This is mainly because ABCA1, as a transmembrane transporter, can use the energy provided by ATP hydrolysis to boost the efflux of free cholesterol within cells (Chen et al. 2022a, b, c). The accumulation of cholesterol facilitates the proliferation of cancer cells, which is particularly prevalent in CRC (Chen et al. 2023) and endometrial cancer (Antmen et al. 2024). Consistent with these studies, our data suggested that overexpression of ABCA1 repressed the proliferation, migration, and invasion of LUAD cells by promoting cholesterol efflux, while accelerating apoptosis, thereby inhibiting the progression of LUAD. These results further confirmed the correlation between abnormal expression of ABCA1 and tumor progression, suggesting ABCA1’s potential as a biomarker for multiple cancers, including LUAD. Furthermore, interventions targeting cholesterol metabolism in LUAD may become a new frontier in clinical treatment in the future.

In addition, m6A RNA methylation is instrumental in the metabolic reorganization of tumor cells. It affects the self-renewal, proliferation, and invasion capabilities of cancer cells by regulating the expression of target genes (An and Duan 2022; Liu et al. 2020). The IGF2BP family (IGF2BP 1/2/3) has garnered widespread attention as a key factor in the m6A modification process (Duan et al. 2024). In this work, IGF2BP1 was selected as a candidate gene related to ABCA1 methylation modification through bioinformatics prediction. IGF2BP1 has an obvious cancer-promoting effect, linking the occurrence and progress of many cancers such as PC (Glass et al. 2020), BC (Li and Jiang 2022), and melanoma (Ghoshal et al. 2019). Our data demonstrated that the overexpression of IGF2BP1 in LUAD is positively linked with tumor malignancy, which confirms the molecule’s promoting role in disease progression. Furthermore, IGF2BP1 exerts oncogenic activity by enhancing its binding to target mRNAs and regulating their stability (Xiao et al. 2023). For example, IGF2BP1 boosts tumor cell migration by repressing the translation of mitogen-activated protein kinase 4(MAPK4) mRNA (Stohr et al. 2012). Our research data revealed that IGF2BP1 modulated the expression stability of ABCA1 through an m6A-dependent mechanism, and this modification ultimately accelerates the malignant process of LUAD cells. This discovery not only clarifies the interaction between IGF2BP1 and ABCA1 but also reveals the cancer-promoting mechanism of the IGF2BP1/ABCA1 axis in LUAD.

In summary, we uncovered the key role of IGF2BP1-mediated ABCA1m6A methylation in LUAD malignant progression. However, the work still entails certain queries that require further exploration, such as the precise location of IGF2BP1 and methylation modifications of ABCA1. Moreover, as an m6A reader protein, IGF2BP1 may interact with other m6A methyltransferase-related proteins. An in-depth investigation of the regulatory network between these proteins will help reveal a more complete mechanism. Nevertheless, this work has elucidated the mechanism by which IGF2BP1 regulates LUAD cell behavior. By targeting the functions of IGF2BP1, ABCA1, as well as cholesterol metabolism signaling pathways, there is potential to provide a new research direction and theoretical support for improving the prognosis of LUAD patients.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

S.X. designed the study and wrote the manuscript. Z.C. and K.L. contributed to data collection. Z.C. and W.T. performed the statistical analysis and interpretation of the results. All authors read and approved the final manuscript.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethical approval

This study was approved by the Animal Ethics Committee of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Approval Number SRRSH2025-0014.