
==== Front
Cell Biol Toxicol
Cell Biol Toxicol
Cell Biology and Toxicology
0742-2091
1573-6822
Springer Netherlands Dordrecht

39289194
9918
10.1007/s10565-024-09918-w
Research
TRMT10C-mediated m7G modification of circFAM126A inhibits lung cancer growth by regulating cellular glycolysis
Zhao Qingyun 12
Li Xiaofei 12
Wu Jiaxi 12
Zhang Ruirui 12
Chen Sixian 12
Cai Dunyu 12
Xu Haotian 12
Peng Wenyi 12
Li Gang ligang@gxmu.edu.cn

12
Nan Aruo nanaruo@163.com

12
1 https://ror.org/03dveyr97 grid.256607.0 0000 0004 1798 2653 School of Public Health, Guangxi Medical University, Nanning, 530021 China
2 https://ror.org/03dveyr97 grid.256607.0 0000 0004 1798 2653 Guangxi Key Laboratory of Environment and Health Research, Guangxi Medical University, Nanning, 530021 China
18 9 2024
18 9 2024
2024
40 1 7830 4 2024
4 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
The N7-methylguanosine (m7G) modification and circular RNAs (circRNAs) have been shown to play important roles in the development of lung cancer. However, the m7G modification of circRNAs has not been fully elucidated. This study revealed the presence of the m7G modification in circFAM126A. We propose the novel hypothesis that the methyltransferase TRMT10C mediates the m7G modification of circFAM126A and that the stability of m7G-modified circFAM126A is reduced. circFAM126A is downregulated in lung cancer and significantly inhibits lung cancer growth both in vitro and in vivo. The expression of circFAM126A correlates with the stage of lung cancer and with the tumour diameter, and circFAM126A can be used as a potential molecular target for lung cancer. The molecular mechanism by which circFAM126A increases HSP90 ubiquitination and suppresses AKT1 expression to regulate cellular glycolysis, ultimately inhibiting the progression of lung cancer, is elucidated. This study not only broadens the knowledge regarding the expression and regulatory mode of circRNAs but also provides new insights into the molecular mechanisms that regulate tumour cell metabolism and affect tumour cell fate from an epigenetic perspective. These findings will facilitate the development of new strategies for lung cancer prevention and treatment.

Graphical Abstract

Graphical Headlights

• circRNA can undergo m7G modification. The methyltransferase TRMT10C mediates circFAM126A m7G modification, thereby enhancing circFAM126A stability.

• m7G-modified circFAM126A can perform a biological function in inhibiting lung cancer progression by regulating cellular glycolysis.

• circFAM126A increases ubiquitination of HSP90 and inhibits AKT1 expression to regulate cellular glycolysis.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10565-024-09918-w.

Keywords

circRNA
m7G modification
HSP90
Glycolysis
Lung cancer
National Natural Science Foundation of ChinaNSFC82260652 Nan Aruo Guangxi Science and Technology Base and Talent Special Projectno.AD22080055 Nan Aruo issue-copyright-statement© Springer Nature B.V. 2024
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pmcIntroduction

Lung cancer is a malignant tumour that seriously threatens human health and life and is also the leading cause of cancer-related death worldwide (Bray et al. 2024; Leiter et al. 2023; Sung et al. 2021). With the development of medical technologies such as biotherapy, immunotherapy, and targeted drug therapy, the prognosis of patients with lung cancer has improved, but early biomarkers and effective treatment methods for lung cancer are lacking. Thus, the overall survival time of patients with lung cancer remains very short (Allemani et al. 2018; Saw et al. 2021). One characteristic of tumour cells is that their demand for energy gradually increases during rapid proliferation, leading to metabolic reprogramming (Boroughs and DeBerardinis 2015). Even under aerobic conditions, tumour cells still prefer glycolysis over oxidative phosphorylation for energy (Dominski et al. 2020; Zhang et al. 2020). This abnormal metabolic mode not only provides raw materials to support the rapid proliferation of tumour cells but also shapes a unique tumour microenvironment. These features not only facilitate tumour progression but also increase therapeutic resistance in tumours (Chelakkot et al. 2023; Paul et al. 2022). Recently, epigenetic changes have been recognized to play important roles in the tumorigenesis and progression of tumours. Exploring the molecular mechanisms of lung cancer development from an epigenetic perspective and identifying therapeutic targets that regulate tumour cell metabolism are highly important for the prevention and treatment of lung cancer.

Circular RNAs (circRNAs) are a class of noncoding RNAs with a circular structure. CircRNAs are evolutionarily conserved, exhibit tissue-specific expression, are structurally stable and highly abundant, and have gradually become a popular topic in epigenetics research (Chen and Shan 2021; Patop et al. 2019). As research has progressed, multiple biological functions of circRNAs have been revealed, and circRNAs are believed to be widely involved in various pathological and physiological processes (Kristensen et al. 2019; Yang et al. 2022). Reports indicate that abnormally expressed circRNAs can participate in the tumorigenesis and development of various tumours (Chen and Shan 2021; Kristensen et al. 2018). CircRNAs can perform their biological functions through various mechanisms, such as sponging microRNAs (miRNAs), directly binding to mRNAs, directly binding to proteins or directly encoding functional peptides. In lung cancer patients, circSLC25A16 can act as a sponge for miR-488-3p to regulate glycolysis and promote lung cancer progression through the hypoxia-inducible factor 1α (HIF-1α)/lactate dehydrogenase A (LDHA) axis (Shangguan et al. 2020). circDCUN1D4 directly binds to the thioredoxin-interacting protein (TXNIP) mRNA through base complementation and enhances its stability, which in turn inhibits glycolysis and lung cancer metastasis (Liang et al. 2021). In patients with renal cell carcinoma (RCC), circVAMP3 interacts directly with LDHA and regulates its activity, thereby promoting glycolysis and RCC proliferation (Li et al. 2022a). CircRNAs can target a variety of key metabolic molecules to alter tumour cell fate and thus regulate tumour progression, an ability that is gradually recognized as a promising strategy for tumour treatment.

Currently, research on circRNAs in tumours focuses mainly on the roles of circRNAs as key factors in tumour promotion or suppression, while the molecular mechanisms that control the dysregulation of circRNA expression are unclear. The increase in RNA methylation studies has led to new insights for elucidating the upstream molecular mechanisms of circRNAs and for conducting functional studies in tumours. RNA methylation modifications are epigenetic modifications that are dynamically regulated by methyltransferases and demethylases and can affect various biological metabolic processes and thus participate in disease development processes (Boulias and Greer 2023; Yang et al. 2021). The N7-methylguanosine (m7G) modification, which represents a new frontier in epigenetic regulation, can affect transfer RNA (tRNA) stability (Tomikawa 2018), microRNA (miRNA) maturation (Pandolfini et al. 2019), ribosomal RNA (rRNA) nuclear processing (Haag et al. 2015), and mRNA translation (Dai et al. 2021). With the advancements in m7G-related research, the important role of m7G in tumours has been progressively revealed. m7G-modified lncRNAs were found to be involved in regulating the prognosis of lung squamous cell carcinoma and the tumour immune microenvironment (Pan et al. 2022). The METTL1-mediated tRNA m7G modification was found to promote bladder cancer progression by regulating EGFR/EFEMP1 translation (Ying et al. 2021). Currently, studies related to m7G modification of noncoding RNAs focus on mainly tRNAs, rRNAs, lncRNAs, etc. Recent studies have indicated that the m7G modification exists in circRNAs and plays an important role in malignant tumours (Fu et al. 2023; Sun et al. 2023). However, the m7G modification of circRNAs has not been fully elucidated. In a previous study, we constructed circRNA differential expression profiles using human normal lung epithelial cells (BEAS-2B) and arsenic-induced malignantly transformed cells (BEAS-2B-As) and reported that circFAM126A (hsa_circ_0005251) was significantly downregulated in arsenic-induced malignantly transformed cell lines (Table S5). We subsequently performed an m7G-circRNA epitranscriptomic microarray analysis of lung cancer tissues and paracancerous tissues to explore the role of the m7G modification in lung cancer development, and the results revealed the presence of the m7G modification in circFAM126A and that the abundance of m7G modification was different between lung cancer and paracancerous tissues (Table S3). These findings indicate that m7G-modified circFAM126A may play a critical role in the tumorigenesis and development of lung cancer. Therefore, circFAM126A was selected for in-depth exploration in this study.

In this study, we proposed the novel hypothesis that circFAM126A can undergo m7G modification and that the methyltransferase TRMT10C mediates the m7G modification of circFAM126A. The role of m7G-modified circFAM126A in lung cancer development was clarified via in vivo and in vitro experiments, and this molecule is beneficial for the prevention and treatment of lung cancer. Finally, we elucidated the novel mechanism by which circFAM126A regulates glycolysis, alters tumour cell fate, and thus inhibits the development of lung cancer from an epigenetic perspective.

Methods

Cell culture

BEAS-2B, lung cancer cell lines (A549, H1299, H226, and H2170), and 293 T cells were obtained from the American Type Culture Collection (ATCC) and the Shanghai Cell Bank of the Chinese Academy of Sciences. BEAS-2B cells were cultured in BEGM medium (Lonza, CC-3171). H1299, H226, and H2170 cells were cultured in RPMI-1640 medium (Gibco, C11875500BT). A549 cells were cultured in Ham's F12 medium (Servicebio, G4560-500ML). 293 T cells were cultured in DMEM (Gibco, C11995500BT). All medium were supplemented with 10% foetal bovine serum (Gibco, 10099141C) and 1% penicillin‒streptomycin (Servicebio, G4003-100ML). All cells were incubated at 37 °C incubator with 5% CO2.

Lung cancer tissues

The 102 pairs of clinical lung cancer tissue samples and paracancerous tissues used in this study were obtained from the lung cancer tissue sample bank established by our group. All samples were obtained from the First Affiliated Hospital of Guangxi Medical University. All research subjects signed an informed consent form. The age, sex, height, weight, smoking history, drinking history, disease history, pathological classification, tumour diameter, tumour stage and other clinical medical data of the patients were collected within 15 d after surgery (Table S1). The study was approved by the Ethics Committee of The First Affiliated Hospital of Guangxi Medical University.

RNA extraction and RT‒qPCR

Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, 15,596,018) and quantified with a Nanodrop One spectrophotometer (Thermo Fisher Scientific, ND-ONEC-W). The RNA was reverse transcribed to cDNA using a GoScript™ Reverse Transcription System (Promega, A5002) kit. The primers used for quantitative PCR (qPCR) were synthesized by Sangon Biotech (Shanghai, China). For detailed primer sequences, refer to Table S2. A GoTaq® qPCR Master Mix (Promega, A6001) kit was used for qPCR, GAPDH was used as an internal reference, and relative expression was calculated using the 2−ΔΔCt method.

m7G-circRNA epitranscriptomic microarray

Total RNA was extracted from lung cancer tissues and quantified with a NanoDrop ND-1000 spectrophotometer, and then, microarray hybridization was performed according to Arraystar's standard protocols. Immunoprecipitation was performed using an anti-m7G antibody to enrich m7G-modified RNA; unmodified RNA markers were extracted from the supernatant. In accordance with the principle of Arraystar RNA labelling, RNA was treated with RNase R and labelled as complementary RNA (cRNA) with Cy5 and Cy3. The cRNAs were combined and hybridized to the Arraystar Human circRNA Epitranscriptomic Microarray (8 × 15 K, Arraystar) (GSE232281).

Plasmid construction and transfection

The circFAM126A, HSP90, and AKT1 overexpression plasmids and the corresponding control empty vectors (Fenghui Biotechnology, Changsha, China) were constructed, and the plasmid sequences were validated by sequencing. Plasmid extraction was performed with a Midi Prep Kit (QIAGEN, 12,145). Overexpression was performed using Lipofectamine® 3000 Transfection Reagent (Invitrogen, L3000015). First, 3.5 × 105 cells were seeded in 6-well plates and cultured for 12 h. When the cells reached 70%-80% confluence, 1 µg of plasmid was added to the 6-well plates for transfection, and the medium was replaced with new complete medium 8 h later. Subsequent experiments were performed after 48 h of culture. In addition, small interfering RNAs (siRNAs) were used for transient silencing. All siRNAs and the corresponding scrambled controls were synthesized by RiboBio Co., Ltd. (Guangzhou, China) (Table S2). siRNAs were transfected into cells using a riboFECT CP transfection kit (RiboBio, C10511-05). 2.5 × 105 cells were seeded in 6-well plates and cultured for 12 h. When the cells reached 30%-40% confluence, 20 nM siRNA was added to each well for transfection, and culture was continued for 48 h before subsequent experiments were performed.

Establishment of cell lines stably transfected with circFAM126A

The circFAM126A stable overexpression plasmid (circFAM126A OE) was constructed in our laboratory. After the plasmid was constructed successfully, the lentivirus was packaged in 293 T cells, and the lentivirus-containing supernatant was collected 48 h after transfection. Then, 500 µL of the virus-containing supernatant was added to A549 cells and incubated for 48 h. The fluorescence intensity was evaluated under a fluorescence microscope (AMG EVOS, Mill Creek, WA, USA). Stably transfected cell clones were selected using a monoclonal method.

Verification of the circular structure of circFAM126A

Total RNA (1 µg) was digested with 3 U/µg RNase R at 37 °C, and the digestion product was subjected to RT‒qPCR. These results indicated that circFAM126A was more resistant to RNase R digestion than FAM126A. A549 cells (4.5 × 105) and H1299 cells were seeded in a 6-well plate and cultured for 12 h. The medium was replaced with fresh complete medium containing 2 µg/mL actinomycin D (MedChemExpress, HY-17559), and the cells were collected at 0, 4, 8, and 12 h. Total RNA was extracted, and the expression of circFAM126A and FAM126A mRNA was measured via RT‒qPCR.

FISH

The subcellular localization of circFAM126A was determined in A549 cells and H1299 cells using a FISH kit (RiboBio, C10910). The specific circFAM126A fluorescence in situ hybridization (FISH) probe was labelled with FAM and synthesized (Sangon Biotech, China) (Table S2). A549 cells and H1299 cells were seeded on round coverslip, fixed with 4% paraformaldehyde (PFA), permeabilized with precooled permeabilization solution (0.5% Triton X-100 in PBS) for 5 min at 4 °C, blocked with prehybridization buffer for 30 min at 37 °C, and then hybridized with the circFAM126A-specific FISH probe at 42 °C in the dark for 16 h. Then, the nuclei were stained with 10 µL of DAPI for 10 min in the dark, and the samples were covered with coverslips. Fluorescence imaging of the circFAM126A subcellular localization was performed using an LSM800 confocal microscope (Zeiss).

Nucleocytoplasmic separation experiment

A PARIS™ (Invitrogen, AM1921) kit was used to conduct nucleocytoplasmic separation to further analyse the subcellular localization of circFAM126A. A total of 1 × 107 A549 cells and H1299 cells were collected, and cytoplasmic and nuclear RNA were extracted with a nuclear‒cytoplasmic fractionation kit. The expression of circFAM126A was measured by RT‒qPCR, with GAPDH serving as a cytoplasmic marker and U6 serving as a nuclear marker.

Cell viability assay

A Cell Counting Kit-8 (CCK-8; Dojindo, CK04) was used to evaluate cell viability. A549 cells and H1299 cells (5 × 103 cells/well) were seeded in a 96-well plate and cultured for 12 h prior to siRNA transfection for 48 h. A549 cells and H1299 cells (8 × 103 cells/well) were seeded in a 96-well plate, cultured for 12 h, transfected with the expression vector and incubated for 48 h. CCK-8 reagent was mixed with complete culture medium at a ratio of 1:10, and the mixed medium was added. After an incubation at 37 °C for 1.5 h, the absorbance was measured at 450 nm.

EdU incorporation assay

A Cell-Light EdU Apollo 567 In Vitro Kit (RiboBio, C10310-1) was used to evaluate cell proliferation. A549 and H1299 cells (5 × 103 cells/well) were seeded in a 96-well plate. After cultured for 12 h perform siRNA transfection. A549 and H1299 cells (8 × 103 cells/well) were seeded in a 96-well plate, cultured for 12 h, transfected with the expression vector and incubated for 48 h. After an incubation for 48 h, the samples were labelled with EdU, fixed and stained. Cell imaging was performed using the EVOS® FL Auto Imaging System, and DAPI-positive cells and EdU-positive cells were then counted using ImageJ.

Detection of apoptosis by flow cytometry

An Annexin V-FITC/propidium iodide (PI) double-staining apoptosis detection kit (KeyGen Biotech, KGA107) was used to detect apoptosis. A549 cells and H1299 cells (2.5 × 105 cells/well) were seeded in a 6-well plate, cultured for 12 h prior to siRNA transfection, and incubated for 48 h. A549 cells and H1299 cells (3.5 × 105 cells/well) were seeded in a 6-well plate, cultured for 12 h, transfected with the expression vector and incubated for 48 h. The cells were collected by detachment with EDTA-free trypsin (Solarbio, T1350), centrifuged, resuspended in binding buffer, and then stained with Annexin V-FITC (5 µL) and PI (5 µL). Apoptosis was quantified using a CytoFLEX flow cytometer (Beckman Coulter).

Wound healing assay

A549 cells and H1299 cells (2.5 × 105 cells/well) were seeded in a 6-well plate and cultured for 12 h prior to siRNA transfection for 48 h. A549 cells and H1299 cells (3.5 × 105 cells/well) were seeded in a 6-well plate, cultured for 12 h. Transfected with the expression vector and incubated for 48 h. When the cell confluence reached nearly 100%, a 200 μL RNase-free pipette tip was used to make a straight and even scratch on the cell surface. Then, 0 h and 48 h after scratching, images were captured at the same position using a microscope (Olympus, Japan). After image acquisition, the wound areas at 0 h and 48 h after wounding were compared and statistically analysed.

Transwell migration assay

A549 cells and H1299 cells (2.5 × 105 cells/well) were seeded in a 6-well plate and cultured for 12 h prior to siRNA transfection for 48 h. A549 cells and H1299 cells (3.5 × 105 cells/well) were seeded in a 6-well plate, cultured for 12 h, transfected with the expression vector and incubated for 48 h. Then, the cells were detached and resuspended in serum-free medium. A migration chamber (Corning, USA) was filled with serum-free medium and equilibrated in a cell culture incubator for 1 h. Then, complete medium (700 μL) was added to the lower compartment, and 400 μL (1 × 104 cells) of the cell suspension was added to the upper compartment and incubated for 24 h. The cell samples were fixed with methanol and stained with crystal violet. The cells were removed from the upper surface of the membrane, and the number of migrated cells was determined using the EVOS® FL Auto Imaging System.

Subcutaneous tumour formation assay in nude mice

Ten 4-week-old female BALB/c nude mice were randomly divided into two groups (the stable circFAM126A overexpression group and the control group). After acclimation in a specific pathogen-free (SPF) environment for one week, 2 × 107 cells were injected subcutaneously into the right axillary region of each nude mice. The tumour volume was measured using callipers every 3 days (volume = length × height2 × 0.5). After 28 days, the nude mice were photographed and then sacrificed, and the tumours were harvested. A portion of the tissue was fixed with 4% PFA for immunohistochemistry, and a portion was frozen at -80 °C for subsequent Western blot (WB) analysis. All animal experiments performed in this study were approved by the Ethics Committee of Guangxi Medical University.

Dot blot (DB) assay

Total RNA was extracted from A549 cells with TRIzol reagent (Invitrogen, 15,596,018). One microgram of RNA was applied to nylon membranes (Beyotime, FFN10), dried, and crosslinked with 1500 J of UV radiation for 5 min. The membranes were blocked with 5% skim milk for 30 min and incubated with an anti-m7G antibody overnight at 4 °C. Nylon membranes were washed with TBST and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Proteintech, SA00001-1) for 1 h prior to chemiluminescence imaging using the Clinx S6 system. The membranes were washed with TBST and incubated with 0.1% methylene blue (Solarbio, G1300) for 5 min before imaging.

WB analysis

Cells were lysed in cell lysis buffer (10 mM Tris–HCl (pH 7.4), 1% SDS, and 1 mM Na3VO4) to extract total protein. Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23,227). Equal amounts of protein were separated on 8‒10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes at a constant voltage of 25 V for 4.5 h or a constant current of 200 mA for 1‒2 h. Membranes were blocked with 5% skim milk for 1 h and incubated overnight at 4 °C with the primary antibody. After washing, an anti-rabbit or anti-mouse secondary antibody was added, and the membranes were incubated at room temperature for 1 h. Chemiluminescence imaging was then conducted with the Clinx S6 system. Grayscale values of protein bands were analysed with ImageJ. The primary antibodies used were as follows: anti-HSP90 (Proteintech, 60,318–1-Ig), anti-AKT1 (SANTA, sc-5298), anti-p-AK (S473) (Cell Signaling Technology, 4060 T), anti-HK2 (Proteintech, 22,029–1-AP), anti-PKM2 (60,268–1-Ig), anti-LDHA (19,987–1-AP), anti-β-actin (Affinity, AF7018), and anti-β-tubulin (Affinity, AF7011). The secondary antibodies used were as follows: anti-rabbit IgG (Cell Signaling Technology, 7074S) and anti-mouse IgG (Proteintech, SA00001-1).

Glucose uptake and lactate production assays

A549 cells and H1299 cells (2.5 × 105 cells/well) were seeded in a 6-well plate and cultured for 12 h prior to siRNA transfection for 48 h. A549 cells and H1299 cells (3.5 × 105 cells/well) were seeded in a 6-well plate, cultured for 12 h, transfected with the expression vector and incubated for 48 h. Glucose uptake and lactate production were measured using a glucose assay kit (Beyotime, S0201S) and lactate assay kit (Solarbio, BC2230). Glucose and lactate concentrations were normalized to the protein concentration.

Protein stability assays

A549 cells stably overexpressing circFAM126A (4.5 × 105 cells/well) were seeded in a 6-well plate. After 12 h, fresh complete medium containing 100 µg/mL cycloheximide (CHX; Mdbio, Inc., C012-1 g) was added, total cellular proteins were collected at 0, 6, 12 and 24 h, and WB experiments were performed. A549 cells stably overexpressing circFAM126A (4.5 × 105 cells/well) were seeded in a 6-well plate. After 12 h, fresh complete medium containing 10 µM MG132 (Sigma, M7449) was added, and total cellular protein was collected 24 h later, followed by WB analysis.

CLIP

Crosslinking immunoprecipitation (CLIP) experiments were performed using a CLIP kit (BersinBio, Guangzhou, China, Bes3014). Prior to the experiment, the medium was replaced with fresh medium containing 100 µM 4-thiouridine (Sigma, T4509), and the culture was continued for 16 h to enhance the binding between proteins and RNAs. Then, the samples were irradiated with 365 nm ultraviolet light for 10 min. Afterwards, the cells were lysed, and m7G-RNA complexes were captured with an anti-m7G antibody. The complexes were subjected to sequential digestion with DNase I, RNase T1, and proteinase K, and RNA was eluted. Primers were designed separately based on the full-length circFAM126A sequence, which was truncated into 14 segments of 50 bp each, and the primer information can be found in Table S2. The enrichment efficiency of the protein‒RNA binding sites was determined by qPCR.

MeRIP

Methylated RNA immunoprecipitation (MeRIP) experiments were performed using a MeRIP kit (IEMed, Guangzhou China, IEMed-K305). The cells were lysed with TRIzol reagent, total cellular RNA was extracted, the RNA was fragmented via sonication and immunoprecipitated with an anti-m7G antibody, the precipitated RNA was collected and washed, and the antibody enrichment efficiency was assessed using RT‒qPCR.

TRAP

Tagged RNA affinity purification (TRAP) experiments were performed using a TRAP Kit (BersinBio, Guangzhou, China, Bes5101). The GST-MS2 fusion expression vector and circFAM126A-MS2 stem‒loop structure tandem repeat vector used for the experiments were constructed by IEMed Biomedical Technology (Guangzhou, China). The MS2, circRNA-MS2, and GST-MS2 vectors were cotransfected into A549 cells, and the GST-MS2-circFAM126A complexes were collected after cell lysis. The complexes were subsequently pulled down by glutathione-labelled magnetic beads, and the collected and eluted complexes were subjected to silver staining and mass spectrometry (MS) analysis.

RIP

RNA immunoprecipitation (RIP) experiments were performed using a RIP Kit (BersinBio, Guangzhou, China, Bes5101) to further analyse the interactions between circFAM126A and downstream proteins. First, 2 × 107 A549 cells were cultured and collected, lysed with polysome lysis buffer, equilibrated with Protein A/G magnetic beads, and subjected to immunoprecipitation with an anti-HSP90 antibody to pull down HSP90 and its associated bound RNAs. Finally, the RNA precipitates were eluted and collected, and circFAM126A enrichment was examined via RT‒qPCR.

Immunofluorescence (IF) staining

A549 cells and H1299 cells (2 × 104 cells/well) were seeded in a 12-well plate containing coverslips and cultured for 12 h. After fixation, the cells were blocked with blocking solution at room temperature for 1 h. Added the primary antibody and incubated overnight at 4 °C. Then, added fluorescently labelled secondary antibody and incubated in the dark with for 1 h at room temperature. DAPI (Boster Biological Technology, AR1176) was added to stain the nuclei, and fluorescence imaging was conducted with an LSM800 confocal microscope (Zeiss). The following primary and secondary antibodies were used: anti-HSP90 (Proteintech, 60,318–1-Ig), anti-p-AKT (S473) (Cell Signaling Technology, 4060 T), anti-mouse IgG (Cell Signaling Technology, Inc., 4408S) and anti-rabbit IgG (Cell Signaling Technology, Inc., 8890S).

Co-IP

Coimmunoprecipitation (Co-IP) experiments were performed using a Co-IP kit (BersinBio, Guangzhou, China; Bes3011) to investigate protein‒protein interactions. Briefly, 2 × 107 cells were collected and lysed with cell lysis buffer to obtain total cellular protein. Then, 2 µg of antibody was added, and the cells were incubated overnight at 4 °C with vertical mixing. Protein A/G-MagBeads were added to capture the antibody‒protein complexes, the enriched proteins were eluted, and WB was performed to detect the target proteins.

Immunohistochemical (IHC) analysis

Tissues from tumours transplanted into nude mice were embedded in paraffin, sectioned, deparaffinized, and rehydrated. After sequential incubations with primary and secondary antibodies, the sections were stained with haematoxylin and eosin (H&E) (Solarbio, China). Images were acquired at 200 × and 400 × magnification under a light microscope (Leica, Mannheim, Germany). The antibodies used for IHC analysis were specific for Ki67 (Servicebio, GB121142), Bcl-2 (Cell Signaling Technology, Inc., 15071 T), and RhoA (Servicebio, GB115176).

Statistical analysis

The data in this study are presented as the means ± standard deviations. Comparisons of quantitative data between two groups were conducted using paired sample t tests (normally distributed data) or rank-sum tests (nonnormally distributed data). A receiver operating characteristic (ROC) curve was constructed to evaluate the potential diagnostic value of circFAM126A. Pearson’s correlation analysis was performed to evaluate correlations between variables. Statistical analyses were performed using SPSS 25.0 (IBM, Chicago, USA), and GraphPad Prism 7.0 (San Diego, USA) was used for graphing. All tests were two-tailed, and a P value < 0.05 was considered statistically significant. All experiments were performed with three biological replicates, and the data presented represent the results of at least three independent experiments.

Results

m7G-modified circFAM126A is significantly downregulated in lung cancer tissues and cells

In our previous study, we constructed a circRNA differential expression profile using BEAS-2B and BEAS-2B-As to explore the mechanism of circRNAs in lung cancer development (Li et al. 2022b). The significantly lower expression of the circRNA circFAM126A in arsenic-induced malignantly transformed cell lines attracted our attention (see Table S5 for the detailed sequencing results). Studies have shown that the m7G modification is one of the most common forms of base modifications involved in posttranscriptional regulation, but the regulatory mechanism of m7G modifications associated with circRNAs remains unknown (Ramanathan et al. 2016). We analysed an m7G-circRNA epitranscriptomic microarray and successfully determined the differential expression profile of m7G-modified circRNAs to explore the biological function and underlying mechanism of m7G-modified circRNAs in lung cancer (see Table S3 for the detailed sequencing results) (Fig. 1a). circFAM126A was found to be expressed at lower levels in lung cancer cell lines (A549, H226, H460, H1299, and H2170) than in BEAS-2B cells by qPCR (Fig. 1b). An analysis of the UCSC Genome Browser (https://genome.ucsc.edu/) revealed that circFAM126A is an exonic circRNA formed by backsplicing of exons 2–7 of the FAM126A precursor mRNA (pre-mRNA) and has a length of 654 nt. Specific primers were designed to target the backsplicing junction site of circFAM126A, which was amplified by qPCR and sequenced via Sanger sequencing to clarify the cyclization site (Fig. 1c). A series of experiments was subsequently conducted to verify the circular structure of circFAM126A. First, total RNA was extracted and treated with linear RNA digestion enzyme (RNase R). The results showed that, compared with linear RNA (FAM126A), circFAM126A is resistant to linear digestion enzyme (Fig. 1d). RT‒qPCR results obtained after Act D treatment revealed that circFAM126A was more stable than the FAM126A mRNA was (Fig. 1e-f). According to the above experiments, circFAM126A has a closed circular structure.Fig. 1 m7G-modified circFAM126A is significantly downregulated in lung cancer tissues and cells. a Hierarchical clustering heatmap of differentially expressed m7G-modified circRNAs. b qPCR analysis of circFAM126A expression levels in lung cancer cell lines (A549, H226, H460, H1299, and H2170) and BEAS-2B cells. c Sanger sequencing results indicating the backsplicing site in circFAM126A and a schematic representation of the circFAM126A structure (https://genome.ucsc.edu/). d RNase R treatment of total RNA, followed by qPCR, were used to evaluate the expression of circFAM126A and FAM126A. e‒f qPCR analysis of the expression of circFAM126A and FAM126A in A549 cells and H1299 cells treated with actinomycin D (2 µg/mL) for 0 h, 4 h, 8 h and 12 h. g MeRIP‒qPCR analysis of circFAM126A expression after m7G antibody enrichment. h Schematic diagram of the CLIP experiment. i CLIP‒qPCR analysis of the enrichment of different regions of circFAM126A. j TRAP assay for protein enrichment, followed by silver staining. k qPCR analysis of circFAM126A expression after the overexpression of TRMT10C in A549 cells. l The overall level of the m7G modification was determined by performing an m7G dot blot assay after the silencing of TRMT10C in A549 cells. m MeRIP‒qPCR analysis of m7G levels on circFAM126A after the silencing of TRMT10C in A549 cells. n qPCR analysis of circFAM126A expression after the silencing of TRMT10C in A549 cells treated with actinomycin D (2 µg/mL) for 0 h, 12 h, 24 h and 36 h. o qPCR analysis of the circFAM126A expression level in lung cancer tissues and paraneoplastic tissues. p Comparison of circFAM126A expression levels in lung cancer tissues and paraneoplastic tissues with different N stages. q Comparison of circFAM126A expression levels in lung cancer tissues and paraneoplastic tissues with different T stages. r Comparison of circFAM126A expression levels in lung cancer tissues and paraneoplastic tissues with different TNM stages. s Correlation analysis of circFAM126A expression with the lung tumour size. t A ROC curve analysis was performed to evaluate the diagnostic value of circFAM126A. * and ** indicate significant differences (P < 0.05 and P < 0.01, respectively)

The results of the m7G-circRNA epitranscriptomic microarray analysis indicated the presence of the m7G modification in circFAM126A (Table S3). Therefore, we further explored the m7G modification of circFAM126A through a series of experiments. MeRIP-qPCR was first performed using an m7G-specific antibody. circFAM126A was significantly enriched in the m7G-specific antibody group compared with the IgG group (Fig. 1g), indicating the presence of the m7G modification in circFAM126A. We conducted CLIP-qPCR to clarify the m7G modification regions in circFAM126A. The full-length sequence of circFAM126A was truncated into 14 segments, each with a length of 50 bp, and 14 primer pairs were designed by segmentation (Fig. S1a). The results showed that the m7G modification occurred mainly in the first and third segments of circFAM126A (Fig. 1h-i).

We conducted TRAP-MS to further explore the circFAM126A m7G modification mechanism (Fig. 1j, Fig. S1b and Table S4). After analysing the MS results, the reported methylation-related enzymes (AKR1B10, CBX5, NSUN2, NCBP2, JMJD6, and TRMT10C) were screened for subsequent studies. Specific siRNAs were designed and synthesized based on the sequences of these methylation-related enzymes, and the silencing efficiency of these siRNAs was assayed in A549 cells (Fig. S1c). Each of these methylation-related enzymes was silenced separately in A549 cells, and the expression level of circFAM126A decreased after TRMT10C was silenced but did not significantly change after any other methylation-related enzyme was silenced (Fig. S1d). Previous studies have reported that TRMT10C is a methyltransferase for the N1-methyladenosine (m1A) modification (Safra et al. 2017; Wang et al. 2020b), and whether TRMT10C can function as a methyltransferase for the m7G modification deserves further exploration. First, we showed through qPCR that TRMT10C was expressed at low levels in lung cancer cells (A549, H226, H460, H1299, and H2170) and lung cancer tissues (Fig. S1e-f). We constructed a TRMT10C overexpression plasmid (Fig. S1g) and observed that the expression level of circFAM126A increased after TRMT10C was overexpressed (Fig. 1k), indicating that TRMT10C could regulate circFAM126A expression. We subsequently performed a dot blot assay, and the results revealed that the level of the m7G modification decreased after TRMT10C was silenced (Fig. 1l) but increased after TRMT10C was overexpressed (Fig. S1h), suggesting that TRMT10C can regulate the intracellular level of the m7G modification. We further investigated the effect of TRMT10C on the m7G modification level of circFAM126A by conducting MeRIP. The MeRIP results revealed that TRMT10C expression was positively correlated with the level of circFAM126A m7G modification (Fig. 1m, Fig. S1i), indicating that TRMT10C can mediate the m7G modification of circFAM126A. In our previous study, we reported that TRMT10C expression correlated with the expression of circFAM126A and that the m7G modification affected RNA stability (Rong et al. 2022; Tomikawa 2018). Therefore, we further explored whether the TRMT10C-mediated m7G modification affects the stability of circFAM126A. After the silencing and overexpression of TRMT10C in A549 cells, an RNA stability assay revealed that TRMT10C could positively regulate the stability of circFAM126A (Fig. 1n, Fig. S1j). In summary, we identified the presence of the m7G modification in circFAM126A and revealed that TRMT10C can mediate the circFAM126A m7G modification and increase the stability of circFAM126A.

We next measured the expression levels of circFAM126A in lung cancer tissues from a lung cancer cohort via qPCR to further explore the relationship between circFAM126A and lung cancer. circFAM126A was significantly downregulated in lung cancer tissues from the cohort (Fig. 1o). A correlation analysis of the clinical data of patients with lung cancer revealed that circFAM126A was expressed at low levels in tumours with different pathological stages (Fig. 1p-r); In addition, the expression of circFAM126A was negatively correlated with the tumour diameter (Fig. 1s). The ROC curve analysis revealed that circFAM126A had clear diagnostic value for lung cancer (AUC: 0.7659) (Fig. 1t). These results suggest that circFAM126A is closely related to the development of lung cancer and is a potential marker for lung cancer.

circFAM126A significantly inhibits lung cancer development in vitro

We first performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis based on circFAM126A to explore its biological functions in lung cancer and found that circFAM126A can participate in the regulation of many cancer-related signalling pathways, indicating that circFAM126A has the potential to regulate the development of lung cancer (Fig. S2a). We then synthesized specific siRNAs and constructed overexpression plasmids based on the sequence of circFAM126A, transiently transfected them into A549 cells and verified their silencing and overexpression efficiency to further explore the function of circFAM126A (Fig. S2b). Transient silencing or overexpression of circFAM126A did not affect the expression of the circFAM126A host gene (Fig. S2c). circFAM126A siRNA1 and siRNA2 had the highest silencing efficiencies and were thus selected for follow-up experiments. After constructing the circFAM126A transient transfection system, we investigated the biological function of circFAM126A in the progression of lung cancer in terms of cell proliferation, viability, migration and apoptosis. First, we performed a cell proliferation assay. Transient silencing or overexpression of circFAM126A in A549 and H1299 cells was performed to detect changes in the cell proliferation capacity via an EdU incorporation assay. The proliferation capacity of circFAM126A-silenced lung cancer cells was increased, whereas that of circFAM126A-overexpressing cells was reduced (Fig. 2a-b). Subsequently, cell viability was examined with a CCK-8 assay. circFAM126A silencing increased and circFAM126A overexpression decreased the viability of A549 and H1299 cells (Fig. 2c). Flow cytometry detection of apoptosis revealed that circFAM126A silencing inhibited and circFAM126A overexpression promoted apoptosis (Fig. 2d-e). We examined cell migration by performing a wound healing assay and a Transwell assay to further elucidate the function of circFAM126A in lung cancer. The silencing of circFAM126A promoted and the overexpression of circFAM126A inhibited cell migration (Fig. 2f-i). In summary, circFAM126A significantly inhibits the development of lung cancer in vitro.Fig. 2 circFAM126A significantly inhibits lung cancer development in vitro. Transient silencing/overexpression of circFAM126A in A549 cells and H1299 cells. a-b EdU incorporation assay of cell proliferation. c CCK-8 assay of cell viability. d-e Flow cytometry analysis of apoptosis. f-g Wound healing assay of cell migration. h-i Transwell assay of cell migration. * and ** indicate significant differences (P < 0.05 and P < 0.01, respectively)

circFAM126A significantly inhibits the development of lung cancer in vivo

Through cell-based experiments, we found that circFAM126A can perform the biological function of inhibiting lung cancer development in vitro. We next explored the roles of circFAM126A in vivo by conducting a subcutaneous tumorigenesis assay in nude mice. First, we constructed an A549 cell line with stable circFAM126A overexpression (Fig. S3a-b) via lentiviral transduction. After one week of feeding, the right dorsal surface of nude mice was injected subcutaneously with vector-transfected or circFAM126A-OE cells. During the tumorigenesis period, the nude mice body weight and tumour volume were recorded. After subcutaneous tumours had formed, they were observed for an additional 4 weeks. The nude mice were then sacrificed, and the tumour bulks were removed (Fig. 3a-b). An analysis of tumour growth and tumour weight data collected during tumorigenesis revealed that stable overexpression of circFAM126A resulted in a significantly decreased tumour volume and weight and slower growth compared with those in the control group (Fig. 3c-d). The expression of proteins related to lung cancer development was subsequently examined via immunohistochemistry, and the results revealed that the expression of the proliferation-related protein Ki67, the metastasis-related protein Rho A and the apoptosis-related protein Bcl2 was reduced in tumour tissues stably overexpressing circFAM126A (Fig. 3e-f). These results indicate that circFAM126A can significantly inhibit the growth of lung cancer in vivo.Fig. 3 CircFAM126A significantly inhibits the development of lung cancer in vivo. a-b Tumour size measured 27 days after the subcutaneous injection of cells into nude mice. c Tumour weight. d Tumour volume growth curve. e–f Immunohistochemical analysis of tumour-related indicators. * and ** indicate significant differences (P < 0.05 and P < 0.01, respectively)

circFAM126A promotes the ubiquitination-mediated degradation of HSP90 by directly binding to HSP90

The intracellular localization of a circRNA is closely related to its mechanism. The subcellular localization of circFAM126A was first determined to investigate the mechanism by which circFAM126A inhibits the development of lung cancer. The results of the nucleocytoplasmic separation and FISH assays revealed that circFAM126A was distributed in both the cytoplasm and nucleus but was distributed mainly in the cytoplasm (Fig. 4a-b). We further explored the molecular mechanisms by which circFAM126A functions by analysing the previously collected TRAP-MS data and identified many binding proteins that potentially interact with circFAM126A. A KEGG pathway enrichment analysis of these binding proteins revealed that circFAM126A was closely related to the PI3K-AKT pathway and the glycolysis/glycogenesis pathway (Fig. 4c). In addition, preliminary TRAP‒PAGE silver staining results revealed that the molecular weights of circFAM126A-binding proteins may be in the 70–100 kDa range (Fig. 1j). The TRAP-MS results revealed a high abundance of bound HSP90, with a molecular weight of 90 kDa; moreover, this protein was significantly associated with the PI3K-AKT signalling pathway (Giulino-Roth et al. 2017; Liu et al. 2020; Park et al. 2017). Therefore, we selected the HSP90 protein as a downstream target of circFAM126A for subsequent mechanistic studies. We performed RIP‒qPCR to further explore the binding of circFAM126A and HSP90 and found that HSP90 was significantly enriched with circFAM126A, indicating that circFAM126A could bind directly to HSP90 (Fig. 4d). An analysis of TCGA-LUAD data (http://gepia.cancer-pku.cn/) revealed that HSP90 was closely associated with the overall survival and disease-free survival of patients with LUAD; specifically, patients with high HSP90 expression had less favourable overall survival and disease-free survival, suggesting that HSP90 promotes the development of lung cancer (Fig. S4a-b).Fig. 4 CircFAM126A promotes the ubiquitination-mediated degradation of HSP90 by directly binding to HSP90. a Nucleoplasmic separation was performed to determine the distribution of circFAM126A in A549 and H1299 cells. U6 was used as a marker of nuclear localization, and GAPDH was used as a marker of cytoplasmic localization. b FISH was performed to determine the distribution of circFAM126A in A549 cells and H1299 cells. Nuclei were stained with DAPI, and circFAM126A was specifically labelled with FAM. c KEGG pathway enrichment analysis of binding proteins identified by TRAP-MS. D RIP‒qPCR analysis of the expression of circFAM126A. e–f WB analysis of HSP90 expression levels after the transient silencing/overexpression of circFAM126A in A549 and H1299 cells. g-h WB analysis of HSP90 expression levels in tumours. i‒j Immunohistochemical analysis of HSP90 expression in tumours. k-l WB analysis of HSP90 expression levels at 0 h, 6 h, 12 h and 24 h after CHX treatment following the stable overexpression of circFAM126A. m WB analysis of HSP90 expression levels 24 h after MG132 treatment following the stable overexpression of circFAM126A. n Co-IP and WB analyses were performed to determine the expression levels of the total and ubiquitinated HSP90 protein after stable overexpression of circFAM126A. * and ** indicate significant differences (P < 0.05 and P < 0.01, respectively)

We next sought to explore the effect of direct binding of circFAM126A to HSP90. The WB analysis after the silencing and overexpression of circFAM126A in A549 and H1299 cells indicated that circFAM126A negatively regulated HSP90 expression (Fig. 4e-f). The HSP90 protein expression level in tumours from nude mice was subsequently evaluated via WB and immunohistochemistry, and the results revealed that stable overexpression of circFAM126A significantly decreased the expression of HSP90 in tumours (Fig. 4g-j). These results indicate that circFAM126A can affect the HSP90 protein expression level both in vitro and in vivo. However, neither the silencing nor the overexpression of circFAM126A affected the mRNA expression level of HSP90 (Fig. S4c), indicating that circFAM126A did not affect the HSP90 mRNA expression level. Previous studies have shown that circRNAs can alter protein stability by binding to proteins, thereby affecting their degradation (Li et al. 2021). We therefore performed a series of experiments to explore whether circFAM126A can regulate the stability of the HSP90 protein. First, we treated cells with CHX, and the WB results revealed that with increasing CHX treatment time, stable overexpression of circFAM126A significantly increased the degradation rate of the HSP90 protein (Fig. 4k-l). Currently, protein degradation is believed to occur through two main pathways: the lysosomal pathway and the ubiquitin-mediated proteasomal degradation pathway (Dikic 2017; Paudel et al. 2023). The ubiquitin-mediated pathway is a specific protein degradation pathway that is strictly spatiotemporally regulated. We treated cells with MG132 to clarify whether circFAM126A can mediate HSP90 protein degradation via the ubiquitination pathway and found by WB analysis that MG132 inhibited the degradation of HSP90 caused by circFAM126A overexpression (Fig. 4m), suggesting that circFAM126A may affect HSP90 protein stability via the ubiquitination pathway. Subsequently, an anti-ubiquitin antibody was used for the HSP90 Co-IP experiments, and stable overexpression of circFAM126A was found to increase the ubiquitination of HSP90 (Fig. 4n). These results indicate that circFAM126A promotes the ubiquitination of HSP90, accelerates its degradation, and thus affects its stability.

circFAM126A regulates the HSP90/AKT1 pathway

Numerous studies have revealed that the PI3K-AKT pathway is closely related to the tumorigenesis and development of lung cancer (Chen et al. 2020). A preliminary KEGG enrichment analysis revealed that the proteins bound to circFAM126A could participate in the PI3K-AKT signalling pathway (Fig. 4c). Therefore, we speculated that circFAM126A could regulate the PI3K-AKT signalling pathway. First, we transiently silenced and overexpressed circFAM126A in A549 and H1299 cells and WB analysis revealed that circFAM126A negatively regulated the levels of the AKT1 and p-AKT (S473) proteins (Fig. 5a-c). WB and immunohistochemical analyses revealed that stable overexpression of circFAM126A significantly reduced the levels of the AKT1 and p-AKT (S473) proteins in tumours (Fig. 5d-g). These results indicate that circFAM126A may be involved in regulating the PI3K-AKT pathway. Previous studies have shown that HSP90 is an important component of the PI3K-AKT signalling pathway and that AKT can serve as an important client protein of HSP90 (Sato et al. 2000). Therefore, we conducted an in-depth exploration of the binding relationships between HSP90 and AKT1 as well as p-AKT (S473). First, a Co-IP assay revealed that HSP90 bound directly to AKT1 and p-AKT (S473) (Fig. 5h). Subsequently, immunofluorescence staining showed that HSP90 and p-AKT (S473) were colocalized mainly in the cytoplasm (Fig. 5i). We first constructed HSP90 overexpression plasmids and verified their overexpression efficiency via WB analysis to determine whether HSP90 alters the levels of AKT1 and p-AKT (S473) (Fig. S4d). A WB analysis after the overexpression of HSP90 in A549 and H1299 cells revealed that HSP90 can positively regulate the levels of the AKT1 and p-AKT (S473) proteins (Fig. 5j-k). These results indicate that HSP90 can regulate its expression upstream of the AKT pathway by directly binding to AKT1 and p-AKT. In previous studies, we reported that circFAM126A can directly bind to the HSP90 protein and regulate AKT1 and p-AKT protein levels. Therefore, we conducted HSP90 rescue assays in cell lines with stable circFAM126A overexpression to determine whether the regulation of AKT1 and p-AKT levels by circFAM126A depends on HSP90. Transient overexpression of HSP90 in A549 cells stably overexpressing circFAM126A was performed via transfection. WB analysis revealed that HSP90 overexpression partially reversed the reduced levels of AKT1 and p-AKT (S473) induced by stable overexpression of circFAM126A (Fig. 5l-n). These results indicate that circFAM126A can target and regulate the HSP90/AKT1 pathway.Fig. 5 CircFAM126A regulates the HSP90/AKT1 pathway. a‒c AKT1 and p-AKT (S473) protein levels were measured via WB analysis after the transient silencing/overexpression of circFAM126A in A549 and H1299 cells. d-e WB analysis of AKT1 and p-AKT (S473) protein levels in tumours. f‒g Immunohistochemical analysis of AKT1 and p-AKT (S473) protein levels in tumours. h Interactions of HSP90 with AKT1 and of HSP90 with p-AKT (S473) in A549 cells and H1299 cells were detected by Co-IP and WB analyses. i Immunofluorescence analysis of p-AKT (S473) (red) colocalization with HSP90 (green) in A549 and H1299 cells. j-k Measurement of HSP90, AKT1 and p-AKT (S473) protein levels via WB analysis after the overexpression of HSP90 in A549 and H1299 cells. l-n AKT1 and p-AKT (S473) protein levels were measured via WB after the transient overexpression of HSP90 in A549 cells stably overexpressing circFAM126A.* and ** indicate significant differences (P < 0.05 and P < 0.01, respectively)

circFAM126A inhibits glycolysis by targeting HSP90/AKT1

The previous KEGG pathway enrichment analysis revealed that circFAM126A was involved in regulating the PI3K-AKT/glycolysis signalling pathway. We first examined whether circFAM126A affects the expression of HK2, PKM2, and LDHA, three key rate-limiting enzymes that regulate glycolysis, to explore whether circFAM126A regulates glycolysis by targeting HSP90/AKT1. The WB analysis after transient silencing or overexpression of circFAM126A in A549 and H1299 cells revealed that circFAM126A negatively regulated HK2 protein expression but did not affect PKM2 or LDHA protein expression (Fig. 6a-b, Fig. S4e-f). WB and immunohistochemical analyses revealed that stable overexpression of circFAM126A significantly reduced HK2 protein expression in tumours (Fig. 6c-f). Transient silencing and overexpression of circFAM126A in A549 and H1299 cells revealed that circFAM126A negatively regulated glucose uptake and lactate production (Fig. 6g-h). We tested whether the regulation of the glycolysis rate-limiting enzyme HK2 by circFAM126A depends on the HSP90/AKT1 signalling pathway by performing AKT1 rescue assays. We first constructed an AKT1 overexpression plasmid and examined its overexpression efficiency via WB analysis (Fig. S4g). Subsequently, AKT1 was transiently overexpressed in A549 cells stably overexpressing circFAM126A. The WB results showed that the overexpression of AKT1 partially reversed the decrease in the HK2 protein level caused by the overexpression of circFAM126A (Fig. 6i-j). Moreover, measurements of glucose uptake and lactate production revealed that the overexpression of AKT1 partially reversed the inhibition of glucose uptake and lactate production caused by the overexpression of circFAM126A (Fig. 6k-l). These results indicate that circFAM126A targets and regulates the HSP90/AKT1 pathway to inhibit glycolysis.Fig. 6 CircFAM126A inhibits glycolysis by targeting HSP90/AKT1. a HK2, PKM2 and LDHA protein expression levels were measured via WB analysis after the transient silencing/overexpression of circFAM126A in A549 and H1299 cells. b WB and grayscale analyses of HK2 protein expression. c‒d WB analysis of HK2 protein expression levels in tumours. e–f Immunohistochemical analysis of HK2 protein expression levels in tumours. g Measurement of glucose uptake after the transient silencing/overexpression of circFAM126A in A549 and H1299 cells. h Measurement of lactate production after the transient silencing/overexpression of circFAM126A in A549 and H1299 cells. i-j Measurement of HK2 protein expression levels via WB analysis after the transient overexpression of AKT1 in A549 cells stably overexpressing circFAM126A. k Measurement of glucose uptake after the transient overexpression of AKT1 in cells stably overexpressing circFAM126A. l Measurement of lactate production after the simultaneous transient overexpression of AKT1 and stable overexpression of circFAM126A. * and ** indicate significant differences (P < 0.05 and P < 0.01, respectively)

circFAM126A inhibits the development of lung cancer by regulating cellular glycolysis

The aforementioned findings suggest that circFAM126A can inhibit glycolysis by targeting the HSP90/AKT1 signalling pathway. Therefore, we sought to elucidate the mechanism by which circFAM126A inhibits the development of lung cancer by modulating the HSP90/AKT1 pathway to inhibit glycolysis through rescue experiments. AKT1 was transiently overexpressed in A549 cells stably overexpressing circFAM126A. First, we examined cell proliferation by conducting an EdU incorporation assay. The overexpression of circFAM126A inhibited cell proliferation, the overexpression of AKT1 promoted cell proliferation, and the simultaneous overexpression of AKT1 and circFAM126A reversed the trend towards reduced cell proliferation (Fig. 7a-b). Subsequently, cell viability was examined via a CCK-8 assay, which revealed that circFAM126A overexpression reduced cell viability, that AKT1 overexpression increased cell viability, and that simultaneous overexpression of AKT1 and circFAM126A reversed the trend towards reduced cell viability (Fig. 7c). Flow cytometry quantification of numbers of apoptotic cells revealed that the overexpression of AKT1 partially reversed the increase in apoptosis caused by the overexpression of circFAM126A (Fig. 7d-e). In addition, the results of wound healing and Transwell assays revealed that cell migration was diminished after circFAM126A overexpression and enhanced after AKT1 overexpression and that AKT1 overexpression partially reversed the attenuation of cell migration caused by circFAM126A overexpression (Fig. 7f-i). In summary, we elucidated the mechanism by which circFAM126A mediates the ubiquitination of HSP90 and participates in regulating the HSP90/AKT1 pathway through direct binding to HSP90, thereby inhibiting glycolysis and, ultimately, the development of lung cancer.Fig. 7 CircFAM126A inhibits the development of lung cancer by regulating cellular glycolysis. a-b AKT1 was transiently overexpressed in A549 cells stably overexpressing circFAM126A. EdU incorporation assay of cell proliferation. c AKT1 was transiently overexpressed in A549 cells stably overexpressing circFAM126A. CCK-8 assay of cell viability. d-e AKT1 was transiently overexpressed in A549 cells stably overexpressing circFAM126A. Flow cytometry analysis of apoptosis. f-g AKT1 was transiently overexpressed in A549 cells stably overexpressing circFAM126A. Wound healing assay of cell migration. h-i AKT1 was transiently overexpressed in A549 cells stably overexpressing circFAM126A. Transwell assay of cell migration. j Graphical Abstract: circFAM126A can undergo the m7G modification, and TRMT10C can mediate the m7G modification of circFAM126A, thereby positively modulating circFAM126A stability; circFAM126A increases the ubiquitination level of HSP90 and reduces its stability, thereby inhibiting AKT1 expression and regulating cellular glycolysis to ultimately inhibit the development of lung cancer. * and ** indicate significant differences (P < 0.05 and P < 0.01, respectively)

Discussion

In this study, differential expression profiles of m7G-modified circRNAs were constructed by analysing an m7G-circRNA epitranscriptomic microarray. We detected the significant downregulation of circFAM126A in lung cancer cell lines and tissues and found that circFAM126A expression correlated significantly with the lung cancer stage and tumour size, suggesting its ability to serve as a potential molecular marker for lung cancer. Based on the results of a series of molecular biology experiments, we are propose that circFAM126A can undergo the m7G modification. The m7G modification is one of the most common RNA modifications and, after the m6A modification, is another hot topic in epigenetic research. Although a variety of noncoding RNAs can undergo the m7G modification and play important roles in tumour development, the m7G modification of circRNAs has not been fully elucidated (Huang et al. 2023; Pan et al. 2022; Pandolfini et al. 2019).

Here, we focused on exploring the m7G modification of circFAM126A and the mechanism of m7G-modified circFAM126A in lung cancer development. By constructing m7G-circRNA epitranscriptomic microarray and performing MeRIP experiments, we found that circFAM126A can undergo the m7G modification, and functional experiments revealed that m7G-modified circFAM126A significantly inhibited lung cancer growth. Previous studies have shown that m7G modifications are widely present at the 5' end of mRNAs, and with increasing research, m7G modifications have also been recognized as important internal RNA modifications (Zhang et al. 2019a). With respect to ncRNAs, current m7G-related studies have focused on tRNAs, and tRNA m7G modifications are involved in the regulation of mRNA translation (Lin et al. 2018; Ma et al. 2021). In addition, the presence of m7G modifications in miRNAs has also been reported and can affect lung cancer cell migration (Pandolfini et al. 2019). Recent studies have revealed that the circRNA m7G modification may be associated with the process of AML drug resistance via MeRIP-seq and bioinformatics analyses (Fu et al. 2023). Another study constructed m7G-modified circRNA expression profiles using oral squamous cell carcinoma (OSCC) and normal tissues and suggested that m7G-modified circRNAs may affect OSCC progression (Sun et al. 2023), indicating that m7G-modified circRNAs may play crucial roles in tumours. Here, although we clarified the m7G-modified region in circFAM126A, the specific “motif” needs in-depth exploration in subsequent studies, and an analysis of the m7G modification "motif" may be helpful in identifying which bases in circRNAs are more susceptible to m7G methylations and thus in deriving specific targeting strategies.

The m7G modification is a dynamic process regulated by methyltransferases and demethylases. Among the identified m7G methyltransferases, METTL1 is the most extensively studied. METTL1 binds to the cofactor WD repeat structural domain 4 (WDR4) and mediates the m7G modification of miRNAs, tRNAs and mRNAs. The METTL1-mediated m7G modification of miRNAs can regulate miRNA structure and biogenesis and thus affect the migration of lung cancer cells (Pandolfini et al. 2019). The METTL1/WDR4-mediated m7G modification of tRNA promotes mRNA translation and thus affects lung cancer progression (Ma et al. 2021). In addition, the WBSCR22/TRMT112 complex and RNMT/RAM methyltransferase complex also mediate the m7G modification (Cowling 2010; Haag et al. 2015). In the present study, we revealed that TRMT10C can mediate the m7G modification of circFAM126A and positively regulate its stability. TRMT10C is involved in the formation of the RNase P complex, which is associated with the maturation of mitochondrial tRNAs (Metodiev et al. 2016; Vilardo et al. 2023). In addition, TRMT10C is an m1A methyltransferase, whereas its role in the m7G modification has not been documented in previous studies(Safra et al. 2017). In our previous experiments, knockdown of TRMT10C altered the expression of circFAM126A; therefore, we further explored the effect of the m7G modification on the stability of circFAM126A, and the results of RNA stability experiments indicated that the TRMT10C-mediated m7G modification can increase the stability of circFAM126A. Here, we were unable to determine whether TRMT10C acts independently or forms a complex with other factors to regulate the level of the m7G modification. We examined the expression of TRMT10C in tissue samples from a lung cancer cohort and found that TRMT10C was significantly downregulated in lung cancer tissues. TCGA database also revealed a negative correlation between TRMT10C expression and the survival of patients in the lung cancer cohort. In summary, we concluded that TRMT10C is downregulated in lung cancer, resulting in reduced levels of circFAM126A m7G methylation and decreased circFAM126A stability; thus, circFAM126A is downregulated in lung cancer. Current studies suggest that circRNA posttranscriptional modifications can be involved in regulating the multiple molecular fates of circRNAs. For example, m6A regulates circRNA stability (Chen et al. 2022; Du et al. 2022), nuclear export(Chen et al. 2019) and translation(Yang et al. 2017). In this study, we focused on the effect of the m7G modification on circRNA stability. Given the biological function of m7G, we will continue to explore the effects of m7G on circRNA translation and other metabolic programs in subsequent studies. Exploring the erasers and readers associated with the circFAM126A m7G modification is necessary in future studies to understand this modification more fully. Here, we have again expanded the knowledge of epigenetic modifications, providing new insights into the regulation of circRNA expression.

Aberrant expression of circRNAs is closely associated with the development of lung cancer, but the mechanism of action has not been fully elucidated. Recently, circRNAs have received increasing attention because their biological functions are mediated through interactions with proteins (Du et al. 2017; Huang et al. 2020b; Luo et al. 2019). RNA‒protein interactions are based mainly on hydrogen bonds, electrostatic forces, base aggregation and hydrophobic interactions and are also influenced by the tertiary structure of RNA (Huang et al. 2020a; Koh et al. 2011). Because of their covalent closed loop and tertiary structure, circRNAs have a stronger protein binding capacity than linear transcripts (Loughlin et al. 2019). In the present study, we found that circFAM126A targets HSP90. HSP90 is a ubiquitous and highly conserved intracellular ATP-dependent molecular chaperone that is expressed at higher levels in tumour cells than in normal cells (Hoter et al. 2018). HSP90 binds to various substrate proteins (client proteins) and assists in the repair and folding of different types of client proteins to allow their maturation, activation, and stability (Schopf et al. 2017). In addition to the tumour suppressor p53 and oncoprotein SRC, the client proteins of HSP90 include many signal transduction molecules, such as protein kinases, HIF1α and telomerase, which play important roles in tumour formation and growth (Vartholomaiou et al. 2016). HSP90 is considered a key promoter of oncogene addiction and cancer cell survival (Whitesell and Lindquist 2005).

Numerous studies have shown that circRNAs can regulate binding proteins through various mechanisms. circRNAs can act as protein sponges, thus blocking protein interactions with other molecules, recruiting proteins to specific substrate regions or altering protein interactions. circPABPN1 can sequester HuR, thereby reducing the stability of the PABPN1 mRNA and inhibiting the proliferation of HeLa cells (Abdelmohsen et al. 2017). circRHOT1 recruits TIP60 to the NR2F6 promoter to activate transcription, thereby inhibiting the development of hepatocellular carcinoma cells (Wang et al. 2019). circHIPK3 acts as a scaffold that enhances the binding of the E3 ubiquitin ligase β-TrCP and HuR and reduces p21 activity, thereby exerting antiaging and cardioprotective effects (Ding et al. 2022). Since we found that circFAM126A negatively regulates HSP90 expression and that the ubiquitin–proteasome pathway is an important regulator of protein stability, we investigated whether circFAM126A affects the level of HSP90 ubiquitination. We found that circFAM126A promotes the ubiquitination of HSP90 and reduces its stability; however, we did not perform in-depth analyses to determine whether circFAM126A enhances the interaction of HSP90 with an E3 ubiquitin ligase or identify the structural domain of HSP90 that binds to circFAM126A. We will explore more specific mechanisms of circRNA–protein interactions in subsequent studies.

HSP90 is an important member of the PI3K-AKT pathway, and AKT is an important client protein of HSP90 (Sato et al. 2000). In this study, HSP90 bound directly to AKT1 and p-AKT (S473) and positively regulated the expression levels of the AKT1 and p-AKT (S473) proteins. It has been shown that HSP90 increases the level of phosphorylated AKT1 and regulates apoptosis in lung cancer cells (Niu et al. 2021); moreover, artemether treatment of Cal27 leads leads to simultaneous increases in HSP90 and p-Akt levels, indicating that the HSP90/AKT pathway may be involved in artemether-induced apoptosis in Cal27 cells (Wu et al. 2019). In addition, through a bioinformatic analysis, we observed a significant association between circFAM126A and the PI3K-AKT pathway. Therefore, in the follow-up study, we focused mainly on this pathway. AKT, also called protein kinase B, is a key regulator of several metabolic enzymes and pathways, and AKT1 is the predominant subtype (Song et al. 2019). AKT increases the activity of glucose transport proteins and promotes glycolysis by activating several glycolytic enzymes, including hexokinase (HK) and phosphofructokinase (PFK). Studies have shown that targeting the PI3K-AKT pathway can effectively modulate the level of cellular glycolysis; in breast cancer, PD and 2-DG treatment effectively inhibited glycolysis by inhibiting the PI3K/AKT/HIF-1α/HK2 axis (Zhang et al. 2019b), and inhibition of the PI3K/Akt/HK2 axis reduced oxygen-mediated glycolysis in HPCs and decreased their proliferation (Wang et al. 2020a). In this study, we found that circFAM126A reduced the expression of hexokinase II (HK2), whereas AKT1 reversed the reduction in HK2 expression. Moreover, circFAM126A negatively regulated glucose uptake and lactate production, and AKT1 reversed these effects. Therefore, we propose that circFAM126A can regulate HSP90/AKT1 and inhibit cellular glycolysis. Most tumour cells have an altered metabolic pattern of reliance on aerobic glycolysis for energy, a phenomenon called the "Warburg effect". Although glycolysis produces ATP less efficiently than does oxidative phosphorylation, high aerobic glycolytic flux confers additional advantages to tumour cells. Targeting glycolytic enzymes to modulate the metabolic state of tumour cells is considered a promising therapeutic strategy for treating malignancies (Pouyssegur et al. 2022; Stine et al. 2022). As research progresses, the important roles of circRNAs in regulating glycolytic activity and influencing tumour progression have gradually been revealed. For example, circ-ENO1 can act as a sponge for miR-22-3p and upregulate ENO1 expression to promote glycolysis and lung adenocarcinoma progression (Zhou et al. 2019). circSLC25A16 accelerates glycolysis and the proliferation of lung cancer cells via the miR-25-16p/HIF-488α/LDHA axis (Shangguan et al. 2020). However, the mechanism by which circRNAs regulate glycolysis and promote lung cancer development by targeting key tumour proteins has not been fully elucidated. In this study, circFAM126A was shown to regulate HSP90 ubiquitination and inhibit AKT1 expression, thereby reducing glycolytic activity and inhibiting lung cancer development. These findings provide a new strategy for targeting cancer cell metabolism, identifying specific therapeutic targets and developing anticancer drugs for lung cancer.

In summary, this study reveals that circFAM126A can undergo the m7G modification, and we propose the hypothesis that TRMT10C mediates the m7G modification of circFAM126A, thereby positively modulating circFAM126A stability. Low expression of circFAM126A in lung cancer is associated with the lung cancer stage and progression and has potential utility as a molecular target for lung cancer. Mechanistically, circFAM126A increases HSP90 ubiquitination, which in turn reduces cellular glycolysis through the HSP90/AKT1 pathway, affects cell fate, and ultimately inhibits lung cancer development (Fig. 7j). This study not only broadens our understanding of methylation modifications but also provides new insights into the epigenetic mechanisms affecting tumour cell metabolism, which will facilitate lung cancer prevention and treatment.

Supplementary Information

Below is the link to the electronic supplementary material. Supplementary file1 (PDF 118 KB)

Supplementary file2 (PDF 119 KB)

Supplementary file3 (XLSX 3657 KB)

Supplementary file4 (XLSX 212 KB)

Supplementary file5 (XLSX 2000 KB)

Supplementary file6 (PDF 603 KB. Fig. S1. Identification of methyltransferases. a Schematic diagram of the CLIP primer design. b Schematic diagram of the TRAP experiment. c Transient silencing of methylation-related enzymes in A549 cells. qPCR assay of their silencing efficiency. d Transient silencing of methylation-related enzymes in A549 cells and qPCR assays for circFAM126A expression. e qPCR analysis of TRMT10C expression levels in lung cancer cell lines (A549, H226, H460, H1299, and H2170) and BEAS-2B cells. f qPCR analysis of the expression level of TRMT10C in lung cancer tissues and paraneoplastic tissues. g TRMT10C was transiently transfected into A549 cells, and its overexpression efficiency was verified. h The overall level of the m7G modification was determined by performing an m7G dot blot assay after the overexpression of TRMT10C in A549 cells. i MeRIP‒qPCR analysis of m7G levels on circFAM126A after the overexpression of TRMT10C in A549 cells. j qPCR analysis of circFAM126A expression after the overexpression of TRMT10C in A549 cells treated with actinomycin D (2 µg/mL) for 0 h, 12 h, 24 h and 36 h. Fig. S2. KEGG pathway enrichment analysis of circFAM126A-targeted mRNAs and qPCR analysis of the expression levels of circFAM126 and FAM126A mRNA. a KEGG pathway enrichment analysis of circFAM126A-targeted mRNAs. b circFAM126A was transiently silenced or overexpressed in A549 cells and circFAM126A expression levels were detected via qPCR. c circFAM126A was transiently silenced or overexpressed in A549 cells and FAM126A expression levels were detected via qPCR. Fig. S3. Construction of A549 cell lines with stable circFAM126A overexpression. a A549 cells were infected with a lentivirus and cell fluorescence was observed after 48 hours. b The efficiency of stable circFAM126A overexpression in the A549 cell line was verified. Fig. S4. Survival curves of patients based on HSP90 expression and glycolysis-related protein assays. a Overall survival curves of patients stratified based on HSP90 expression. The number of HSP90AA1 low expression group is 120, The number of HSP90AA1 high expression group is 120. (http://gepia.cancer-pku.cn/). b Disease-free survival of patients stratified based on HSP90 expression. The number of HSP90AA1 low expression group is 120, The number of HSP90AA1 high expression group is 120 (http://gepia.cancer-pku.cn/). c Detection of HSP90 mRNA expression levels via qPCR after the transient silencing/overexpression of circFAM126A in A549 and H1299 cells. d Overexpression of HSP90 in A549 cells and WB analysis of the efficiency of HSP90 overexpression. e WB and grayscale analyses of PKM2 protein expression. f WB and grayscale analyses of LDHA protein expression. g Overexpression of AKT1 in A549 cells and WB analysis of the efficiency of AKT1 overexpression.)

Abbreviations

circRNA Circular RNA

CHX Cycloheximide

CLIP Cross-linking Immunoprecipitation

Co-IP Co-Immunoprecipitation;

HSP90 protein Heat Shock Protein 90 protein

HK2 Hexokinase 2

IF Immunofluorescence

IP Immunoprecipitation

MeRIP Methylated RNA Immunoprecipitation

MG132 Carbobenzoxyl-L-leucyl-L-leucyl-L-leucine

m7G N7-methylguanosine

METTL1 Methyltransferase-like 1

MS Mass Spectrometry

PKM2 Pyruvate Inase Isozyme Type M2

TRAP Tagged RNA Affinity Purification

Acknowledgements

We thank Huafu Zhou and Jun Liu from the first Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China for their support and assistance in the collection of clinical samples.

Author contributions

ARN conceived and designed this study. GL supervised and guided the study. QYZ, XFL and JXW performed the experiments, wrote the article and results analysis. RRZ, SXC, DYC, HTX and WYP collected clinical samples and analyzed the data. ARN guided the revision of this article. All authors reviewed and approved the final manuscript.

Funding

This work was partially supported by the National Natural Science Foundation of China (NSFC82260652) and Guangxi Science and Technology Base and Talent Special Project (AD22080055).

Data availability

The datasets used and/or analyzed in the present study are available from the corresponding author on reasonable request. The circRNA sequencing results have been uploaded to the GEO database (PRJNA971588), and the m7G-circRNA Epitranscriptomic Microarray (GSE232281).

Declarations

Ethics approval and consent to participate

This study was approved by the Ethical Committee of The First Affiliated Hospital of Guangxi Medical University (NO.2022-KY-E-(289)). All animal experiments conducted was compliant with the Ethics Committee of Guangxi Medical University (Nọ: 202401203).

Competing interest

The authors declare no competing interests.

Qingyun Zhao and Xiaofei Li are co-first authors.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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