Highlights
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This study identifies miR-125b as an inflammation-responsive miRNA in NSCLC.
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DAZAP2 is validated as a direct target of miR-125b.
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The miR-125b/DAZAP2 axis contributes to NSCLC progression and may represent a potential molecular target.
Keywords: Lung cancer, Inflammation, DAZAP2, miR-125b
Abstract
Background
Lung cancer remains the primary cause of cancer-related mortality globally, despite significant advancements in therapeutic strategies. Overall survival rates remain unsatisfactory. Chronic inflammation and microRNAs both play pivotal roles in cancer development.
Methods
This study aimed to elucidate the roles of key microRNAs in inflammation-associated non-small cell lung cancer (NSCLC) development.
Results
Our findings reveal a significant reduction in miR-125b expression within NSCLC cell lines when stimulated by IL-10. Furthermore, when stimulated by IFN-γ, the expression levels of miR-125b markedly increase. Enforced expression of miR-125b markedly bolstered cell proliferation, migration, and invasion, while diminishing cell apoptosis. Conversely, inhibition of miR-125b produced opposing effects. Mechanistically, DAZAP2 was identified as a direct regulatory target of miR-125b However, because both miR-125b inhibition and DAZAP2 knockdown suppressed malignant phenotypes, DAZAP2 may represent one component of a broader miR-125b-associated regulatory network rather than the sole mediator of miR-125b function. Combined inhibition of miR-125b and DAZAP2 produced more pronounced tumor-suppressive effects both in vitro and in vivo.
Conclusions
Our data suggest that miR-125b and DAZAP2 are involved in the cytokine-responsive regulatory network of inflammation-related NSCLC progression.
Introduction
Lung cancer ranks as the leading cause of cancer-related death worldwide [1], of which non-small cell lung cancer accounts for about 85% of lung cancer [2]. Though tremendous therapeutic advances have been gained over the last decade, the overall survival (OS) of lung cancer patients were only improved slightly, and the five-year overall survival rate remained poor (<15%) [3], which seriously threatens human life and health. Chronic inflammation may induce a series of inflammatory diseases including malignant tumors [4], and under the influence of chronic inflammation, abnormal cloning of cells of human tissues and organs will escape the monitoring of the immune system and eventually progress to aggressive tumors [5]. Understanding how inflammation influences tumor cell behavior may help clarify the mechanisms underlying inflammation-associated tumor progression.
IL10 (interleukin 10) also known as an inhibitor of human cytokine synthesis (CSIF), plays an important role in maintaining tissue homeostasis during infection and inflammation by inhibiting excessive inflammatory responses and promoting tissue repair mechanisms [6,7]. In recent years, studies have found that IL10 has two sides in the development of tumors, some have found that low IL-10 expression levels predict a worse prognosis in patients with early NSCLC [8], and studies have found that in patients with advanced NSCLC, patients with high expression IL-10 have a worse prognosis after treatment [9]. At the same time, variants in the IL-10 gene have also been found to be significantly associated with the risk of NSCLC [10]. Therefore, the role of IL10 in tumor initiation, development, and progression in non-small cell lung cancer is still unclear. miRNAs are a class of non-coding single-stranded RNAs encoded by endogenous genes with a length of about 21–25 nucleotides, which inhibit gene expression by partially complementary binding to 3′ non-coding regions (3′ UTRs) of the target mRNA. The correlation between miRNA and tumor has been widely demonstrated [11,12], such as miRNA promoting tumor growth and metastasis [13]. Previous studies have confirmed that miRNAs play an important regulatory role in inflammation-associated tumors, mainly miR-146a, miR-21, miR-101, miR-196, and miR-223 [[14], [15], [16]].
In our study, we found that miR-125b was markedly downregulated after IL-10 treatment, whereas it was upregulated after IFN-γ treatment. Inhibition of miRNA-125b expression suppressed cell proliferation, induced cell apoptosis and decreased cell migration and invasion in NSCLC cell lines, while transfection with the miRNA-125b mimic have opposite effects. DAZAP2 was identified as a direct regulatory target of miR-125b However, considering that both miR-125b inhibition and DAZAP2 knockdown suppressed malignant phenotypes, DAZAP2 should be interpreted as one component of a broader miR-125b-associated regulatory network rather than the sole downstream mediator of miR-125b function. Simultaneous inhibition of miR-125b and DAZAP2 produced more pronounced tumor-suppressive effects both in vitro and in vivo. Together, these findings suggest that miR-125b and DAZAP2 are involved in inflammation-related NSCLC progression through a cytokine-responsive regulatory network.
Materials and methods
Cell lines and culture
The human NSCLC cell lines A549, H460 and 293T were obtained from American Type Culture Collection (ATCC). All the cell lines were examined by certified laboratories for authenticity using Short Tandem Repeat (STR) analysis, and were maintained under the recommended culture conditions. 10 ng/mL IL-10 and 10ng/mL IFN-γ were used in cell culture for cell treatment.
Lentiviral transduction
Lentiviral LV-has-miRNA-125b expression and NC clones, LV-has-miRNA-125b Inhibitor and INC, DAZAP2 shRNA and shNC were obtained from Genechem (China). A549 or H460 cells were seeded in 6-well plates at density of 2 × 105 cells the prior day, transduced with lentiviruses for 72 h and selected with 1 μg/mL puromycin. The efficacy of transduction was verified by qRT-PCR and western blotting.
RNA isolation and qRT-PCR assays
Total RNAs were extracted from cells or tissues with TRIzol reagent (Invitrogen, USA) following the manufacturer’s instructions. The levels of miRNA and mRNA were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) using SYBR Green (Takara, Japan) in a 7500 Real-Time PCR System (ABI, USA), GAPDH was used as internal references, respectively. The qRT-PCR results were analyzed and shown as fold changes relative to GAPDH and were calculated using the 2-△△CT method.
Cell migration and invasion assay
Cells were seeded in the upper chamber with serum-free medium after being transfected with indicated mimic or inhibitor or their corresponding negative control. Complete medium containing 10% FBS were added to the lower chamber. After incubation for 24 h, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. Chambers were washed with PBS and cells on the upper surface were removed. Cells adhering to the bottom of the chamber were imaged and counted by fluorescence microscope (IX71, OLYMPUS, Japan).
Cell proliferation assay
Cells were seeded in 96-well plates at density of 4000 cells per well. Cell proliferation assays were assessed at 24, 48, 72 and 96 h using Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) and MTT Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, China), respectively. The operation was carried out according to manufacturer’s instructions.
Cell apoptosis assay
Cell apoptosis assay was performed using the apoptosis detection kit (Ebioscience, USA). Briefly, cells were trypsinized and resuspended in 1 × binding buffer at a density of 1 × 106 cells/ml. The cell suspension was incubated with 5 uL fluorescein isothiocyanate (FITC)-Annexin V and 5uL propidium iodide (PI) for 15 min in the dark at room temperature. The reaction was examined using flow cytometry (C6 PLUS, BD, USA). The experiments were independently repeated three times.
Luciferase reporter assay
Wide-type or mutant 3′-UTR of DAZAP2 were cloned downstream of Rluc sequence in pGL4 luciferase reporter vector (Promega, China). Cells were plated in 24-well plates at density of 5 × 104 per well. The next day, cells were co-transfected with luciferase reporters, Renilla luciferase (Promega, China) and miRNA-125b mimic or NC for 48 h. The Dual-Luciferase Reporter Assay System (Promega, China) was used to measure luciferase activity according to manufacturer’s instructions.
Immunofluorescence staining assay
Cells grown in glass-bottomed dishes, washed with PBS, fixed with 4% paraformaldehyde for 10 min, then permeabilized with 0.2% Triton X-100 for 5 min, blocked with 5% BSA for 30 min at room temperature, incubated with anti-DAZAP2 antibody (Thermo Fisher Scientific, PA5–98,445) overnight at 4 °C. The next day, cells were incubated with Goat anti-Rabbit IgG (H+L) Alexa Fluor 555 secondary antibody (Thermo Fisher Scientific, A21429) for 1 h, stained with DAPI for 15 min and imaged on CQ1 Confocal Quantitative Image cytometer (CQ1, YOKOGAWA, Japan).
Western blotting
Cells were lysed in RIPA buffer with 1 mM PMSF and protein concentration were measured by BCA protein assay kit (Thermo Fisher Scientific, 23,227). After denaturation, equal amounts of protein were separated by 10% SDS-PAGE gel and transferred to PVDF membrane. The membrane was blocked with 5% non-fat milk in TBST buffer for 1 h and incubated with primary antibody overnight at 4 °C. After incubation with HRP-conjugated secondary antibodies, the membrane was subjected to ECL substrate and scanned by ChemiDoc Touch Imaging system (Bio-Rad, 1708,370). The primary antibodies information were as follows: DAZAP2 (Thermo Fisher Scientific, PA5–98,445), β-actin (Thermo Fisher Scientific, MA5–15,452).
In vivo xenograft assay
A549 cells stably transfected with miR-125b inhibitor or shDAZAP2 or their combination were suspended in PBS buffer and injected subcutaneously (2 × 107 cells/ml) into both flanks of athymic nude mice (BALB/c, 5 weeks old). Tumor volume was measured by vernier calipers every two days and calculated as length × width2/2. When tumor volume of control group reached ∼1500 mm3, mice were euthanized and the tumor were harvested. The experiment was approved by ethics committee of Shanghai Chest Hospital, Shanghai Jiaotong University School of Medicine (No.: KS(Y)1828). All protocols involving animal treatments complied with the national guidelines for the care and use of laboratory animals (Ministry of Health, P.R. China, 1998).
Bioinformatic analysis based on public database
TargetScan (http://www.mirdb.org/cgi-bin/search.cgi), miRcode (http://www.mircode.org/index.php) and miRDB (http://www.mirdb.org/cgi-bin/search.cgi) were used to predict the candidate genes containing miR-125b binding site(s) in their 3′-UTR. We obtained data from The Cancer Genome Atlas (TCGA) database, including gene expression levels and clinical information from 515 cases of lung adenocarcinoma and 59 adjacent cancer samples. Patients were divided into high and low expression groups based on the median expression of DAZAP2. The R package 'survminer' was utilized for survival analysis and visualization of different patient groups. The Log-rank test was employed to evaluate the statistical significance of differences in overall survival time and progression-free survival time among patient groups, with a significance level set at p < 0.05.
Statistical analysis
Data are presented as the mean ± SEM. Statistical analyses were performed using Wilcoxon rank-sum test, Student’s t-test, Pearson’s X2 test or Fisher’s exact test, where appropriate (GraphPad Prism 9.0). P < 0.05 was considered statistically significant difference.
Results
miRNA-125b was associated with inflammation and promote tumor progression
To investigate the potential microRNAs, which may play a critical role in inflammation-related NSCLC, we treated NSCLC cell line A549 with IFN-γ (pro-inflammatory cytokine) and IL-10 (anti-inflammatory cytokine) respectively. Changes in the levels of 24 miRNAs associated with inflammation were then measured. Our results showed that miRNA-125b was the most dramatically up-regulated miRNA and miR-191 was the most down-regulated miRNA after treatment with IFN-γ (Fig. 1A). Whereas after stimulation with IL-10, miRNA-125b was the most strikingly down-regulated ones and miR-429 became the most up-regulated ones (Fig. 1B). Based on the above results, miRNA-125b may be an important mediator linking inflammation and NSCLC.
Fig. 1.
miRNA-125b is associated with inflammation and promotes tumor progression.
A-B Expression of inflammation-related miRNAs were measured after treatment with IFN-γ or IL-10 for 48 h in A549 cells. C-F A549 and H460 cells were transiently transfected with miR-125b mimic, negative control (NC), miR-125b inhibitor, or inhibitor negative control (INC) as indicated for 72 h. Cell viability was measured by CCK8 and MTT assay. G-J Cell apoptosis was measured by flow cytometric apoptosis assay after transfection with miR-125b mimic and inhibitor and their corresponding negative control for 48 h in A549 and H460 cells. K-Q Transwell migration and invasion assays of A549 and H460 cells transiently transfected with miR-125b mimic (mimic), negative control, miR-125b inhibitor (inhibitor) or inhibitor negative control (INC) as indicated for 48 h. * p < 0.05; ** p < 0.01; *** p < 0.001.
To investigate the effects of miRNA-125b in inflammation-related NSCLC, we modulated miRNA-125b levels in cells (Figure S1) and then assessed cell proliferation by cell viability assays, apoptosis by flow cytometry, and migration and invasion by Transwell assays. First, we found that miRNA-125b mimic increased A549 and H460 proliferation, whereas miRNA-125b inhibitor decreased A549 and H460 proliferation (Fig. 1C-F). We also found that miRNA-125b inhibitor promoted A549 (Fig. 1G-H) and H460 (Fig. 1I-J) apoptosis. Migration and invasion assays demonstrated that miRNA-125b inhibitor suppressed A549 migration and invasion, but miRNA-125b mimic enhanced A549 migration and invasion, similar results were found in H460 cells (Fig. 1K-Q). These findings suggested that miRNA-125b might promote tumor progression in inflammation related NSCLC.
DAZAP2 is directly regulated by miR-125b in NSCLC cells
To further explore the regulatory network associated with miR-125b in inflammation-related NSCLC, we identified prospective targets of miR-125b Genes contained miR-125b binding sites in their 3′-UTR, as predicted by TargetScan (http://www.mirdb.org/cgi-bin/search.cgi), miRcode (http://www.mircode.org/index.php) and miRDB (http://www.mirdb.org/cgi-bin/search.cgi), six candidate genes ANKRD29, DAZAP2, ITSN1, KCNC3, KCNMA1 and SDC1 were selected and validated by RT-qPCR. Among six Candidate genes, DAZAP2 and ITSN1 showed the most significant increases after IL10 treatment (Fig. 2A-F). Next, to determine which gene was a target of miR-125b, we modulated the miR-125b levels by transiently transfecting A549 cells with miR-125b inhibitor. DAZAP2 levels were upregulated after inhibition of miR-125b (Fig. 2G). Western blot results also demonstrated that DAZAP2 was upregulated after IL10 treatment and after miR-125b inhibition (Fig. 2I). To determine whether DAZAP2 is directly regulated by miR-125b, first we performed 3′-UTR luciferase reporter assays containing DAZAP2, ITSN1, SDC1 and KCNC3 wild-type or mutated 3′-UTR fragments (Fig. 2J-K and Figure S2). miR-125b mimic decreased the luciferase activity of DAZAP2 3′-UTR wild-type construct (WT) in HEK293T cells and A549 cells. The suppression of luciferase activity by miR-125b mimic was completely abolished in DAZAP2 3′-UTR mutant construct (MUT) (Fig. 2J-K), which suggested that miR-125b directly binds to the predicted site within the DAZAP2 3′-UTR. These results support that DAZAP2 is a direct regulatory target of miR-125b in NSCLC cells. Next, we examined whether knockdown of miR-125b and DAZAP2 affected IL10 and other inflammation-related cytokine levels (Fig. 2L-M and Figure S3). Combined inhibition of miR-125b and DAZAP2 increased IL10 and CXCL10 levels in A549 and H460 cells (Fig. 2L-M and Figure S3I-J). Collectively, these findings indicate that DAZAP2 is directly regulated by miR-125b and responds to inflammatory cytokine stimulation in NSCLC cells. Given the parallel tumor-suppressive effects of miR-125b inhibition and DAZAP2 knockdown, DAZAP2 may represent one component of a broader miR-125b-associated regulatory network rather than the sole downstream mediator of miR-125b function.
Fig. 2.
DAZAP2 is a direct regulatory target of miR-125b.
A-F The expression of miR-125b target candidates were measured by real-time PCR assays after A549 cells were treated with IL10 for 48 h or 72 h. G-H DAZAP2 and ITSN1 mRNA levels were determined in A549 cells after cells were transiently transfected with miR-125b inhibitor or inhibitor negative control as indicated for 72 h. I western blot analysis of DAZAP2 expression of A549 cells after treatment with IL10 for 48 h or 72 h or transfected with miR-125b inhibitor or inhibitor negative control (INC) for 72 h. J-K Luciferase activity was measured in HEK293T cells and A549 cells co-transfected with miR-125b mimic and wild type or mutant 3′-UTR of DAZAP2 for 48 h. l-M IL-10 levels were detected after cells were transfected with miR-125b inhibitor, DAZAP2 shRNA or their combination. * p < 0.05; ** p < 0.01; *** p < 0.001.
Combined inhibition of miR-125b and DAZAP2 further suppresses malignant phenotypes in NSCLC cells
To further confirm the connection between miR-125b and DAZAP2, A549 and H460 cells were transfected with miR-125b inhibitor and DAZAP2 shRNA (Figure S4). CCK8 (Fig. 3A-B) and MTT (Fig. 3C-D) cell viability results suggested that combined transfection with miR-125b inhibitor and DAZAP2 shRNA decreased cell viability more markedly than either single treatment. Combined inhibition of miR-125b and DAZAP2 induced greater apoptosis compared with their single effects (Fig. 3E-F). Transwell migration and invasion assay results demonstrated that DAZAP2 knockdown further enhanced the inhibitory effects of the miR-125b inhibitor on migration and invasion in A549 and H460 cells (Fig. 3G-N). These data show that combined inhibition of miR-125b and DAZAP2 suppresses malignant phenotypes more strongly than either single intervention. However, since both miR-125b inhibition and DAZAP2 knockdown reduced these phenotypes, DAZAP2 is unlikely to be the sole downstream mediator of miR-125b Rather, miR-125b and DAZAP2 may promote NSCLC progression through partially distinct or compensatory mechanisms.
Fig. 3.
Combined inhibition of miR-125b and DAZAP2 exerts enhanced tumor-suppressive effects.
A-D Cells were transfected with miR-125b inhibitor, DAZAP2 shRNA or their combination as indicated for 72 h. Cell viability was assessed using CCK8 and MTT assay. E-F Cell apoptosis was measured by flow cytometric apoptosis assay after transfected with miR-125b inhibitor, DAZAP2 shRNA or their combination as indicated for 48 h in A549 cells. G-N Transwell migration and invasion assays were determined in A549 and H460 cells after transfected with miR-125b inhibitor or DAZAP2 shRNA or their combination for 48 h.
Combined inhibition of miR-125b and DAZAP2 further suppresses tumor growth in vivo, and high expression of DAZAP2 predicts poor prognosis
To evaluate the combined effect of miR-125b inhibition and DAZAP2 knockdown in vivo, a subcutaneous xenograft tumor experiment was performed. Whole animal bioluminescence imaging results showed that DAZAP2 knockdown suppressed tumor growth and the combination produced a stronger inhibitory effect (Fig. 4A-B). Tumor growth curves and tumor weights showed the similar results (Fig. 4C-D), representative tumor images are shown in Fig. 4E. These findings indicate that combined targeting of miR-125b and DAZAP2 further suppresses tumor growth in vivo. Clinical dataset analysis suggested that high expression of DAZAP2 predicts poor prognosis in patients with NSCLC, especially for early-stage patients (Fig. 4F-G).
Fig. 4.
Combined inhibition of miR-125b and DAZAP2 suppresses tumor growth, and high expression of DAZAP2 predicts poor prognosis.
A-B Bioluminescence imaging of tumor in immunocompromised mice subcutaneously injected with luciferase-labeled A549 cells stably transfected with miR-125b inhibitor or shDAZAP2 or their combination. C-D Tumor growth curve and tumor weight of subcutaneous A549 xenografts treated as indicated. E Photos of different groups of tumor tissues. F-G High DAZAP2 levels predict poor prognosis of NSCLC patients. H 10 genes interacting with DAZAP2 from the StringDB database. I Lasso regression to extract non-redundant feature genes. J 7 candidate marker genes.
In order to comprehensively analyze the role of DAZAP2 in tumor occurrence and development, and construct a prognostic model related to lung adenocarcinoma, we extracted 10 genes interacting with DAZAP2 from the StringDB database, with active interaction sources set to Experiments (Fig. 4H). Lasso regression was used to extract non-redundant feature genes (Fig. 4I). The 'cv.glmnet' function in the 'glmnet' package of R was employed to determine the hyperparameters of the Lasso regression model. Using the Lasso regression, we obtained a total of 7 candidate marker genes. Employing Cox multivariate regression analysis and stepwise regression, we identified the optimal prognostic model and marker genes (Fig. 4J).
Discussion
Despite significant advances in treatment strategies, the prognosis of patients with lung cancer remains poor. Therefore, understanding the mechanism of the occurrence and progression of lung cancer could lead to the new treatment options. Chronic inflammation promotes the development of tumors [17,18], which has been widely reported. It has been reported that miRNAs are related with tumor development [19] and metastasis [20]. To investigate the potential microRNAs, which may play critical role in inflammation-related NSCLC, we treated the NSCLC cell line with IFN-γ (pro-inflammatory cytokine) and IL-10 (anti-inflammatory cytokine) respectively. Our results showed that miR-125b was markedly upregulated after IFN-γ treatment but downregulated after IL-10 treatment, suggesting that miR-125b may be an inflammation-associated miRNA in NSCLC and may respond differently to distinct inflammatory cytokine contexts.
The biological function of miR-125b appears to be highly context dependent. Previous studies have reported that miR-125b may act either as an oncogene or as a tumor suppressor depending on tumor type, cellular state, and the dominant downstream target network [21,22]. For example, miR-125b has been reported to promote prostate cancer progression by suppressing the pro-apoptotic gene Bak1 [23], whereas miR-125b-5p was recently shown to suppress colorectal cancer metastasis through the TRAF6 axis [24]. These findings indicate that the functional output of miR-125b is determined by the tumor-specific molecular context. In the present study, miR-125b displayed a tumor-promoting phenotype in NSCLC cells, as miR-125b overexpression increased cell viability, migration, and invasion and reduced apoptosis, whereas miR-125b inhibition produced opposite effects. Therefore, our results do not necessarily contradict the tumor-suppressive role of miR-125b reported in other cancers, but rather support the context-dependent functional plasticity of miR-125b in cancer.
IL-10 also has context-dependent roles in cancer biology [25]. As an anti-inflammatory cytokine, IL-10 can suppress excessive inflammatory responses and may limit inflammation-driven tumor initiation in certain settings [25,26]. Conversely, in established tumors, IL-10 may contribute to immune suppression or tumor progression depending on disease stage, immune microenvironment, and the responding cell type [27,28]. In our NSCLC cell models, IL-10 downregulated miR-125b, whereas IFN-γ upregulated miR-125b, indicating that miR-125b is differentially regulated by inflammatory cytokines. Together, these findings suggest that miR-125b is subject to cytokine-dependent regulation in NSCLC cells, and that the IL-10/miR-125b/DAZAP2 axis identified in this study may represent one component of the inflammatory regulatory network in inflammation-associated NSCLC.
To further characterize the miR-125b-associated regulatory network in inflammation-related NSCLC, we screened potential miR-125b targets and identified DAZAP2 as a cytokine-responsive candidate regulated by IL-10 and miR-125b modulation. Luciferase reporter assays confirmed that miR-125b directly binds the DAZAP2 3′-UTR, supporting DAZAP2 as a direct target of miR-125b Although DAZAP2 has been reported to exert context-dependent functions in different cancers [[29], [30], [31]], our data showed that DAZAP2 knockdown reduced cell viability, migration, invasion, and tumor growth while promoting apoptosis, suggesting a tumor-promoting role in the NSCLC models used here. However, DAZAP2 should not be interpreted as the sole downstream mediator of miR-125b function. Since both DAZAP2 knockdown and miR-125b inhibition suppressed malignant phenotypes, miR-125b likely regulates NSCLC progression through additional targets. Thus, the stronger inhibitory effect observed after combined miR-125b inhibition and DAZAP2 knockdown may reflect simultaneous disruption of partially distinct pro-tumor mechanisms rather than a simple linear miR-125b/DAZAP2 pathway.
Several limitations should be noted. Although our study included in vitro assays, xenograft experiments, and TCGA-based analysis, clinical validation was limited to public datasets. Independent clinical cohorts and patient-derived tumor samples were not available in the present study. Therefore, the clinical relevance of the IL-10-responsive miR-125b/DAZAP2 regulatory network requires further validation in larger NSCLC cohorts. In addition, given the molecular heterogeneity of NSCLC, future studies are needed to identify additional miR-125b targets and interacting pathways involved in inflammation-related tumor progression. Finally, this study did not assess the druggability, delivery feasibility, safety, or clinical applicability of targeting miR-125b or DAZAP2. Thus, our findings should be interpreted primarily as mechanistic insights into inflammation-related NSCLC biology rather than as direct evidence supporting therapeutic intervention.
Conclusions
In summary, our study found that miR-125b was markedly downregulated after IL-10 treatment and upregulated after IFN-γ treatment. Inhibition of miR-125b suppressed malignant phenotypes, and DAZAP2 was identified as a direct regulatory target of miR-125b However, DAZAP2 should be interpreted as one component of a broader miR-125b-associated regulatory network rather than the sole mediator of miR-125b function. Combined inhibition of miR-125b and DAZAP2 produced enhanced tumor-suppressive effects in vitro and in vivo. Our study provides new insights into the cytokine-dependent regulatory network involved in inflammation-related NSCLC progression.
Ethics approval and consent to participate
This study was approved by the Ethics Committee of Shanghai Chest Hospital.
CRediT authorship contribution statement
Yanwei Zhang: Conceptualization, Resources, Data curation, Funding acquisition, Writing – review & editing. Beibei Sun: Investigation, Resources. Yabin Tang: Methodology, Formal analysis, Visualization. Yuqing Lou: Resources, Data curation. Keji Liu: Resources, Data curation. Fangfei Qian: Investigation, Resources. Lele Zhang: Investigation, Resources. Hongyu Liu: Resources. Rong Qiao: Resources. Bo Zhang: Resources. Wenrui Xia: Resources. Wei Zhang: Supervision, Project administration, Validation. Hua Zhong: Supervision, Project administration, Validation. Jun Lu: Supervision, Project administration, Validation. Baohui Han: Conceptualization, Writing – review & editing, Supervision, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the following sources: National Multi-disciplinary Treatment Project for Major Diseases (2020NMDTP), National Science Foundation of China (81802265 and 82002941), the foundation of Shanghai Chest Hospital (2021YNZYB01).
Footnotes
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2026.102902.
Contributor Information
Wei Zhang, Email: zhwei2002@126.com.
Hua Zhong, Email: eddiedong8@hotmail.com.
Jun Lu, Email: lujun512@yahoo.com.
Baohui Han, Email: xkyyhan@gmail.com.
Appendix. Supplementary materials
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.