The NCI-H460 and NCI-A549 human NSCLC cell lines were obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd., and Procell Life Science & Technology Co., Ltd., respectively, while the BEP2D human bronchial epithelial cell line was sourced from Otwo Biotech. All cell lines were authenticated through short tandem repeat profiling and mycoplasma testing. The cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 µg/ml). All cells were maintained in a 5% CO₂ incubator at 37°C. The STAT3 inhibitor S3I 201 (97%; cat. no. ab146606; Abcam) was stored at room temperature, and the cells were treated with S3I 201 (10 µM) at 37°C for 24 h before subsequent experiments.

To study sphere formation, H460 and A549 cells were cultured in stem-cell culture medium at a density of 5,000 cells per well in ultra-low attachment 6-well plates until spheres containing >20 cells formed (12,13). Stem-cell culture medium (DMEM/F12; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 2% B27, 1% penicillin/streptomycin, basic fibroblast growth factor (20 ng/ml), epidermal growth factor (20 ng/ml) and insulin (4 µg/ml), was used. The inhibitory effects of DFOG on sphere formation were assessed by incubating SFCs derived from H460 and A549 cells with varying concentrations of DFOG (1, 5 and 10 µM) at 37°C for 72 h. Subsequently, the cells were reseeded at a density of 1,000 cells per well in ultra-low attachment 24-well plates and cultured until spheres reformed in the absence of DFOG. Subsequently, the number and status of spheres were evaluated manually under a light microscope (Leica Microsystems GmbH). The sphere formation rate was calculated using the following formula: Number of spheres/number of cells seeded ×100%. Experiments were performed in triplicate.

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Inc.). Single-cell suspensions were seeded at 1,000 cells per well in 96-well plates for 24 h and treated with varying concentrations of DFOG (1, 5 and 10 µM) at 37°C. After 72 h, the cells were incubated with 10 µl CCK-8 solution per well for 2 h, and the optical density (OD₄₅₀) was measured using a microplate reader (BioTek; Agilent Technologies, Inc.).

The Superscript IV RT kit and SYBR Green fluorophore were purchased from Thermo Fisher Scientific, Inc. The RT reaction conditions were incubation at 37°C for 5 min, 50°C for 15 min and 75°C for 5 min. Total RNA was extracted from H460 cells, A549 cells or SFCs (1×10⁵ cells) using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For RNA extraction from tissue samples, grinding using a tissue homogenizer (Beyotime Institute of Biotechnology) was first performed. cDNA synthesis was conducted according to the supplier's instructions (Thermo Fisher Scientific, Inc.). The 2^−ΔΔCq method (33) was used for qPCR, with U6 as the internal control for miR-152 and GAPDH as the internal control for STAT3. To identify candidate miRNAs affected by DFOG treatment, H460 cells, A549 cells or SFCs were treated with DFOG (5 µM) at 37°C for 24 h, followed by total RNA extraction and cDNA synthesis. PCR amplification was performed using specific primers (Table I), with the following thermocycling conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 30 sec, 55°C for 30 sec and 70°C for 30 sec. For miRNA quantification, 2 µg total miRNA was transcribed and amplified using the All-in-One™ miRNA qRT-PCR Detection Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the TaqMan MicroRNA Assay (GeneCopoeia, Inc.), with U6 (Sangon Biotech Co., Ltd.) as the reference gene. Data analysis was conducted using the 2^−ΔΔCq method. All experiments were conducted in triplicate independently.

For the colony formation assay, a bottom agar layer was prepared by mixing 1.2% agarose (Invitrogen; Thermo Fisher Scientific, Inc.) with DMEM in equal proportions, and 500 µl of this mixture was added to each well of a 24-well plate. The top agar layer was prepared by mixing H460 cells, A549 cells or SFCs (1,000 cells) with 0.7% agarose and 500 µl of 20% FBS-supplemented DMEM. After 12 h of cell seeding, different concentrations of DFOG (1, 5 and 10 µM) were added according to the needs of each group. The drug was continuously administered at 37°C until the end of the experiment. Images of colony formation were captured under a light microscope (Leica Microsystems GmbH) and colonies were counted manually. More than 20 cells were defined as a colony. The cells were incubated for 14 days at 37°C, and colonies were counted to calculate the colony formation rate per 1,000 cells based on triplicate experiments.

The RIPA protein extraction kit was purchased from Thermo Fisher Scientific, Inc. BCA was used to quantitatively determine the protein concentration. Each lane was loaded with 20 µg of protein. The gel concentration used was 10%. After electrophoresis, protein was transferred to a PVDF membrane. Blocking was performed using 5% skimmed milk at 37°C for 1 h. Membranes were incubated with the primary antibody at 4°C for 6 h, and membranes were incubated with the horseradish peroxidase-conjugated IgG secondary antibody (1:1,000 dilution; cat. no. RGAR011; Proteintech Group, Inc.) at room temperature for 1 h. Antibodies against α-tubulin (1:1,000 dilution; cat. no. 2125; Cell Signaling Technology, Inc.), STAT3 (1:1,000 dilution; cat. no. 12640; Cell Signaling Technology, Inc.), phosphorylated-STAT3 (p-STAT3; 1:2,000 dilution; cat. no. 9145; Cell Signaling Technology, Inc.), CD133 (1:1,000 dilution; cat. no. 64326; Cell Signaling Technology, Inc.), CD44 (1:1,000 dilution; cat. no. 37259; Cell Signaling Technology, Inc.), Oct4 (1:1,000 dilution; cat. no. 2890; Cell Signaling Technology, Inc.) and Sox2 (1:1,000 dilution; cat. no. 3579; Cell Signaling Technology, Inc.) were used as previously described (12). Finally, the chemiluminescent substrate (ECL) was purchased from Beyotime Institute of Biotechnology, and ImageJ 1.37 (National Institutes of Health) was used for gray-scale analysis.

MicrON™ miR-152-3p mimic (5′-UCAGUGCAUGACAGAACUUGG-3′) and micrOFF™ miR-152-3p inhibitor (5′-CCAAGUUCUGUCAUGCACUGA-3′), miR-152-3p mimic negative control (5′-UUGUACUACACAAAAGUACUG-3′) and miR-152-3p inhibitor negative control (5′-GGAACUUAGCCACUGUGAAUU-3′), were purchased from Guangzhou RiboBio Co., Ltd., and transfected into SFCs using transfection reagent iboFECT™ CP (Guangzhou RiboBio Co., Ltd.) at a concentration of 50 nM, according to the manufacturer's instructions. The small RNA complexes were incubated with cells for 2 h before the medium was replaced, then the culture was continued at 37°C for 48 h, and cells were used for subsequent experiments.

The binding sites of miR-152-3p and STAT3 were predicted using RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid). For the luciferase reporter assays, SFCs were co-transfected with miR-152-3p or miR-control and the pGL3 luciferase vector (Guangzhou RiboBio Co., Ltd.) containing the firefly luciferase reporter, along with the wild-type (WT) or mutant (MUT) 3′-untranslated region (UTR) sequence of STAT3. After 48 h, luciferase activity was measured using a luciferase assay kit (Promega Corporation), and normalized to Renilla luciferase activity in triplicate experiments.

Transfection was performed using 10 µg nucleic acid with a concentration of 1 µg/µl at 37°C for 48 h, and subsequent experiments were performed 48 h after transfection. For STAT3 overexpression, cells were transfected with pcDNA3.1-Control or pcDNA3.1-STAT3 plasmids obtained from Invitrogen; Thermo Fisher Scientific, Inc., using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.), according to the manufacturer's guidelines.

A total of 18 female pathogen-free nude BALB/c mice (aged 4–5 weeks; weight, 18–22 g) were sourced from GemPharmatech Co., Ltd., and housed in a specific pathogen-free facility [SYXK (Xiang) 2020-0012] under a standard 12-h light/12-h dark cycle, at 20–26°C, with an atmospheric pressure of 20–50 Pa and a relative humidity of 40–70%, and ad libitum access to regular mouse chow and water. The site of cell injection was the armpit of the upper limb. When the tumor grew to ~100 cm³, the mouse was treated with drug treatment for 21 days. The time interval between the injection of cells and the end of the experiment was 6 weeks, and tumor volume was detected every 2 days until the end of the experiment. The ethical approval (approval no. D2023045) was granted by the Ethics Committee of Hunan Normal University (Changsha, China).

To evaluate the effects of DFOG in the xenograft mouse model, 1×10⁶ SFCs were suspended in PBS and mixed with 100% Matrigel at a 1:1 ratio (BD Biosciences). A 100 µl mixture was subcutaneously injected into each mouse. When the xenograft volume reached ~100 mm³, mice in the control group received 200 µl of 2% DMSO every 2 days, while those in the experimental groups were orally administered DFOG (10 and 50 mg/kg) for 3 weeks every 2 days. Each group consisted of 6 mice. Tumor volume was calculated using the following formula: V (mm³)=(L × W²)/2, where L is the longest diameter and W is the shortest diameter of the xenograft, measured using a Vernier caliper. At the end of the experiment, xenograft-bearing mice were euthanized using CO₂ asphyxiation (CO₂ replacement rate of 30%), and the xenografts were collected, weighed, and snap-frozen in liquid nitrogen, and tumor tissues were fixed in 4% paraformaldehyde for subsequent H&E staining and immunohistochemistry. Tumor tissues for qPCR analysis were preserved in RNAlater.

For H&E staining, tumor tissues were fixed in 4% paraformaldehyde at 4°C for 24 h, and the slice thickness was 4 µm. Hematoxylin staining was performed for 5 min, followed by eosin staining for 1 min, and these were performed at 25°C. Staining was observed under a light microscope (Leica Microsystems GmbH).

Tumor tissues were fixed in 4% paraformaldehyde at 4°C for 24 h. Tissue sections (thickness, 4 µm) from paraffin-embedded and fixed samples were subjected to deparaffinization in citrate buffer. Sections were heated in an oven at 90°C for 20 min, followed by a series of ethanol washes (anhydrous ethanol, 95, 85 and 75% ethanol). Subsequently, the slices underwent three consecutive washes with PBS. After blocking endogenous peroxidase activity with 3% H₂O₂ and nonspecific binding with 5% goat serum (Beyotime Institute of Biotechnology) for 15 min at 25°C, the sections were incubated overnight at 4°C with the primary antibody against p-STAT3 (1:200 dilution; cat. no. 9145; Cell Signaling Technology, Inc.). As a negative control, PBS was used in place of the primary antibody. The sections were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibodies (1:500 dilution; cat. no. RGAR011; Proteintech Group, Inc.) for 20 min at 25°C. Staining was developed using the 3,3′diaminobenzidine substrate (Fuzhou Maixin Biotechnology Development Co., Ltd.). The results were observed and images were captured under a light microscope (Leica Microsystems GmbH). The signal intensity was evaluated as previously described (34), and semi-quantitatively analyzed using ImageJ 1.37.

All statistical analyses were performed using GraphPad Prism Software 9 (Dotmatics). Data are presented as the mean ± SD. Comparisons between groups were performed using one-way analysis of variance with Tukey's post hoc test or an unpaired Student's t-test. For in vitro analyses, experiments were performed in triplicate. P<0.05 was considered to indicate a statistically significant difference.

The effect of DFOG on cell viability was assessed in BEP2D, H460 and A549 cell lines using the CCK-8 assay, following treatment at various concentrations. A reduction in cell viability was observed in H460 and A549 cells compared with the control group (0 µM), yielding an IC₅₀ of ~10 µM (Fig. 1A). Noncytotoxic concentrations of DFOG (1, 5 and 10 µM) were selected for subsequent experiments.

Natural phytochemicals have been reported to modulate miRNA-mediated suppression of stemness characteristics in hepatocellular carcinoma cells (34). Therefore, it was detected whether tumor suppressor miRNAs in NSCLC cells, including miR-671-5p (31), miR-148a-3p (35), miR-340-5p (36), miR-342-3p (37), miR-34a-5p (38) and miR-152-3p (39), are regulated by DFOG (5 µM). DFOG treatment (5 µM) led to significant upregulation of miR-152-3p in both H460 and A549 cell lines, with the most notable increase among the tested miRNAs (Fig. 1B and C).

To examine the role of miR-152-3p and STAT3 expression in self-renewal and tumor growth in NSCLC, miR-152-3p expression was compared between H460 cells and H460-derived SFCs. The results revealed lower miR-152-3p levels in SFCs compared with H460 cells (Fig. 1D). Additionally, SFCs exhibited elevated STAT3 mRNA expression and p-STAT3 levels (Fig. 1E and F).

The sphere formation and colony formation were significantly increased in H460-derived SFCs compared with H460 cells (Fig. 1G and H). Furthermore, the expression levels of stem cell-associated markers, including CD133, CD44, Oct4 and Sox2, were elevated in H460-derived SFCs compared with H460 cells (Fig. 1I and J).

DFOG treatment at noncytotoxic concentrations (1, 5 and 10 µM) induced a dose-dependent increase in miR-152-3p expression in H460-derived SFCs (Fig. 2A). miR-152-3p has been recognized for its role in suppressing carcinogenesis by inhibiting carcinogenic transcription factors and signaling pathways in colon cancer, breast cancer, prostate cancer and ovarian cancer (22–25). Subsequently, the effects of DFOG on STAT3 mRNA expression and p-STAT3 protein levels were assessed using RT-qPCR and western blotting. DFOG treatment resulted in a decrease in STAT3 mRNA expression (Fig. 2B) and a significant reduction in p-STAT3 protein levels (Fig. 2C).

To evaluate the effect of DFOG on self-renewal and tumor growth in NSCLC, sphere formation and colony formation assays were performed. DFOG treatment led to reduced sphere (Fig. 2D) and colony (Fig. 2E) formation rates in H460-derived SFCs. Additionally, the protein levels of the stem cell markers CD133 and CD44 were significantly decreased (Fig. 2F). Consistent with these results, DFOG treatment substantially decreased the protein levels of Oct4 and Sox2 (Fig. 2G) in H460-derived SFCs.

For in vivo assessment, DFOG was orally administered to mice with H460-derived SFC xenograft tumors, with DMSO administered in the control group. The volumes of xenografts are shown in Table II and the maximum diameter is shown in Table III. As shown in Fig. 2H-1, −2 and −3, DFOG significantly suppressed the growth of xenograft tumors. Mechanistically, DFOG exerted a dual effect by reducing the p-STAT3 levels (Fig. 2H-4 and −5) and enhancing miR-152-3p expression (Fig. 2H-6). These results suggested that DFOG inhibited the self-renewal and tumor growth of H460-derived SFCs both in vitro and in vivo, likely through modulation of miR-152-3p and its target, STAT3.

To further elucidate the role of miR-152-3p regulation in DFOG-mediated suppression of self-renewal, H460-derived SFCs were transfected with a miR-152-3p mimic. As shown in Fig. 3A, the miR-152-3p mimic, in combination with DFOG (5 µM), elevated miR-152-3p expression. Furthermore, both DFOG (5 µM) and the miR-152-3p mimic synergistically reduced STAT3 mRNA levels and p-STAT3 protein levels (Fig. 3B and C). Notably, miR-152-3p overexpression enhanced the inhibitory effects of DFOG on self-renewal, leading to a reduction in sphere (Fig. 3D) and colony (Fig. 3E) formation rates compared with the control group (0 µM). The combined action of miR-152-3p and DFOG resulted in decreased expression of the stemness markers CD133 and CD44 (Fig. 3F), as well as the pluripotent factors Oct4 and Sox2 (Fig. 3G). These results suggested that DFOG elevated miR-152-3p expression, which in turn suppressed STAT3 transcription and activity, thereby inhibiting self-renewal in H460-derived SFCs.

To further validate the role of miR-152-3p regulation in DFOG-induced suppression of self-renewal, H460-derived SFCs were transfected with miR-152-3p inhibitor or miR-inhibitor-NC, followed by treatment with or without DFOG (5 µM). As shown in Fig. 4A, miR-152-3p inhibitor transfection effectively reversed the DFOG-induced increase in miR-152-3p expression. Additionally, miR-152-3p inhibitor counteracted the reduction in STAT3 mRNA expression and p-STAT3 protein levels induced by DFOG (Fig. 4B and C). Notably, miR-152-3p inhibitor mitigated the inhibitory effects of DFOG on self-renewal, as evidenced by an increase in sphere (Fig. 4D) and colony formation rates (Fig. 4E). miR-152-3p inhibitor transfection also increased the expression of the stemness-associated markers CD133 and CD44 (Fig. 4F), and the pluripotent factors Oct4 and Sox2 (Fig. 4G), reversing the suppressive effect of DFOG. These results suggested that inhibiting the expression of miR-152-3p could counteract the inhibition of p-STAT3 activity and self-renewal induced by DFOG treatment in H460-derived SFCs.

To investigate whether the DFOG-induced suppression of self-renewal was linked to the regulation of STAT3 mRNA expression and activity, H460-derived SFCs were treated with S3I 201, a specific STAT3 inhibitor. As shown in Fig. 5A, S3I 201 (10 µM) treatment did not affect the DFOG-induced elevation of miR-152-3p expression. However, as illustrated in Fig. 5B and C, S3I 201 (10 µM), in combination with DFOG (5 µM), effectively decreased STAT3 mRNA expression and p-STAT3 protein levels. Inhibition of STAT3 activity enhanced the suppressive effects of DFOG on self-renewal compared with S3I 201 (10 µM) or DFOG (5 µM) treatment alone, leading to a significant reduction in sphere (Fig. 5D) and colony (Fig. 5E) formation rates. Furthermore, the combination of S3I 201 and DFOG resulted in decreased protein levels of the stemness markers CD133 and CD44 (Fig. 5F), as well as the pluripotent factors Oct4 and Sox2 (Fig. 5G). These results highlighted that modulation of STAT3 mRNA expression and activity by DFOG contributed to the suppression of self-renewal in H460-derived SFCs.

To verify the upstream and downstream relationship between miR-152-3p and STAT3, H460-derived SFCs were transfected with STAT3 cDNA and pcDNA3.1, followed by treatment with or without DFOG (5 µM). As shown in Fig. 6A, transfection with STAT3 cDNA did not affect the miR-152-3p expression induced by DFOG. However, STAT3 cDNA transfection significantly counteracted the reduction in STAT3 mRNA and protein levels caused by DFOG (Fig. 6B and C).

To determine whether miR-152-3p directly targets STAT3, a luciferase reporter assay was conducted in H460-derived SFCs to identify the specific binding site of the miR-152-3p seed sequence on the 3′-UTR of STAT3 mRNA. RNAhybrid predicted binding sites for miR-152-3p and STAT3 (Fig. 7A). As shown in Fig. 7B, luciferase activity was reduced in cells co-transfected with the miR-152-3p mimic and STAT3-3′-UTR-WT, while no change in luciferase activity was observed following co-transfection with STAT3-3′-UTR-MUT. Additionally, the luciferase activity was further decreased in NSCLC cells co-transfected with miR-152-3p mimic and STAT3-3′-UTR-WT after DFOG (5 µM) treatment, compared with cells treated with miR-152-3p mimic or DFOG alone (Fig. 7C). These results demonstrated that DFOG inhibited self-renewal by upregulating miR-152-3p, which directly suppressed STAT3 expression by disrupting its transcriptional activity (Fig. 7D).

The inhibitory effect of DFOG on self-renewal was also assessed in A549-derived SFCs. Consistently, DFOG dose-dependently increased miR-152-3p expression, and downregulated STAT3 mRNA expression and p-STAT3 protein levels in A549 cells (Fig. 8A-C). Additionally, sphere formation (Fig. 8D) and colony formation (Fig. 8E) rates, as well as the expression of the stem cell markers CD44 and CD133 (Fig. 8F), and the pluripotent factors Oct4 and Sox2 (Fig. 8G), were all suppressed. These results indicated that DFOG-induced inhibition of self-renewal in A549-derived SFCs was mediated through the upregulation of miR-152-3p, and the suppression of STAT3 mRNA and activity.