Opsin3 regulates cell proliferation, migration, and apoptosis in lung adenocarcinoma via GPX3 pathway

Abstract

Despite recent progress in understanding lung adenocarcinoma (LUAD) and the emergence of new therapeutic strategies, LUAD continues to be one of the deadliest lung cancer types, with a five-year survival rate of under 5%. Opsin3 (OPN3), a member of the G protein-coupled receptor superfamily, has been linked to various cancer-related processes, including tumor progression and therapy resistance. However, its specific role in LUAD remains insufficiently investigated. This study aimed to explore OPN3’s regulatory functions in LUAD and evaluate its potential as a therapeutic target. OPN3 expression in LUAD cells was assessed using quantitative PCR, Western blotting, and immunohistochemistry. The effects of OPN3 on cell migration and invasion were evaluated through wound healing and transwell assays. Additionally, the influence of OPN3 on cell cycle progression and signaling pathways in vivo—critical for cellular responses to external stimuli—was examined. Pathway enrichment analysis revealed significant disruption of genes associated with glutathione metabolism. Notably, a strong correlation between OPN3 expression and the regulation of Glutathione Peroxidase 3 (GPX3), a key enzyme in this metabolic pathway, was identified. Our results demonstrate that OPN3 is markedly overexpressed in LUAD tissues relative to normal lung tissues. Silencing OPN3 via siRNA significantly diminished the malignant features of LUAD cells, including proliferation, migration, and invasion. In contrast, OPN3 overexpression enhanced these malignant characteristics, indicating its involvement in tumor progression. Moreover, an inverse relationship between OPN3 expression and GPX3 levels was observed, suggesting that OPN3 may drive LUAD progression through the GPX3 pathway. This study offers new insights into the function of OPN3 in LUAD and suggests that targeting the OPN3-GPX3 axis could provide a promising therapeutic strategy for LUAD patients.

Introduction

Lung cancer (LC) is a leading cause of cancer-related death worldwide, characterized by its high prevalence and significant heterogeneity [1, 2]. It is traditionally categorized into two main histological types: small cell lung carcinoma and non-small cell lung carcinoma (NSCLC) [3]. NSCLC is further divided into several histological subtypes, including lung adenocarcinoma (LUAD), lung adenosquamous carcinoma, lung squamous cell carcinoma, and large cell carcinoma [3]. LUAD, the most common subtype, accounts for approximately 40% of all LC cases [4, 5]. The efficacy of anti-cancer treatments in LUAD is often compromised by chemotherapy resistance and apoptosis evasion, resulting in tumor recurrence and poor prognosis. Metastasis significantly affects survival outcomes in LUAD patients, with metastatic cells frequently displaying key characteristics of epithelial-mesenchymal transition (EMT) [6,7,8,9].

Opsins, a diverse group of G-protein-coupled receptors, were first identified in the eye and include both canonical and noncanonical forms [10]. Canonical opsins, such as OPN1SW, OPN1MW, OPN1LW, and rod opsins like OPN2, are localized in classical photoreceptors and play a critical role in phototransduction during vision [11]. Noncanonical opsins, including Opsin3 (OPN3) (encephalopsin, panopsin), OPN4 (melanopsin), and OPN5 (neuropsin), are involved in both light-dependent and light-independent processes, with photons activating photoreceptors in cells unrelated to visual perception [12,13,14].

OPN3, originally recognized as an extraocular opsin, is expressed in adipocytes and various tissues, with particularly high levels in the brain, testis, and skin [15, 16]. The OPN3 gene encodes a transmembrane protein featuring seven α-helical transmembrane domains, a C-terminal region rich in serine and threonine, and a glycosylated N-terminal region [16]. OPN3 is integral to modulating UVA-induced photoaging in human dermal fibroblasts and serves as a key sensor in the formation of supranuclear caps in keratinocytes following UVA exposure [17]. Its function is vital for the skin's protection against UV radiation. Additionally, OPN3 has been shown to enhance tyrosinase activity in human epidermal melanocytes, particularly in co-culture with keratinocytes in vitro, highlighting its role in visible light-induced hyperpigmentation [18]. Recent research has further implicated OPN3 in various cancer-related processes. For instance, it is linked to resistance to 5-fluorouracil in hepatocellular carcinoma cells through activation of anti-apoptotic signaling pathways [19]. Furthermore, analysis of The Cancer Genome Atlas (TCGA) data reveals a significant upregulation of OPN3 in LUAD and other cancers, including breast, cervix, colon, ovary, pancreas, rectum, skin, and uterus, relative to normal tissues [20]. This increased expression of OPN3 contributes to enhanced LUAD metastasis and promotes EMT.

This study aimed to investigate the function and regulatory mechanisms of OPN3 in LUAD. OPN3 expression in LUAD cells was assessed through quantitative polymerase chain reaction (qPCR), Western blot (WB), and immunohistochemistry (IHC). The effects of OPN3 on cellular behaviors such as migration and invasion were evaluated using wound healing and transwell assays. Furthermore, microarray analysis identified a negative correlation between OPN3 and Glutathione Peroxidase 3 (GPX3), suggesting that OPN3 may influence LUAD progression via the GPX3 pathway.

Methods

Cell culture and transfection

The LUAD cell lines NCI-H1944, NCI-H1650, NCI-H1437, NCI-H1395, and NCI-H1299 were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) in a 37 °C incubator with 5% CO₂. The cells were seeded in 24-well plates at a density of 5 × 10⁴ cells per well and incubated overnight. OPN3 knockdown and overexpression plasmids, along with their respective control vectors, were obtained from Genechem (Shanghai, China). Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For OPN3 overexpression in NCI-H1299 cells, adenoviral vectors (Ad-OPN3, MOI 1:100) were employed along with polybrene (Millipore Sigma Aldrich Corporation, 107689, final concentration 8 ng/mL). OPN3 knockdown was achieved using mouse OPN3-specific siRNA, with non-specific siRNA serving as a negative control.

Tumor formation assay in nude mice

Female BALB/c nude mice, aged 4 to 5 weeks, were housed in a pathogen-free facility. NCI-H1437 cells, transfected with either shNC or shOPN3, were harvested, washed, and resuspended in phosphate-buffered saline (PBS). A 150 μL suspension containing 5 × 10⁶ cells was subcutaneously injected into the left inguinal region of each mouse, with three mice per experimental group. Thirty-five days post-injection, the mice were euthanized, and the tumors were surgically removed for further analysis. Tumor images were captured, weights recorded, and volumes determined using the formula: (length × width²) × 0.5.

RNA extraction and real-time PCR

Total RNA was isolated using TRIzol reagent (Invitrogen, 15596026), and cDNA synthesis was carried out using the PrimeScript reverse transcription kit, following the manufacturer’s instructions. Real-time PCR was performed with the SYBR Green PCR Kit (Applied Biosystems, 4309155). Gene expression was quantified by calculating the Ct (threshold cycle) values and normalizing them to GAPDH levels. All samples were processed in triplicate. The primer sequences for OPN3 were sourced from Jiao et al. [19].

Western blot analysis

Cells were lysed in ice-cold RIPA buffer (Solarbio, R0010) supplemented with protease and phosphatase inhibitors (Abcam, GR304037-28). Protein concentrations were determined using the BCA protein assay kit. Proteins were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, and blocked with 10% non-fat milk. Primary antibodies against Phosphorylated Nuclear Factor Kappa B (P-NF-κB), Nuclear Factor Kappa B (NF-κB), CyclinB1, OPN3, Phosphorylated Extracellular Signal-Regulated Kinase (p-ERK), Extracellular Signal-Regulated Kinase (ERK), and Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) were incubated overnight at 4 °C. Following incubation with appropriate secondary antibodies, bands were visualized and analyzed using Bio-Rad’s Image Lab 3.0 software. Anti-GAPDH antibody (Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436) served as the loading control.

Cell counting kit 8 (CCK8) assay

After OPN3 knockdown or overexpression, cells were seeded in 96-well plates at a density of 1 × 10³ cells per well. CCK8 (Vazyme) was added at 24-h intervals for 4 days, and optical density at 450 nm was measured using a Bio-Rad iMark plate reader following a 2-h incubation.

Transwell assay

The migratory and invasive capabilities of H1437 cells were assessed using 24-well polyester membrane inserts (pore size: 8.0 μm, JET, Guangzhou, China). Cells were seeded in the upper chamber in serum-free medium at densities of 2 × 10⁴ or 4 × 10⁴ cells per well. In invasion assays, the upper chamber was pre-coated with Matrigel. After 16 to 24 h, cells that had migrated to the lower chamber were fixed with methanol and stained with 0.5% crystal violet. Migrating or invading cells were quantified microscopically.

Plate clone formation assay

OPN3 knockdown or overexpressing cells were seeded into plates at densities of 500 or 10,000 cells per well. After 14 days of incubation, cells were fixed with 4% paraformaldehyde (PFA) and stained with Giemsa stain (Leagene). Colony formation was then quantified and analyzed.

TUNEL staining

Apoptotic cells were assessed using the TUNEL assay kit (MA 0224, Meilun Biology, China). After fixation with 4% PFA, cells were permeabilized with 0.1% Triton X-100. The TUNEL reaction mixture was applied, and the cells were incubated at 37 °C for 60 min. DAPI was used for nuclear counterstaining, and apoptosis was visualized using fluorescence microscopy.

Flow cytometry

NCI-H1437 and siOPN3-H1437 cells were washed with PBS, subjected to trypsinization, and then collected by centrifugation at 1000 × g for 5 min. The cells were resuspended in PBS, and 2 × 10⁵ cells were incubated with 195 μL of Annexin V-FITC binding solution. After treatment with Annexin V-FITC and propidium iodide staining solutions, the cells were gently mixed and incubated in the dark for 20 min at 37 °C. Apoptosis was evaluated by flow cytometry.

Wound healing assay

Cells (1 × 10⁵) were seeded in 6-well plates, scratched with a pipette tip, and washed three times with PBS. The wound area was monitored, and images were captured and analyzed using a microscope.

Immunohistochemistry

Two days after transfection, cells were harvested and transferred to laser confocal dishes for a 12-h incubation, followed by two washes with PBS. The cells were then fixed in 4% PFA containing 2 mg/mL glycine for 15 min and permeabilized with 0.2% Triton X-100 for 5 min. After blocking with 5% BSA for 30 min, the cells were incubated overnight at 4 °C with anti-OPN3 primary antibodies. Subsequently, the cells were treated with Alexa Fluor-conjugated secondary antibodies (Earthox, Millbrae, CA, USA) and DAPI (Beyotime, Biotechnology).

H&E staining

For H&E staining, lung tissue was extracted from mice, washed with 1 × PBS, and fixed overnight in 4% PFA. The tissue was then embedded in paraffin, sectioned into 4 μm-thick slices, and stained with H&E. Images were captured using an optical microscope (Olympus, Tokyo, Japan).

Statistical analysis

OPN3 expression across various human cancers was assessed using data from the TCGA database. Protein expression of OPN3 in LUAD was determined through IHC staining results from the Human Protein Atlas (HPA) database. The relationship between OPN3 expression and LUAD progression stages was analyzed via the GEPIA2 platform. Each experiment was performed in triplicate, with results presented as mean ± standard deviation. Statistical analysis involved an independent two-sample t-test to compare means between two groups and one-way ANOVA to assess differences among three or more groups. Statistical significance was denoted as * for P < 0.05, ** for P < 0.01, *** for P < 0.001, and **** for P < 0.0001.

Results

Expression profiles of OPN3 in normal tissues and LUAD

OPN3 expression levels in tumor and normal tissues from the TCGA database were analyzed using the TIMER 2.0 platform, revealing a significant upregulation of OPN3 transcription in LUAD (Fig. 1a). Additionally, protein expression was assessed through the HPA database, and IHC staining confirmed elevated OPN3 levels in LUAD tissues (Fig. 1b). The relationship between OPN3 expression and LUAD progression was further explored using GEPIA2, demonstrating a strong correlation between OPN3 expression and disease stages (Fig. 1c). High OPN3 expression was associated with poor prognosis in LUAD patients, suggesting its potential as a LUAD-specific biomarker (Fig. 1d). These results indicate that OPN3 is overexpressed in LUAD tissues compared to normal lung tissues and may contribute to cancer diagnostics. The study also analyzed OPN3 expression in five LC cell lines, showing varying expression levels, with the highest in H1437 and relatively lower levels in H1299 (Fig. 1e).

Fig. 1

Expression profiles of OPN3 in normal tissues and LUAD. a OPN3 expression differences between LUAD and normal tissues in OPN3 from the TCGA database. b The expression intensity of OPN3 in LUAD tissues and normal tissues was accomplished through IHC using the Human Protein Atlas (HPA) database, utilizing the antibody CAB013682. c The correction between OPN3 expression and the pathological stages of LUAD using the GEPIA2 database. d Survival analysis of patients with high expression of OPN3 and low expression of OPN3 in LUAD on TIMER 2.0 website. Overall survival between the OPN3 high- and low-expression group in patients with LUAD by Kaplan–Meier analysis. e The basic endogenous expression of OPN3 in five distinct LUAD cell lines, *P < 0.05, **P < 0.01; means + SD

To investigate the association between OPN3 expression and clinical-pathological characteristics in lung adenocarcinoma, IHC was performed on tissue microarrays containing 92 paraffin-embedded lung adenocarcinoma samples and 88 adjacent tissues. Figure S1 A-B demonstrates significantly higher OPN3 staining scores in cancer tissues compared to adjacent tissues (P < 0.01). Patients were then stratified based on OPN3 expression, and Kaplan–Meier survival analysis revealed that high OPN3 expression correlated with reduced overall survival (OS) (P < 0.001) (Figure S1 C). Subsequent analysis of clinical-pathological features revealed that OPN3 expression was associated with age, survival status, and T stage (Table 1). Cox univariate and multivariate analyses (Figure S1D-E) identified pathological stage (HR = 3.096, P = 0.001) and OPN3 expression (HR = 2.399, P = 0.004) as independent poor prognostic factors in operable LUAD patients. Additionally, a correlation analysis between OPN3 expression and various clinical and pathological features in these groups further confirmed these associations, as detailed in Table S1.

Table 1 The difference of the composition ratio between OPN3 high or low expression and age, survival status, T stage in LUAD in TMA

Effect of OPN3 knockdown in NCI-H1437

OPN3-specific siRNA was used to investigate the role of OPN3 in modulating the biological functions of H1437 cells in vitro. Transfection efficiency was confirmed through WB analysis, which verified successful suppression of OPN3 expression (Fig. 2a-b). A CCK8 assay assessed tumor cell proliferation, revealing a marked decrease in the growth rate of siOPN3-H1437 cells following OPN3 knockdown (Fig. 2c). Additionally, OPN3 knockdown impaired the colony-forming ability of these cells (Fig. 2d-e). Increased DNA fragmentation, observed via the TUNEL assay, suggested enhanced apoptotic cell death in siOPN3-H1437 cells (Fig. 2f-g). These results were corroborated by flow cytometry, which aligned with the TUNEL assay findings (Fig. 2h-i).

Fig. 2

Silencing OPN3 in LUAD cells markedly inhibited their proliferation, migratory, and invasive behaviors. a and b Transfection efficiency was validated using qPCR assays and western blot analysis, ***P < 0.001; means + SD. c The CCK8 assay was utilized to evaluate the proliferation of H1437 and siOPN3-H1437 cells following transfection with siOPN3. d and e Post-OPN3 knockdown, colony formation was assessed in H1437 and siOPN3-H1437 cells, *P < 0.05; means + SD. f and g The TUNEL assay was conducted, and three photographs representing different cellular fields are shown, **P < 0.01; means + SD. h and i The percentage of apoptotic H1437 and siOPN3-H1437 cells at specified time points in suspension was determined by dual staining with annexin V-FITC and PI, *P < 0.05, **P < 0.01; means + SD. EA: Early apoptotic cells; LA: Late apoptotic cells; TA: Total apoptotic cells. j and k Wound-healing assays were performed to measure the migration of H1437 cells after OPN3 knockdown, *P < 0.05, ***P < 0.001; means + SD. l and m A Transwell assay was employed to determine the impact of OPN3 knockdown on the migration and invasion of H1437 cells, ***P < 0.001; means + SD

The effect of OPN3 knockdown on the migration and invasion of LUAD cells was further examined. Wound-healing assays revealed a significant reduction in migration rate following OPN3 knockdown (Fig. 2j-k). Likewise, Transwell assays confirmed these findings, showing a notable decrease in both migration and invasion capacities (Fig. 2l-m). Collectively, these results demonstrate that OPN3 knockdown impairs the migration and invasion of LUAD cells in vitro.

Overexpression of OPN3 in LUAD

To investigate the role of OPN3 in LUAD, OPN3 overexpression was induced in H1299 cells (Fig. 3a-b). This led to a significant increase in cellular proliferation, as evidenced by higher CCK-8 assay results and enhanced colony formation (Fig. 3c-e). The TUNEL assay revealed a reduction in apoptotic cell death following OPN3 overexpression in H1299 cells (Fig. 3f-g). Additionally, flow cytometry analysis of both detached NCI-H1299 and H1299 cells demonstrated a notable decrease in apoptosis after OPN3 overexpression (Fig. 3h-i).

Fig. 3

Enforced OPN3 expression inhibits proliferation and metastasis in H1299 cells. a and b Over-expression of OPN3 in H1299 cells was confirmed by qPCR assays and western blot analysis, ***P < 0.001; means + SD. c CCK-8 assays of NCl-H1299 and H1299 cells after over-expression of OPN3. d and e the clonogenic potential of NCl-H1299 and H1299 cells after over-expression of OPN3 was assessed, ***P < 0.001; means + SD. f and g The TUNEL assay was conducted, and three photographs representing different cellular fields are shown, *P < 0.05; means + SD. h and i The percentage of apoptotic H1299 and H1299 cells after over-expression of OPN3 at specified time points in suspension was determined by dual staining with annexin V-FITC and PI, **P < 0.01; means + SD. EA: Early apoptotic cells; LA: Late apoptotic cells; TA: Total apoptotic cells. j and k The wound-healing assays quantified the migratory response of NCl-H1299 and H1299 cells upon OPN3 over-expression, ***P < 0.001; means + SD. l and m The Transwell assay was applied to evaluate the influence of elevated OPN3 levels on H1299 cell migration and invasiveness, **P < 0.01, ***P < 0.001; means + SD

Overexpression of OPN3 also significantly promoted cellular migration, as shown by wound-healing assays (Fig. 3j-k). This migratory enhancement was further confirmed by transwell migration assays, which revealed substantial increases in both migration and invasion in H1299 cells (Fig. 3l-m). Collectively, these results suggest that OPN3 plays a key role in driving the malignant properties of LUAD cells in vitro.

OPN3 silencing suppresses xenograft tumor development

To assess the impact of OPN3 knockdown on tumorigenesis, a lentiviral vector was designed to stably silence OPN3 in H1437 cells (Fig. 4a-b). These modified cells, along with control shNC cells, were subcutaneously injected into nude mice. Tumor growth was monitored, showing a moderate reduction in tumor size in the OPN3 knockdown group (Fig. 4c). After 35 days, the mice were euthanized, and tumor weights were measured, revealing a significant reduction in tumor mass in animals injected with shOPN3-H1437 cells (Fig. 4d-f). Furthermore, the TUNEL assay demonstrated a substantial increase in apoptosis in the OPN3 knockdown group compared to controls (Fig. 4g-h). Hematoxylin and eosin (H&E) staining of organ tissues from both groups highlighted significant alterations in lung cell morphology (Fig. 4i). IHC analysis revealed decreased expression of Ki-67 and OPN3 in shOPN3 cells (Fig. 4j-k). These results collectively suggest that OPN3 silencing inhibits tumorigenesis in vivo.

Fig. 4

OPN3 knockdown suppresses tumor growth in nude mice. a and b A lentiviral vector was constructed for the stable knockdown of the OPN3 gene, with expression levels confirmed through qPCR and Western blot analysis, *P < 0.05, ***P < 0.001; means + SD. c Tumor volume in xenograft models, initiated with shNC and shOPN3, was monitored from day 0 to 35, ***P < 0.001; means + SD. d Photographs of the xenograft tumors derived on day 35. e and f Tumor weight was measured for xenografts on day 35, **P < 0.01; means + SD. g and h Apoptosis was assessed using the TUNEL assay, ***P < 0.001; means + SD. i The histological alterations of tumors induced in nude mice. Histological features of tumors were analyzed following H&E staining. j Ki67 expression was analyzed by the IHC assay. k Reduced OPN3 protein levels in the tumors induced by OPN3-knockdown in nude mice. OPN3 expression in tumor tissues was analyzed by the IHC assay

Microarray analysis and WB validation of OPN3-regulated genes and pathways in LUAD cells

Microarray assays were employed to identify dysregulated genes in siOPN3-H1437 cells infected with lentivirus expressing NC or siOPN3, aiming to elucidate the molecular mechanisms associated with OPN3. A total of 832 genes exhibited differential expression following OPN3 knockdown, with 398 genes upregulated and 434 downregulated. Pathway enrichment analysis revealed that these dysregulated genes were predominantly involved in four pathways, including glutathione metabolism, the longevity regulating pathway, and homologous recombination (Fig. 5a-b). Notable alterations were detected in GPX3 and other genes related to the GPX3 pathway. To validate these findings, WB analysis was conducted. OPN3 overexpression in H1299 cells led to decreased GPX3 expression, increased activation of P-NF-κB and p-ERK, and upregulation of CyclinB1 (Fig. 5c-f). In contrast, siOPN3-H1437 cells displayed diminished activation of these pathways and elevated GPX3 expression, suggesting a downregulation of OPN3-associated signaling pathways (Fig. 5g-j). These results indicate that OPN3 likely modulates cell cycle progression and cellular responses to external stimuli through the regulation of P-NF-κB and p-ERK activity in LUAD. The reduced p-ERK levels observed after OPN3 knockdown may reflect a negative feedback mechanism or a direct impact on the ERK signaling cascade, warranting further investigation to elucidate the underlying molecular mechanisms.

Fig. 5

Microarray Analysis and Western Blot Validation of OPN3-Regulated Genes and Pathways in LUAD Cells. a and b The enriched pathways was revealed by the microarray assay, with a heatmap illustrating the relative expression levels of key genes under control and siOPN3 conditions. c and d The expression levels of target protein GPX3 as detected by western blotting in H1299 cells over-expressing OPN3, *P < 0.05; means + SD. e and f The levels of phosphorylated NF-κB (p-NF-κB), total NF-κB p65, CyclinB1, phosphorylated extracellular signal-regulated kinase (p-ERK), total ERK, and OPN3 proteins were measured in NCL-H1299 and H1299 cells over-expressing OPN3, *P < 0.05, ***P < 0.001, ****P < 0.0001; means + SD. g and h The expression levels of target protein GPX3 as detected by western blotting in siOPN3-H1437 cells, ***P < 0.001; means + SD. (i and j) The levels of p-NF-κB, total NF-κB p65, CyclinB1, p-ERK, total ERK, and OPN3 proteins were assessed in NCL-H1437 and siOPN3-H1437 cells using the same method, *P < 0.05, ***P < 0.001; means + SD

Discussion

This study revealed that OPN3 expression was significantly elevated in LUAD tissues relative to normal lung tissues, and its overexpression correlated with poor prognosis in LUAD patients. Knockdown of OPN3 via siRNA technology notably diminished LUAD cell proliferation, migration, and invasion, suggesting that OPN3 serves as a key promoter of malignancy in these cells. Conversely, OPN3 overexpression enhanced these traits, further emphasizing its role in tumor progression. Additionally, a negative correlation between OPN3 and GPX3 expression was observed. While OPN3 overexpression appears to activate cell survival and proliferation pathways, it concurrently suppresses GPX3 expression. Considering the essential role of GPX3 in maintaining antioxidant defenses and redox balance, its reduced expression could impair the cell’s ability to manage oxidative stress, potentially contributing to the more aggressive phenotype associated with OPN3 overexpression [21,22,23]. The decrease in GPX3 levels in response to heightened OPN3 activity may have significant implications for LUAD cell biology, influencing not only proliferation and survival but also treatment outcomes, including chemotherapy resistance.

To clarify the context-dependent role of OPN3 in LUAD progression, its expression patterns were analyzed in relation to key molecular markers. OPN3 expression was upregulated in BRAF-mutant cell lines (e.g., NCI-H1437), indicating a potential interaction with the BRAF signaling pathway (Table S2), while lower expression was observed in K-ras-mutant cell lines (e.g., NCI-H1944 and NCI-H1395), suggesting a context-dependent function in LUAD progression. This differential expression suggests OPN3's complex involvement, possibly modulating pathways such as BRAF and GPX3. The inverse correlation between OPN3 and GPX3 further implies that OPN3 may disrupt redox homeostasis, contributing to tumor aggressiveness. Further investigation is required to elucidate the regulatory mechanisms of OPN3 and its therapeutic potential, particularly targeting the OPN3-GPX3 axis or BRAF signaling. Understanding these interactions may lead to personalized therapies based on molecular profiles, thereby improving outcomes for LUAD patients.