Introduction
Non-small cell lung cancer (NSCLC) is the dominant form of lung cancer, constituting around 85% of all reported lung cancer instances [
1]. It is further divided into a variety of subtypes, with adenocarcinoma, squamous cell carcinoma, and large cell carcinoma being the most widespread [
2]. Even with progress in early detection and treatment techniques, NSCLC is commonly diagnosed at an advanced stage, greatly reducing treatment alternatives and resulting in a bleak prognosis [
3]. The 5-year survival rate for late-stage NSCLC patients remains dishearteningly low, highlighting the urgency for sustained investigation and enhanced treatment modalities. The etiology of NSCLC is multifaceted and involves a combination of genetic and environmental influences [
4]. A host of genetic modifications have been found in NSCLC, such as anomalies in the epidermal growth factor receptor (EGFR) and KRAS genes or changes concerning the anaplastic lymphoma kinase (ALK) gene, leading to the advent of targeted therapeutic interventions [
5]. Moreover, immunotherapy, specifically immune checkpoint inhibitors targeting the PD-1/PD-L1 axis, has surfaced as an encouraging treatment avenue for NSCLC, conferring significant survival improvements for some patients [
6]. Despite this, not all patients react favorably to these treatments, and the development of resistance is a possibility, instigating ongoing research to detect biomarkers that can forecast patient prognosis and formulate tactics to surmount resistance.
Circular RNAs (circRNAs) are a unique subclass of non-coding RNAs distinguished by their covalently closed loop structures, unlike their linear counterparts [
7]. Notable for their robust stability, conservation, and often tissue or development-stage-specific expression, circRNAs have recently garnered considerable interest for their implicated roles across various diseases, prominently including cancer [
8,
9]. Within the landscape of tumorigenesis, circRNAs demonstrate regulatory capacity at diverse stages—spanning from transcription to translation [
10]. They exhibit functionality as microRNA sponges, engage in the formation of RNA–protein complexes through interaction with RNA-binding proteins, modulate transcription of parent genes, and can even give rise to functional peptides through translation [
11]. Across various cancer types, a dysregulation of circRNA expression is commonly observed, often with ties to the progression and prognosis of the disease. Certain circRNAs act as oncogenes, fostering tumor growth, metastasis, and resistance to therapy, while others assume the role of tumor suppressors, curbing these processes [
12]. Given the multifaceted roles that circRNAs play in the cancer biology, they hold substantial promise as innovative diagnostic biomarkers and therapeutic targets for cancer. For example, Li et al. discovered that circNDUFB2 can break the stability of IGF2BP through the TRIM25/circNDUFB2/IGF2BPs complex, further suppressing NSCLC progression [
13]. Xu et al. found that the circRNA hsa_circ_0000326 can enhance lung cancer progression by regulating the miR-338-3p/RAB14 axis [
14]. Moreover, Liu et al. found that N6-methyladenosine-modified circIGF2BP3 impairs CD8 + T-cell responses and encourages tumor immune evasion by enhancing the deubiquitination of PD-L1 in NSCLC [
15].
In this paper, we have shed light on the significant role that circATP9A plays in NSCLC. We established that circATP9A can drive NSCLC progression. On a mechanistic level, circATP9A engages with the HuR protein, forming an RNA–protein complex and subsequently enhancing both the mRNA and protein expression levels of its target gene, NUCKS1. Importantly, we identified the PI3K/AKT/mTOR signaling pathway as a downstream effector of the circATP9A/HuR/NUCKS1 axis. Furthermore, the protein hnRNPA2B1 has been shown to facilitate the encapsulation of circATP9A into extracellular vesicles (EVs). These EVs, enriched with circATP9A, subsequently induce the M2 phenotype in tumor-associated macrophages (TAMs), thus promoting NSCLC progression. The findings of our study point to circATP9A as a potential diagnostic tool and therapeutic target in NSCLC management.
Methods
Samples collection
The open-accessed circRNAs expression profile and corresponding tissue information were obtained from the Gene Expression Omnibus (GEO), GSE112214 and GSE158695 projects. Detailed, the GSE112214 and GSE158695 projects both provide the circRNAs expression profile of three NSCLC and normal adjacent tissues (NATs), whose annotation platform is GPL19978. Eighty patients with NSCLC and normal adjacent tissues (NATs) underwent surgical resection at the Huadong hospital. Totally, 39 NSCLC samples and 20 NATs were collected fromthe Huadong hospital. The samples were promptly preserved in liquid nitrogen at the Huadong hospital. Each sample underwent independent evaluation by two distinct pathologists. Importantly, no patients had been subjected to any preoperative procedures. We also gathered preoperative serum samples from each participant. The Ethical Committee of Huadong hospital, approved the use of these specimens. Written informed consent was procured from every participating patient. Pan-cancer data was get from the UCSC Xena database (
https://xenabrowser.net/datapages/). Expression profile and clinical data of NSCLC patients in The Cancer Genome Atlas Program (TCGA) database was downloaded from the TCGA-GDC [
16]. Open-accessed clip-seq data was get from the ENCORI database (
http://starbase.sysu.edu.cn/).
Cell lines
The human non-small cell lung cancer (NSCLC) cell lines H522, A549, H1299, H460, the human bronchial epithelium cell line (BEAS-2B),human monocytes cell line THP-1 and mouse Lewis lung carcinoma cell line (LLC), were all obtained from the American Type Culture Collection (ATCC, Manassas, VA). These cell lines were all grown in a controlled environment at 37 °C with a 5% CO2 concentration, using RPMI 1640 medium (Biosharp, Guangzhou, China) or DMEM medium (Biosharp, Guangzhou, China). The media were further supplemented with 10% fetal bovine serum (FBS; Gibco, South America).
Animal experiments
Every animal experiment conducted received approval from the Fudan University (20230304Z). Both subcutaneous xenograft and tail vein lung metastasis models were established using female BALB/c nude mice. For the subcutaneous tumor model, roughly 2 × 106 A549 cells suspended in a 40% Matrigel (BD, San Jose, CA, USA) medium were injected into each mouse's flank. Tumor development was monitored and quantified on a weekly basis, with tumor volume calculated using the formula (length × width2/2). After a period of four weeks, the mice were euthanized and tumor weights were recorded. For the tail vein metastasis model, an approximate of 1 × 106 A549 cells were introduced via the tail vein into the nude mice. Four weeks later, all mice were euthanized. Their lungs were imaged and then harvested for ensuing analysis. LLC cells were utilized to establish a tail vein metastasis model in C57/B6 mice. For the exosome treatment experiments, exosomes produced by both control and A549 cells, which overexpressed circATP9A, were administered intravenously into the tail vein of C57/B6 mice. Each injection contained 2 μg of exosomes, and the mice received two injections per week. After a total of five injections, the mice were sacrificed for tissue collection and further assessments. Ki67 expression in tumor tissues from xenografted nude mice was quantitatively assessed using the H score. Tumor samples were first fixed, paraffin-embedded, and sectioned for immunohistochemical (IHC) analysis. These sections were stained with a Ki67-specific antibody, highlighting proliferating cells. The H score for Ki67 was calculated by evaluating both the intensity of staining (graded as 0, 1 + , 2 + , or 3 + for none, weak, moderate, or strong staining, respectively) and the percentage of Ki67-positive cells in the tissue. The final H score, a product of the intensity score and the percentage of positive staining.
Plasmid construction and cell transfection
For stable circATP9A knockdown, three unique shRNAs targeting circATP9A were engineered into lentiviral vectors, a task completed by GenePharma (Suzhou, China). A nonspecific sh-NC vector was employed as the control. Additionally, a full-length circATP9A lentiviral vector, also synthesized by GenePharma, was employed for circATP9A overexpression, with a control vector lacking any circATP9A sequence used as the reference group. For the generation of stable cell lines, A549 and H1299 cells were infected with respective lentiviruses using 1 µl of Polybrene (5 µg/µl) from GenePharma as an enhancer. After 72 h, the cells were exposed to 5 µg/ml of puromycin in medium for selection. Post a 10-day duration, puromycin-resistant cells were deemed as stably transfected. A similar methodology was applied for siRNAs against HuR and NUCKS1-OE plasmids, both sourced from RiboBio (Guangzhou, China). The transfection of si-RNAs and plasmids was executed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), abiding by the manufacturer’s instructions. The knockdown or overexpression efficacy of specific molecules was validated through qRT–PCR or western blot assays. The sequences of sh-circATP9As and siRNAs are provided in Table S
1.
Quantitative Real-time PCR (qRT–PCR)
Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by cDNA conversion via the PrimeScript RT Reagent Kit (Takara, Tokyo, Japan). The ensuing qRT-PCR analysis was carried out utilizing the Sybr green system, with GAPDH functioning as the internal standard. RNA relative expression levels were computed via the 2–ΔΔCT method. Table S
2 lists the primer sequences used in this study.
Rnase R treatment
In summary, RNA was retrieved from A549 and H1299 cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the provided instructions. This was accompanied by the addition of Rnase R (1.0 U/μg) (Geenseed, Guangzhou, China) to 500-ng RNA, and incubation for 20 min at 37 °C. For control, a similar amount of RNA was processed without Rnase R under identical conditions. The stability of circATP9A versus linear mRNA-ATP9A was then assessed using qRT-PCR.
Electrophoresis analysis
For gel electrophoresis, a 1% agarose gel was prepared by heating 50 ml of 1 × TAE buffer with 0.5-g agarose until boiling. After cooling to 70 °C–80 °C, 5 μl of 4S GelRed (Sangon Biotech, Shanghai, China) was thoroughly mixed in. After solidification of the poured solution in a mold, the gel was placed in an electrophoresis tank filled with 1 × TAE buffer. Subsequently, 10 μl of DNA samples, mixed with loading buffer, were loaded into each well. The electrophoresis ran at 120 V for 30 min, and the resulting bands on the gel were captured using an ultraviolet imaging system.
In situ hybridization (ISH)
For ISH, we used a probe targeting the circATP9A splicing site, tagged with 5′-digoxin (DIG) and 3′-DIG. This probe was custom made by Servicebio (Wuhan, China). Scramble probe (negative) and a U6 probe (internal) served as controls. The procedure started with deparaffinization and rehydration of paraffin sections with xylene and a series of graded ethanol. Subsequently, a 20-min incubation with proteinase K at 37 °C was performed, followed by a 10-min Triton-X100 treatment at 4 °C. The sections were then incubated overnight at 37 °C in a hybridization buffer containing the circATP9A probe, and another overnight incubation with the anti-digoxin antibody at 4 °C followed. Afterwards, sections were treated with 5-Bromo-4-Chloro-3-Indolylphosphate/Nitroblue Tetrazolium (BCIP0/NBT) (Beyotime, Shanghai, China) at room temperature for 30 min, and with Nuclear fast red (Servicebio, Wuhan, China) at room temperature for 3 min. Visualization and imaging were done with an Olympus microscope (Olympus, Tokyo, Japan). The H-score for circATP9A was computed as: H-score = Σ (P × I), where P signifies the percentage of stained cells, and I represents the staining intensity score: 0 for no staining, 1 for weak, 2 for moderate, and 3 for intense staining. Table S
3 provides details of the ISH probes.
RNA fluorescence in situ hybridization (FISH)
To ascertain the subcellular localization of circATP9A in NSCLC cells, we used a Cy3-labeled circATP9A-specific probe from RiboBio (Guangzhou, China). Briefly, NSCLC cells (A549 and H1299) were trypsinized and resuspended in medium. Approximately 2000 cells were seeded on a 48-well plate with a glass cover slip. Upon reaching 70% to 90% confluence, cells were thrice washed with PBS and fixed with 3.7% paraformaldehyde. Cell permeabilization was achieved using 0.5% Triton-100 for 10 min at 4 °C. Cells were then pre-hybridized at 37 °C for 30 min, followed by overnight incubation at 37 °C with the circATP9A-FISH probe in a dark hybridization buffer. After hybridization, cells were washed using SSC solutions and stained with DAPI for 15 min in darkness. The prepared slides were observed and photographed using a confocal fluorescence microscope (Carl Zeiss AG, Jenna, Germany). Table S
3 contains information about the circATP9A-FISH probe utilized in this experiment.
Colocalization of circATP9A with HuR
Fluorescence staining was employed to assess the colocalization of circATP9A and HuR in NSCLC cells. Briefly, around 2,000 NSCLC cells were seeded onto a 48-well plate with cover glass. Upon reaching 70%–90% confluence, the cells were washed three times with PBS, then fixed with 3.7% paraformaldehyde. Subsequently, cells were permeabilized using 0.5% Triton-100 for 10 min at 4 °C. Pre-hybridization for 30 min at 37 °C using pre-hybridization buffer was carried out, followed by an overnight incubation at 37 °C with a Cy3-labeled circATP9A-FISH probe (RiboBio) in hybridization buffer in a dark environment. The cells were then permeabilized again with 0.5% Triton-100 for 10 min at 4 °C, before incubating with an anti-HuR antibody (Abcam) overnight at 4 °C in darkness with gentle rotation. Following this, cells were washed with PBS and nuclei were stained with DAPI for 15 min. Finally, cells were visualized and imaged with a confocal fluorescence microscope (Carl Zeiss AG, Jenna, Germany).
Western blotting
The Total Protein Extraction Kit (KeyGEN, Nanjing, China) was used to extract the total protein in NSCLC cells, and the concentrations were measured by a BCA protein assay kit (KeyGEN, Nanjing, China). Antibodies against HuR (ab200342, 1:1000), NUCKS1 (12,023–2-AP, 1:1000), β-actin (20,536–1-AP, 1:5000), p-AKT (Ser273) (66,444–1-Ig, 1:5000), AKT (60,203–2-Ig, 1:5000), p-mTOR (Ser2448) (67,778–1-Ig, 1:2000), mTOR (66,888–1-Ig, 1:5000), CD9 (20,597–1-AP, 1:1000), CD63 (25,682–1-AP, 1:1000), CD81 (66,866–1-Ig,1:2000), Calnexin (10,427–2-AP, 1:5000), hnRNPA2B1 (14,813–1-AP, 1:2000) were obtained from Abcam and Proteintech. Chemiluminescent signals were detected using Western ECL Substrate (Advansta, Menlo Park, CA, USA) and images were captured with a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).
RNA pull-down assay
The interactions between circATP9A and HuR, as well as circATP9A and hnRNPA2B1, were verified using biotin-coupled circATP9A probes and control probes, furnished by GenePharma (Suzhou, China). The protocol is outlined as follows: Roughly 1 × 10
7 A549 and H1299 cells were lysed and sonicated in a 4 °C water bath for half an hour. A fraction (20 µl) of the lysate was allocated for RNA input, while a major portion (80 µl) was set aside for protein input. Probes were then incorporated into the lysate and stirred at room temperature for 16–24 h. Next, 100 µl of streptavidin magnetic beads (MCE, Monmouth Junction, NJ, USA) were added to the lysate and rotated at room temperature for 2–4 h. Using a magnetic stand, the beads were gathered and washed five times with washing buffer (containing PMSF, Protease inhibitor, and Rnase inhibitor). The cleansed beads were resuspended in 1 ml of washing buffer, with 100 µl of this solution used for RNA purification and the remaining 900 µl allocated for protein purification. For RNA extraction, the 100-µl sample was combined with 5 µl of proteinase K (Sangon Biotech, Shanghai, China) and RNA PK buffer, then gently rotated at 50 °C for 45 min followed by 10-min heating at 95 °C to break the formaldehyde cross-links. The RNA was subsequently purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), converted into cDNA, and stored at -80 °C for future use. For protein extraction, 300 µl of 4 × loading buffer was added to the remaining 900-µl sample, followed by a 10-min incubation at 100 °C. The supernatant with the protein extract was then isolated using the magnetic stand, and the protein was deployed for mass spectrometry (MS) analysis and Western blotting. The sequences of the circATP9A probe and the control probe are presented in Table S
3.
Silver staining
Overall, proteins extracted from the circATP9A RNA pull-down assay were differentiated using a 10% SDS-PAGE gel. The gel was subsequently stained with a Silver Stain kit (BL620A, Biosharp, Beijing, China) in accordance with the producer’s instructions.
RNA immunoprecipitation (RIP) assay
For verification of the link between HuR and circATP9A, and hnRNPA2B1 and circATP9A, a RIP assay was conducted employing a RIP kit (Millipore, MA, USA). In essence, approximately 2 × 10^7 A549 and H1299 cells were harvested and lysed with RIP lysis buffer. Interacting RNAs were then precipitated with anti-HuR antibody (ab200342, Abcam) and anti-hnRNPA2B1 antibody (14,813–1-AP, Proteintech). The anti-IgG antibody (ab172730, Abcam) was utilized as a negative control. The co-precipitated RNAs were then isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and their abundance was evaluated by qRT-PCR.
Co‐culturing system
To mimic the EV-mediated intercellular communication that takes place between tumor cells and TAMs, an in vitro indirect co-culture model was established. Macrophages and LUAD cells were individually planted into the upper and lower chambers of a Corning® Transwell® cell culture insert (4 μm pore size, Corning Inc., Corning, NY, USA), incorporating a polycarbonate membrane. After a 48-h co-culture period, the cells were harvested for further experiments.
Isolation Evs from cell medium
Evs were derived from NSCLC cell media through a series of steps: NSCLC cells were cultured in a medium enriched with 10% EV-free FBS. Following a 72-h incubation at 37 °C with 5% CO2, the medium was collected and centrifuged under various conditions: initially at 2,000 g for 10 min, then at 3,500 g for 20 min, followed by 10,000 g for 1 h, and finally at 120,000 g for 2 h. All centrifugation phases were carried out at 4 °C. The resultant purified Evs were re-suspended in PBS and then stored at -80 °C for subsequent use.
Macrophage induction from monocytes and flow cytometry
THP1 cells, cultured in six-well plates, were treated with 100 ng/mL of phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich) and incubated for 24 to 48 h. After incubation, the medium was swapped with fresh PMA-free medium, and the cells were maintained for another 3 days before use. For the detection of macrophage surface markers, cells in chilled PBS were treated with either anti‐CD206 or anti‐HLA-DR antibodies (both from eBioscience) at 4℃ for 30 min. Following incubation, the cells were washed and subsequently analyzed using a BD Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA, USA) to detect macrophage surface markers CD206 and HLA-DR.
Evs internalization
Evs derived from RCC cells were labeled using the PKH26 Red Fluorescent Cell Linker Kit (Umibio, Shanghai, China). Following this, PMA-stimulated THP-1 cells [THP-1 (Mφ)] were co-incubated with these PKH26-tagged Evs in a dark setting overnight. After staining the cells with 4′,6-diamidino-2-phenylindole (DAPI), the EV uptake process was visualized using a confocal fluorescence microscope (Carl Zeiss AG, Jenna, Germany).
CCK-8 assay, EdU assay, colony formation assay, and transwell assay
The proliferation of NSCLC cells was gauged using the CCK-8 assay, the 5-ethynyl2′deoxyuridine (EdU) assay, and the colony formation assay. The cell invasion and migration capabilities were assessed through transwell invasion and migration assays, executed following the protocols delineated in our previous research [
17].
Statistics analysis
Statistical evaluations were conducted using GraphPad Prism and SPSS Software. A two-tailed Student’s t-test, ANOVA followed by Tukey’s multiple comparisons post-test, and Pearson’s correlation analysis were the methods used for statistical comparisons. All statistical data are expressed as mean ± standard error of the mean. All p values were calculated using a two-sided test, with p values < 0.05 considered statistically significant. Each experimental procedure was performed at least three times for reliability. The survival difference in different groups was compared using the Kaplan–Meier method.
Discussion
Circular RNAs (circRNAs) have garnered substantial interest due to their unique stability and diverse expression profiles, marking them as potential contributors in the field of cancer research [
26]. As potential oncological markers, circRNAs can be utilized for the early identification of cancer, the evaluation of prognosis, and the monitoring of treatment responses [
27]. Given that circRNA expressions within cells are often tissue-specific, it affords the opportunity for precise diagnoses of various cancer types [
28]. Concurrently, certain circRNAs have been found to influence the growth, migration, and invasion of cancer cells, suggesting the possibility of leveraging circRNAs as novel therapeutic targets [
29]. Nevertheless, despite the promising potential of circRNAs as cancer markers, further research is imperative to substantiate and deepen our understanding of their specific roles within oncology [
30]. In the present study, we noticed a considerable amplification of circATP9A in NSCLC tissue samples, particularly in those at more advanced clinical stages. These findings strongly propose the potential role of circATP9A as a meaningful tissue biomarker in NSCLC progression. However, the study is constrained by the limited number of patients involved and the relatively brief inclusion period. We aim to increase patient enrollment and continue patient follow-ups to acquire survival data, thereby evaluating the efficacy of circATP9A as a prognostic indicator in NSCLC. Further, we could probe the expression levels of circATP9A in serum in future studies to ascertain its potential as a liquid biopsy biomarker.
Our study elucidates the functional role of circATP9A in NSCLC. We demonstrated that circATP9A can foster the progression of NSCLC. From a mechanistic standpoint, circATP9A can interact with the HuR protein to form an RNA–protein complex, subsequently amplifying the mRNA and protein levels of the target gene NUCKS1. Further, the PI3K/AKT/mTOR signaling was identified as the downstream pathways of circATP9A/HuR/NUCKS1 axis. More notably, hnRNPA2B1 can mediate the incorporation of circATP9A into EVs. Subsequently, these EVs containing circATP9A induce the M2 phenotype of TAMs, thereby facilitating NSCLC development (Fig.
8F).
Within cells, circRNAs can interact with RNA-binding proteins (RBPs) [
31]. RBPs are a category of proteins that bind to RNA molecules, governing RNA metabolism and functionality [
32]. The association of circRNA with RBP can influen’e RBP's functionality and the stability of RNA [
33]. The complexity and intrigue of circRNA-protein interaction exceed that of circRNA-miRNA interaction, attracting recent research focus [
34]. RBPs exert influence over all phases of the circRNA life cycle, including its biogenesis, localization, functionality, and degradation.
HuR, a key RNA-binding protein, forms complexes with a wide variety of RNA molecules, including mRNA, non-coding RNA, and others, subsequently influencing their stability, localization, and translation [
35]. By recognizing and binding to specific sequences of RNA (usually AU-rich regions), HuR significantly determines the fate of RNA [
36]. In the context of cancer, overexpression or malfunctions of HuR frequently give rise to malignant phenotypes, including amplified cell proliferation, apoptosis resistance, and enhanced migratory ability [
37,
38]. Furthermore, HuR partakes in numerous physiological and pathological processes such as immune response and stress response, positioning itself as a pivotal regulator of RNA metabolism and messenger RNA [
39]. In our research, we identified the interaction between circATP9A and HuR via bioinformatic analysis and substantiated this interplay through subsequent pulldown assays. Moreover, we found that escalated circATP9A expression encourages NSCLC progression through its engagement with HuR, thus amplifying downstream NUCKS1 expression. Yet, the precise mechanistic details warrant further investigation. Concurrently, we cannot disregard other potential mechanisms such as those involving miRNA sponges.
Recently, more and more studies have shown that circRNAs can interact with regulatory RBPs and further affect the fate of their target mRNAs [
40]. Our results suggest that NUCKS1 is regulated by the circATP9A/HuR complex. NUCKS1 is a non-histone chromatin structural protein widely present in a variety of organisms [
41]. NUCKS1 plays an important role in cell proliferation, DNA damage repair and signal transduction. Studies in recent years have shown that NUCKS1 is upregulated in some types of cancer and may be related to tumorigenesis and progression, so it is considered a potential tumor biomarker and therapeutic target [
42]. In NSCLC, Zhao et al. noticed that NUCKS1 can promote NSCLC proliferation, invasion and migration through upregulating CDK1 expression [
43]. Yu et al. demonstrated that circRNA circ_0008037 promotes tumor growth and the Warburg effect by upregulating NUCKS1 through binding to miR-433-3p in NSCLC [
44]. Our investigation demonstrates that circATP9A associates with HuR, thereby enhancing its expression, which in turn results in increased regulation of NUCKS1 mRNA. However, our current experimental data indeed demonstrate an interaction between RNA and protein, but they do not conclusively establish whether this interaction is direct or mediated by other molecules. Meanwhile, it is important to note that the influence of circATP9A on NSCLC cells cannot be entirely attributed to NUCKS1 alone, suggesting the existence of other mechanisms. Our results demonstrate that circATP9A directly binds with HuR to increase NUCKS1 expression, subsequently activating PI3K/AKT/mTOR signaling and promoting cancer progression. Nevertheless, the incomplete reversal of NSCLC malignancy phenotypes by NUCKS1 reintroduction implies additional circATP9A-related pathways or mechanisms that require further exploration.
According to previous studies, non-cancerous cells present within the tumor microenvironment can significantly contribute to tumor progression [
45]. Notably, benign macrophages situated in the tumor microenvironment have been identified to exacerbate malignant progression, facilitating angiogenesis, invasiveness, and migration of cancer cells while hampering antitumor immunity [
46]. Macrophages, being critical immune cells in the tumor milieu, are often polarized into two distinct phenotypes: M1 or M2. These variants execute different roles, with M1 macrophages generally exerting tumor-suppressive functions, while M2 macrophages encourage tumor development. Importantly, Guo et al. noticed that the M2 phenotype induced by THP-1 cells can promote NSCLC cell metastasis [
47].
EVs are a diverse group of minute vesicles, with sizes spanning from 30 to 1000 nm [
48]. They are secreted by cells and are packed with biomolecules including proteins, lipids, RNA, and DNA [
49]. In the context of cancer, EVs hold substantial significance. Cancer cells can secrete EVs that ferry these biomolecules to designated cells, thereby altering the biological activities of these recipient cells. For instance, EVs released by cancer cells can transport growth and metastasis-promoting signaling molecules, thereby influencing the conduct of both neighboring normal cells and distant recipient cells. Moreover, EVs may impact the tumor microenvironment, such as by inhibiting immune responses and boosting angiogenesis [
50]. Therefore, EVs play a pivotal role in the onset and development of cancer. In our research, we discovered that circATP9A can be encapsulated into EVs, subsequently being engulfed by TAMs [
51]. This process fosters their polarization towards an M2 phenotype, further expanding the functional spectrum of circATP9A. Nonetheless, the precise mechanism underlying this polarization warrants further investigation in subsequent studies.
The RNA-binding protein hnRNPA2B1 has been identified to play a role in the encapsulation of RNA into EVs by recognizing specific motifs, such as GGAG/CCCU [
52]. Herein, we identified the presence of the GGAG motif within the sequence of circATP9A. Additionally, we observed a decreased expression of circATP9a in EVs after the knockdown of hnRNPA2B1. These results indicated that EVs-circATP9A may serve as a distinctive tactic for the treatment of NSCLC.
In conclusion, Our study's elucidation of the functional role of circATP9A in NSCLC presents several significant implications for the treatment and management of this disease. By demonstrating that circATP9A fosters the progression of NSCLC through its interaction with the HuR protein and the subsequent amplification of NUCKS1 mRNA and protein levels, we have uncovered a novel regulatory mechanism in lung cancer pathology. The identification of the PI3K/AKT/mTOR signaling pathway as a downstream target of the circATP9A/HuR/NUCKS1 axis further expands our understanding of the molecular underpinnings of NSCLC. From a therapeutic standpoint, these insights provide a foundation for developing new strategies targeting circATP9A. Inhibiting the function or expression of circATP9A could potentially disrupt the circATP9A/HuR/NUCKS1 signaling axis, thereby impeding NSCLC progression. This approach could be particularly effective in tumors where this pathway is upregulated. Moreover, our findings about the role of hnRNPA2B1 in mediating the incorporation of circATP9A into EVs and its subsequent influence on TAMs open up new avenues for therapy. Targeting these EVs or modulating the M2 polarization of TAMs could provide an innovative approach to altering the tumor microenvironment in favor of therapeutic efficacy. The potential of circATP9A as a diagnostic biomarker also cannot be overlooked. Its presence and levels in patient samples could serve as an indicator of disease progression or response to therapy, aiding in personalized treatment planning. In summary, our discoveries highlight circATP9A as a promising candidate for both diagnostic and therapeutic applications in NSCLC. Targeting this molecule could lead to the development of new therapeutic strategies that improve treatment outcomes for patients suffering from this challenging disease.
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