Dihydroartemisinin targets the miR-497-5p/SOX5 axis to suppress tumor progression in non-small cell lung cancer

Abstract

Introduction: Non-small cell lung cancer (NSCLC) remains a lethal malignancy with limited therapeutic options. Although dihydroartemisinin (DHA) exhibits anticancer properties, its mechanisms in NSCLC are incompletely understood. This study investigated the role of the miR-497-5p/SOX5 axis in mediating DHA's effects on NSCLC.

Methods: In vitro experiments utilized A549 and H1299 cells treated with DHA (50 μM). Proliferation, migration, invasion, and apoptosis were assessed. miR-497-5p and SOX5 expression was modulated via genetic silencing. In vivo, A549 xenograft tumor growth in mice was evaluated under DHA treatment (25/50 mg/kg).

Results: DHA significantly suppressed proliferation, migration, and invasion while inducing apoptosis in vitro. Mechanistically, DHA upregulated miR-497-5p and downregulated SOX5-overexpressed in clinical NSCLC. Silencing miR-497-5p attenuated DHA's effects and increased SOX5, whereas SOX5 knockdown reversed miR-497-5p inhibition. In vivo, DHA dose-dependently inhibited tumor growth with miR-497-5p elevation and SOX5 suppression, effects abrogated by miR-497-5p inhibition but rescued by SOX5 knockdown.

Discussion: DHA exerts antitumor activity by activating the miR-497-5p/SOX5 axis, revealing a novel mechanism. Bridging efficacious in vitro concentrations with clinically achievable dosing remains essential for therapeutic translation.

Keywords: SOX5; anticancer activity; dihydroartemisinin (DHA); miR-497-5p; non-small cell lung cancer (NSCLC).

FIGURE 1

Dihydroartemisinin Modulates Viability, Apoptosis, Migration, and Invasion in Non-Small Cell Lung Cancer Cells. (A–C) Concentration- and time-dependent suppression of NSCLC cell viability by DHA CCK-8 assay demonstrating viability of A549 (A), H1299 (B) and BEAS-2B (C) cells treated with DHA (0, 5, 10, 25, 50, 100 µM) for 24, 48, and 72 h (D–E) Induction of apoptosis by DHA treatment (D) Flow cytometry analysis of Annexin V/PI staining in A549 cells after 24 h exposure to 50 µM DHA. Quantification in (E) shows significant increase in total apoptosis (early + late apoptotic cells). (F–G) Inhibition of invasive capacity by DHA. (F) Representative images of Transwell invasion assay (Matrigel-coated) in A549 cells treated with 50 µM DHA for 24 h. (G) Quantitative analysis showing >60% reduction in invaded cells. (H,I) Suppression of migratory capacity by DHA. (H) Wound healing assay images at 0 and 24 h post-treatment with 50 µM DHA. (I) Quantification of wound closure rate demonstrating significant inhibition of migration. Each column represented the mean ± standard deviation (SD) (n = 3 fields per group).

FIGURE 2

Dihydroartemisinin Modulates Expression of Epithelial-Mesenchymal Transition and Apoptosis Markers in A549 Cells. (A) mRNA expression levels of epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, matrix metalloproteinase-2 [MMP-2], matrix metalloproteinase-9 [MMP-9]) determined by quantitative real-time PCR (qRT-PCR) in A549 cells exposed to 50 µM dihydroartemisinin for 24 h. DHA significantly upregulated E-cadherin (p = 0.002) while downregulating N-cadherin (p < 0.001), MMP-2 (p < 0.001), and MMP-9 (p = 0.002) mRNA expression. (B) Western blot validation of EMT and apoptosis marker protein expression in A549 cells exposed to 50 µM dihydroartemisinin for 24 h. Protein-level changes corresponded with transcriptional alterations, showing elevated E-cadherin (p < 0.001) and reduced N-cadherin (p = 0.003), MMP-2 (p < 0.001), and MMP-9 (p < 0.001). (C) mRNA expression levels of apoptosis markers (Bax, Bcl-2) determined by qRT-PCR. DHA significantly upregulated Bcl-2 (p = 0.002) while downregulating Bax (p = 0.002) mRNA expression. (D) Protein expression levels of apoptosis markers (Bax, Bcl-2) determined by Western blot. Protein-level changes corresponded with transcriptional alterations, showing elevated Bax (p < 0.001) and reduced Bcl-2 (p < 0.001). GAPDH/β-actin served as loading controls for all experiments. Data represent mean ± SD of three independent experiments.

FIGURE 3

The Expression of miR-497-5p and SOX5 in LAC cells and tissues. (A–C) SOX5 is over-expressed in LAC tissues: Immunohistochemical analysis of SOX5 levels in five human lung adenocarcinoma patients (LAC) and their respective non-tumor counterparts (N), SOX5 was localized in the cytoplasm (A,B). SOX5 expression is significantly higher in LAC than in non-tumor counterparts (C). (D) Quantitative RT-PCR analysis of miR-497-5p mRNA level in the same patients, normalized versus U6. (E) Quantitative RT-PCR analysis of SOX5 mRNA level in the same patients, normalized versus U6. (F) Western blot analysis of SOX5 levels in four human lung adenocarcinoma patients (LAC) and their respective non-tumor counterparts (N). p < 0.001 (LAC vs. N). (G) Quantitative RT-PCR analysis of miR-497-5p level in several lung cancer cell lines (H1299, H460 and A549) and normal human bronchial epithelial cells (BEAS-2B). Data normalized to U6 snRNA. (H) Quantitative RT-PCR analysis of SOX5 level in several lung cancer cell lines (H1299, H460 and A549) and normal human bronchial epithelial cells (BEAS-2B). Data normalized to U6 snRNA. (I) Western blot analysis of SOX5 level several lung cancer cell lines (H1299, H460 and A549). GAPDH served as endogenous control.

FIGURE 4

DHA Modulates the Expression of miR-497-5p and SOX5 in Non-Small Cell Lung Cancer. (A) Quantitative RT-PCR analysis of miR-497-5p levels in several lung cancer cell lines (H1299, H460, and A549) and normal human bronchial epithelial cells (BEAS-2B) after treatment with 25 and 50 μM DHA for 24 h. Data normalized to U6 snRNA. (B) Quantitative RT-PCR analysis of SOX5 levels in several lung cancer cell lines (H1299, H460, and A549) and normal human bronchial epithelial cells (BEAS-2B) after treatment with 25 μM and 50 μM DHA for 24 h. Data normalized to U6 snRNA. (C,D) Western blot analysis of SOX5 levels in several lung cancer cell lines (H1299, H460, and A549) after treatment with 25 μM and 50 μM DHA for 24 h. GAPDH served as endogenous control. (E) miR-497-5p inhibition efficiency: Transfection with miR-497-5p inhibitor (vs. NC inhibitor) significantly reduced basal miR-497-5p levels in A549 cells, validating functional knockdown. Data normalized to U6 snRNA. (F) SOX5 silencing verification: siRNA-mediated SOX5 knockdown (si-SOX5) significantly decreased SOX5 mRNA versus si-NC controls (p < 0.001), confirming target efficiency. Data normalized to U6 snRNA. All Quantitative RT-PCR data represent mean ± SD of three independent experiments. Western blots show representative images from biological triplicates.

FIGURE 5

SOX5 Silencing Partially Reverses DHA-Mediated Suppression of Malignant Phenotypes via the miR-497-5p/SOX5 Axis. (A) Apoptosis analysis by Annexin V/PI flow cytometry: Representative dot plots showing apoptotic rates in A549 cells under indicated conditions. (B) Transwell invasion assay: Matrix-coated membranes visualize invasive capabilities (crystal violet staining). (C) Wound healing migration assay: Scratched monolayers monitored at 0/24 h demonstrates migratory capacity. (D) Cell viability quantification: CCK-8 absorbance measurements at 450 nm after 24 h treatments. (E–G) Quantitative analyses of apoptosis (E), invasion (F), and migration (G) data presented in (A–C). Data expressed as mean ± SD (n = 3).

FIGURE 6

The Impact of DHA on the Expression of EMT and Apoptosis Mmarkers, Mediated Through the Regulation of the miR-497-5p/SOX5 Aaxis, was Assessed Using qRT-PCR. (A) miR-497-5p regulation hierarchy: qRT-PCR analysis confirmed DHA-induced miR-497-5p upregulation was effectively abolished by miR-497-5p inhibitor, while SOX5 knockdown (si-SOX5) showed no regulatory effect. Normalized to U6 snRNA. (B) Unidirectional targeting validation: miR-497-5p inhibition significantly elevated SOX5 mRNA expression, confirming SOX5 as downstream target. U6 served as endogenous control. (C) Apoptotic marker rescue: miR-497-5p inhibitor reversed DHA-mediated pro-apoptotic effects - suppressing Bax induction (p < 0.001) and restoring Bcl-2 expression. These alterations were rescued by concurrent SOX5 knockdown. (D) EMT marker reprogramming: Inhibition of miR-497-5p antagonized DHA’s anti-EMT effects, elevating mesenchymal markers N-Cadherin/MMP-2/MMP-9 while suppressing epithelial E-Cadherin. SOX5 co-silencing restored the DHA-induced anti-EMT phenotype. Data represent mean ± SD of triplicate experiments.

FIGURE 7

The Impact of DHA on the Expression of EMT and Apoptosis Markers, Mediated Through the Regulation of the miR-497-5p/SOX5 Axis, was Assessed Using Western blot. (A) SOX5 regulation cascade: Representative Western blots demonstrating DHA-induced SOX5 downregulation and its rescue by miR-497-5p inhibition. GAPDH loading control shown. (B) Densitometric quantification of SOX5 protein expression (normalized to GAPDH): Confirmed significant SOX5 reduction by DHA reversed by miR-497-5p inhibitor, while SOX5 knockdown validated targeting efficiency. (C) EMT marker reprogramming: Immunoblotting of epithelial (E-Cadherin) and mesenchymal (N-Cadherin, MMP-2, MMP-9) markers showing DHA-mediated phenotype reversal. (D) Quantitative analysis of EMT markers: miR-497-5p inhibition significantly antagonized DHA’s effects - reducing E-Cadherin while elevating N-Cadherin, MMP-2 and MMP-9. These alterations were rescued by SOX5 co-silencing. (E) Apoptotic regulator modulation: Protein expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 under experimental conditions. (F) Densitometric analysis: miR-497-5p inhibitor reversed DHA-induced Bax upregulation and Bcl-2 downregulation. Concurrent SOX5 depletion restored pro-apoptotic signaling. Data represent mean ± SD of triplicate experiments.

FIGURE 8

Dihydroartemisinin Suppresses Tumor Growth In Vivo via the miR-497-5p/SOX5 Axis. (A). Experimental design of xenograft tumor model. A549 cells transfected with miR-inhibitor NC (negative control), miR-497-5p inhibitor, si-NC (scrambled siRNA), si-SOX5, or miR-497-5p inhibitor + si-SOX5 were subcutaneously injected into nude mice. Mice were treated with 25 or 50 mg/kg DHA (intraperitoneal injection, every 2 days) for 4 weeks. (B) DHA dose-dependently reduces tumor weight. Final tumor weights (measured post-sacrifice) show significant suppression by both 25 mg/kg and 50 mg/kg DHA. This effect was reversed by miR-497-5p inhibition or SOX5 knockdown, while co-treatment with miR-497-5p inhibitor + si-SOX5 restored DHA’s antitumor efficacy. (C) Dynamic tumor volume changes during treatment. Tumor volume was measured every 7 days. 50 mg/kg DHA showed the strongest growth inhibition. Key reversal groups: miR-497-5p inhibitor: Abrogated DHA-induced suppression. si-SOX5: Partially reversed DHA efficacy. miR-497-5p inhibitor + si-SOX5: Synergistically restored tumor suppression. (D) SOX5 mRNA expression in tumor tissues. qRT-PCR analysis confirmed DHA significantly downregulated SOX5, consistent with in vitro data. SOX5 knockdown (si-SOX5) further reduced its expression, validating targeting efficiency. (E) miR-497-5p expression in tumor tissues. qRT-PCR revealed DHA (50 mg/kg) markedly upregulated miR-497-5p. Transfection with miR-497-5p inhibitor effectively blocked this induction, confirming functional antagonism. Data represent mean ± SD of triplicate experiments.

FIGURE 9

Dihydroartemisinin Modulates the Expression of EMT and Apoptosis Markers In Vivo via the miR-497-5p/SOX5 Axis. (A,B) DHA reverses EMT progression in tumor tissues. Western blot analysis of epithelial-mesenchymal transition (EMT) markers. Epithelial marker: E-Cadherin (E-Cad) was upregulated by DHA.Mesenchymal markers: N-Cadherin (N-Cad), MMP-2, and MMP-9 were downregulated by DHA.Reversal effects: miR-497-5p inhibition or SOX5 knockdown attenuated DHA’s modulation of EMT markers, while dual inhibition (miR-497-5p inhibitor + si-SOX5) restored DHA’s efficacy. (C,D) DHA promotes pro-apoptotic signaling. Western blot of apoptosis regulators: Pro-apoptotic Bax was increased by 50 mg/kg DHA. Anti-apoptotic Bcl-2 was suppressed by DHA. Bax/Bcl-2 ratio significantly rose in DHA groups, Bax/Bcl-2 ratio (apoptosis indicator) significantly rose in DHA groups. Key trend: miR-497-5p/SOX5 axis disruption blunted DHA-induced apoptosis, rescued by co-targeting both molecules. Data represent mean ± SD of triplicate experiments.