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International Journal of Nanomedicine logoLink to International Journal of Nanomedicine
. 2026 Aug 1;21:603774. doi: 10.2147/IJN.S603774

Calcium Phosphate Nanoparticle Delivery of siTRIB3 Inhibits EMT and Stemness in NSCLC

Yinqiu Wu 1,2,*, Jixin Song 1,2,*, Sheng He 3,*, Jing Zhang 4, Juqun Xi 1,5, Shengzhe Zhang 1,2, Xuejun He 4,✉, Hua Dai 1,2,✉
PMCID: PMC13440341  PMID: 42559326

Abstract

Background

TRIB3 is upregulated in non-small cell lung cancer (NSCLC) and associates with worse survival, yet targeted therapeutics remain lacking. Calcium phosphate (CaP) nanoparticles offer biocompatible, serum-stable delivery for siRNA therapy. Here, we developed CaP nanoparticles loading siRNA targeting TRIB3 (NPCaP/siTrib3) and evaluated antitumor efficacy and mechanisms in vitro and in vivo.

Methods

NPCaP/siTrib3 was synthesized via biomineralization, and RNase resistance was verified by PAGE. Mouse KP-1 NSCLC cells were used for transfection, apoptosis (Annexin V/7-AAD), migration (scratch), invasion (Matrigel Transwell), qRT-PCR, and Western blot of epithelial–mesenchymal transition (EMT) and stemness markers. C57BL/6 subcutaneous tumor models received tail-vein injections for efficacy and biosafety assessment.

Results

TRIB3 was significantly upregulated in NSCLC tissues compared with normal lung tissues and was associated with poorer overall survival. NPCaP/siTrib3 formed nanosheets with an average size of approximately 159.4 nm and exhibited a more negative zeta potential than bare CaP, indicating successful siRNA loading. The CaP shell effectively protected siTrib3 from RNase degradation. NPCaP/siTrib3 efficiently silenced TRIB3 protein expression in KP-1 cells, with gene knockdown efficacy comparable to Lipo8000 but with lower cytotoxicity. Functionally, NPCaP/siTrib3 significantly promoted apoptosis and inhibited cell migration and invasion. In vivo, intravenously administered NPCaP/siTrib3, which primarily accumulated in tumors through the enhanced permeability and retention (EPR) effect, markedly suppressed tumor growth without affecting body weight. Mechanistically, NPCaP/siTrib3 reversed EMT by upregulating E-cadherin and downregulating N-cadherin and Snail, and reduced cancer stemness by decreasing the expression of Sox2, Nanog, Pou5f1, Klf4, and c-Myc. Biosafety evaluations demonstrated negligible hemolysis, normal serum biochemistry and hematology, and no histopathological damage in major organs.

Conclusion

NPCaP/siTrib3 is an effective and biocompatible siRNA nanoplatform that suppresses NSCLC progression through dual inhibition of EMT and cancer stemness, supporting TRIB3 as a promising therapeutic target.

Keywords: non-small cell lung cancer, the pseudokinase tribbles homolog 3, calcium phosphate nanoparticles, siRNA delivery, epithelial–mesenchymal transition

Graphical Abstract

SiRNA delivery blocks TRIB3, curbing metastasis and self-renewal, inhibiting tumor growth in mice. The diagram illustrates a biochemical pathway for siRNA delivery targeting TRIB3. Calcium and phosphate condense with siRNA to form NP_CaP siTrib3. This complex undergoes cellular uptake, releasing siTrib3 siRNA in a pH-triggered manner. The siRNA associates with RISC, leading to TRIB3 mRNA silencing. This results in decreased epithelial-mesenchymal transition markers (E-cadherin, N-cadherin, Snail, Vimentin) and stemness markers (Sox2, Nanog, Pousf1), inhibiting metastasis and self-renewal. The therapeutic outcome is increased apoptosis and reduced tumor volume and weight in mice, with good biocompatibility and stable body weight, comparing treatment and control groups.

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Introduction

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases and remains the leading cause of cancer-related mortality worldwide.1,2 Despite advances in surgery, radiotherapy, and chemotherapy, the prognosis for patients with advanced or metastatic NSCLC is poor, with a 5-year survival rate of less than 20%.3–5 The major obstacles to effective treatment are acquired drug resistance and distant metastasis, necessitating the exploration of novel therapeutic targets and strategies.3,6–8

The pseudokinase Tribbles homolog 3 (TRIB3) has emerged as a protein of significant interest in oncology.9 As a stress-inducible protein, TRIB3 lacks intrinsic kinase activity but functions as a crucial scaffold protein that modulates multiple signaling pathways involved in cell proliferation, metabolism, and survival.10 A growing body of evidence indicates that TRIB3 is significantly upregulated in various malignancies, including NSCLC. Our analysis of public databases, including The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), confirms that TRIB3 expression is markedly elevated in NSCLC tissues compared to normal lung tissues, and this high expression level is negatively correlated with patient overall survival. Mechanistically, TRIB3 has been shown to drive oncogenic processes in NSCLC through several routes.11,12 It promotes aerobic glycolysis (the Warburg effect) to provide a metabolic advantage for tumor growth, activates the MAPK signaling pathway to enhance malignant cell transformation, and supports cancer stem cell (CSC) properties, which are linked to tumor initiation and metastasis.13–16 Furthermore, recent studies have revealed that TRIB3 can directly interact with and stabilize the epidermal growth factor receptor (EGFR), thereby promoting resistance to tyrosine kinase inhibitors (TKIs), a frontline therapy for EGFR-mutated NSCLC.11,17,18 Metastasis, recurrence, and acquired therapeutic resistance remain major barriers to successful NSCLC treatment and are closely associated with EMT activation and cancer stemness.19 Therefore, the development of effective therapeutic strategies targeting these malignant phenotypes represents an urgent unmet clinical need in NSCLC management. In this context, the multifaceted role of TRIB3 in driving NSCLC progression, epithelial–mesenchymal transition (EMT) activation, and therapy resistance makes it a highly attractive therapeutic target.

Gene therapy using small interfering RNA (siRNA) offers a powerful approach to selectively silence oncogenic drivers like TRIB3 with high specificity, thereby minimizing off-target effects common to conventional chemotherapy.20–22 However, the clinical translation of siRNA faces a significant siRNA delivery challenge. As a naked polyanionic macromolecule, siRNA is susceptible to rapid degradation by ribonucleases (RNases) in the bloodstream, is subject to renal clearance, and cannot efficiently penetrate the negatively charged cell membrane to reach its cytosolic target.23,24 To overcome these barriers, various non-viral delivery vectors, such as cationic lipids and polymers, have been developed.25 While these systems can protect siRNA and facilitate cellular uptake, their application is often limited by issues of systemic toxicity, poor stability in serum, and low transfection efficiency in vivo.26–28

In this context, inorganic nanomaterials such as calcium phosphate (CaP) nanoparticles have garnered increasing attention as promising alternatives for siRNA delivery. Recent studies have shown that CaP-based nanocarriers can efficiently encapsulate and protect nucleic acids while enabling pH-responsive intracellular release in acidic endosomal environments.29–31 Compared with certain cationic polymeric systems, CaP nanoparticles exhibit lower intrinsic cytotoxicity and improved biodegradability, thereby reducing concerns regarding long-term biosafety during systemic administration.32 In addition, recent CaP-based delivery platforms have demonstrated promising therapeutic efficacy for siRNA and gene delivery in multiple tumor models, including lung cancer-related applications, owing to their favorable serum stability, intracellular delivery capability, and tumor-responsive dissolution behavior.33 As delivery vectors, CaP nanoparticles can be readily prepared to efficiently condense and encapsulate siRNA through electrostatic interactions, thereby providing protection against RNase-mediated degradation.34–36 Moreover, acidic endosomal environments promote the nanoparticles dissolution, thereby facilitating intracellular siRNA release.37–39 To date, a dedicated nanotherapeutic platform specifically designed to target TRIB3 for NSCLC treatment has not yet been reported.

Therefore, we hypothesized that a CaP-based nanoplatform loaded with siRNA against TRIB3 (termed NPCaP/siTrib3) could effectively silence TRIB3 expression in NSCLC cells. Since TRIB3 is overexpression in NSCLC compare to the normal tissues, we speculate that NPCaP/siTrib3 would have the ability to suppress tumorigenesis and development in NSCLC. This study aims to develop and characterize NPCaP/siTrib3 and evaluate its therapeutic efficacy and underlying mechanisms both in vitro and in vivo, with the goal of providing a new, safe, and effective strategy for NSCLC treatment.

Methods

Materials

Cell Lines and Culture

The KP-1 murine lung adenocarcinoma cell line used in this study was obtained as a gift from Prof. Guanglei Zhuang’s laboratory (Shanghai Jiao Tong University) and is not commercially available. KP-1 cells were originally established from a primary lung adenocarcinoma arising in the KP genetically engineered mouse model (KrasLSL-G12D/+; Trp53fl/fl after Cre-mediated recombination; tumor cells commonly described as KrasG12D/+; Trp53-deficient). Use of this gifted, non-commercial cell line in the present study was reviewed and approved by the Institutional Research Ethics Committee of Yangzhou University Medical School (approval no. 202403081; approved on March 5, 2024). KP-1 cells were used exclusively for in vitro experiments, including TRIB3 knockdown and functional assays for proliferation, migration, invasion, and apoptosis. All procedures involving KP-1 cells were conducted in accordance with institutional biosafety requirements and regulations governing the use of non-commercial biological materials. The murine lung epithelial cell line TC-1 was purchased from Qisai Biological Co., Ltd. (Shanghai, China; Cat# QS0128). All cells were maintained in growth media according to the manufacturers’ or providers’ instructions and were negative for mycoplasma contamination.

Synthetic siRNA and Transfection Reagents

Three specific small interfering RNA (siRNA) sequences targeting murine Trib3 (siTrib3-1, siTrib3-2, siTrib3-3) and a non-targeting scrambled negative control (siNC) were designed and synthesized by Anhui General Biology Co., Ltd. (Chuzhou, China). The detailed sequences are provided in Supplementary Table S1. SiRNA-mediated gene silencing experiments were performed according to previously established RNA interference strategies.21,22 For sequence screening and comparative transfection experiments, Lipo8000 transfection reagent (Beyotime Biotechnology, Shanghai, China; Cat# C0533) was utilized as a commercial reference, following the manufacturer’s optimized instructions.

Chemical Reagents for Nanoparticle Preparation

The inorganic precursors and buffering agents utilized for the synthesis of NPCaP/siTrib3 nanoparticles were of analytical grade and used without further purification. Specifically, calcium chloride (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; Cat# 10005818), sodium phosphate dibasic (Sinopharm Chemical Reagent Co., Ltd.; Cat# 10020318), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Sinopharm Chemical Reagent Co., Ltd.; Cat# 30101618) were employed to prepare the mineral shell and stabilizing buffers. Tris-HCl buffer (1 mM, pH 7.4) and HEPES/phosphate buffer (140 mM NaCl, 50 mM HEPES, 1.5 mM Na2HPO4, pH 7.4) were freshly prepared using DEPC-treated water (White Shark Biotechnology, Co., Ltd., Hefei, China) and sterilized via filtration through a 0.22 μm membrane prior to use.

Antibodies for Western Blot Analysis

For the quantitative analysis of protein expression, the following primary antibodies were utilized according to standard Western blot protocols: rabbit anti-TRIB3 polyclonal antibody (Proteintech, Chicago, USA; Cat# 13300-1-AP), rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Danvers, USA; Cat# 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology; Cat# 13116), rabbit anti-Snail monoclonal antibody (Cell Signaling Technology; Cat# 3879), mouse anti-Sox2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA; Cat# sc-365823), mouse anti-Nanog monoclonal antibody (Santa Cruz Biotechnology; Cat# sc-374103), and mouse anti-β-actin monoclonal antibody (Proteintech; Cat# 66009-1-Ig), detection was performed using corresponding secondary antibodies, including HRP-conjugated goat anti-rabbit IgG (Proteintech; Cat# SA00001-2) and HRP-conjugated goat anti-mouse IgG (Proteintech; Cat# SA00001-1), followed by enhanced chemiluminescence (ECL) visualization.

Synthesis and Characterization of NPCaP/siTrib3

Nanoparticle Preparation

The NPCaP/siTrib3 nanoparticles were synthesized using a modified calcium phosphate co-precipitation method as previously described with minor modifications.35,38,40 Briefly, 30 μL of CaCl2 solution (2.5 M) was mixed with 30 μL of siTrib3 solution (60 μM). The mixture was then diluted to a total volume of 300 μL with Tris-HCl buffer (1 mM, pH 7.4). After incubation for 1 minute at room temperature, an equal volume (300 μL) of HEPES/phosphate buffer (140 mM NaCl, 50 mM HEPES, 1.5 mM Na2HPO4, pH 7.4) was added, followed by immediate vortexing for 2 minutes. The reaction was allowed to equilibrate for 30 minutes to ensure stable nanoparticle formation. Subsequently, the nanoparticles were harvested by centrifugation at 4°C (2,000 rpm, 10 minutes), washed three times with DEPC-treated water and lyophilized. Control nanoparticles, including plain CaP and NPCaP/siNC (loaded with negative control siRNA), were prepared following the same protocol.

Physicochemical Characterization

The morphology and size of the nanoparticles were examined by transmission electron microscopy (TEM). Hydrodynamic diameter and zeta potential were measured by dynamic light scattering (DLS) using a Malvern Zetasizer.

In vitro Studies

Cell Transfection and Knockdown Efficiency

Cells were transfected with the three siTrib3 variants or siNC using Lipo8000 according to the manufacturer’s protocol to screen for the most effective sequence. For nanoparticle-mediated delivery, cells were treated with CaP, NPCaP/siNC, or NPCaP/siTrib3. TRIB3 knockdown efficiency at both mRNA and protein levels was evaluated by quantitative real-time PCR (qRT-PCR) and Western blot, respectively. The transfection efficiency of NPCaP/siTrib3 was directly compared to that of Lipo8000.

Cell Viability and Apoptosis Assay

Cell viability was assessed using the CCK-8 assay. Apoptosis was quantified by flow cytometry using an Annexin V-FITC/7-AAD apoptosis detection kit according to the manufacturer’s instructions.

Migration and Invasion Assays

Cell migration was evaluated using a wound-healing (scratch) assay. A linear scratch was created in a confluent cell monolayer, and wound closure was monitored and quantified at 0, 12, and 24 hours. Cell invasion was assessed using Transwell chambers coated with Matrigel. Cells that invaded through the Matrigel after 24 hours were fixed, stained with crystal violet, and counted under a microscope.

Mechanistic Protein Analysis

The expression of proteins related to EMT (E-cadherin, N-cadherin, Snail)41,42 and cancer stemness (Sox2, Nanog)16,43,44 was analyzed by Western blot in KP-1 cells following various treatments, based on previously established EMT and cancer stemness-associated markers in NSCLC studies.

In vivo Studies

Animal Model and Treatment

All animal experiments were approved by the Institutional Animal Care and Use Committee of Yangzhou University and conducted in accordance with the relevant guidelines and regulations. A subcutaneous xenograft model was established by injecting KP-1 cells into the right flank of female C57 mice. When tumor volumes reached approximately 80 mm3, mice were randomly divided into four groups (n = 4 per group): (1) PBS, (2) free siTrib3, (3) NPCaP/siNC, and (4) NPCaP/siTrib3. Nanoparticles were administered via tail-vein injection every other day at an siRNA equivalent dose of 1.5 mg/kg. The treatment schedule was designed according to previously reported calcium phosphate-based siRNA delivery systems with favorable therapeutic efficacy and biosafety. Mice were euthanized on day 11, tumors and major organs were harvested for further analysis.45

Biosafety Evaluation

Blood was collected for comprehensive analysis, including routine hematology and blood biochemistry (markers for liver and kidney function). Major organs (heart, liver, spleen, lungs, and kidneys) were fixed, sectioned, and stained with hematoxylin and eosin (H&E) for histopathological examination.

Ethics Statement

All animal experiments were approved by the Animal Welfare and Ethics Committee of Yangzhou University (Approval No. 202502175, approved on February 26, 2025) and conducted in accordance with institutional and national guidelines for the care and use of laboratory animals. Mice were anesthetized prior to procedures, and at the end of the experiment, animals were euthanized under anesthesia.

The collection and use of human lung cancer tissues were approved by the Ethics Committee of the Medical School of Yangzhou University (Approval No. XYLL-2025-16). Written informed consent was obtained from all participants, and the study was conducted in accordance with the Declaration of Helsinki.

Statistical Analysis

All quantitative data are presented as mean± standard deviation (SD). Statistical comparisons between multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. A p-value of less than 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism software (version 9.0).

Results

Bioinformatics Analysis and Characterization of NPCaP/siTrib3 Nanoparticles for Targeted NSCLC Therapy

Bioinformatics analysis using TCGA (LUSC and LUAD datasets) and GEO datasets (GSE30219, GSE31210) revealed that TRIB3 mRNA levels were significantly higher in tumor tissues compared to normal controls (Figure 1A and B). Kaplan-Meier survival analysis indicated that high TRIB3 expression is negatively correlated with overall survival in NSCLC patients (Figure 1C and D). This was validated in clinical specimens, where qRT-PCR confirmed elevated TRIB3 in tumor tissues from 16 NSCLC patients (Figure 1E and Table S2). Furthermore, Western blot screening of cell lines showed high TRIB3 protein expression in mouse NSCLC KP-1 cells compared to normal TC-1 epithelial cells (Figure 1F and G). Collectively, these data confirm that TRIB3 is consistently overexpressed in NSCLC and correlates with poor prognosis, highlighting it as a promising therapeutic target. Therefore, the design of siRNA targeting TRIB3 represents a novel and rational strategy for NSCLC treatment.

Figure 1.

Infographic on TRIB3 expression in NSCLC, survival impact and CaP nanoparticle synthesis for siRNA delivery. This multi-panel research figure explores TRIB3 expression, prognosis and CaP nanoparticle siRNA formulation. Panels A and B show higher TRIB3 expression in tumor tissues compared to normal, with significant p-values. Panels C and D illustrate Kaplan-Meier survival curves, indicating worse survival with higher TRIB3 expression. Panel E presents qRT-PCR validation of elevated TRIB3 mRNA in tumor samples. Panel F shows Western blot analysis of TRIB3 protein in various cell lines, with bands at 40 kDa. Panel G provides relative expression levels with statistical comparisons. Panel H illustrates the synthesis of CaP nanoparticles encapsulating siTrib3. Panel I includes TEM images and elemental mapping of nanoparticles. Panel J displays hydrodynamic size distribution, highlighting peaks at 159.4 nm and 108.7 nm. Panel K shows zeta potential measurements. Panel L demonstrates a gel retardation assay, confirming siTrib3 protection from RNase degradation.

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Identification of TRIB3 as a therapeutic target and characterization of NPCaP/siTrib3 (A and B) Bioinformatics analysis of TRIB3 mRNA expression in NSCLC tumor versus normal tissues using TCGA datasets. (C and D) Kaplan-Meier survival curves comparing overall survival between NSCLC patients with high and low TRIB3 expression. (E) qRT-PCR validation of TRIB3 mRNA levels in clinical NSCLC tissue samples (n=16) compared with adjacent normal tissues. (F) Western blot analysis of TRIB3 protein expression in mouse lung epithelial TC-1 cells and NSCLC KP-1, KP-13, 889-DTC cells. (G) qRT-PCR analysis of TRIB3 mRNA expression in TC-1, KP-1, KP-13 and 889-DTC cell lines. (H) Schematic illustration of the synthesis of NPCaP/siTrib3 via biomineralization.(I) TEM image and corresponding elemental mapping of NPCaP/siTrib3. (J) Hydrodynamic size distribution of NPCaP/siTrib3 measured by DLS. (K) Zeta potential of NPCaP/siTrib3. (L) Gel retardation assay demonstrating the protection of encapsulated siTrib3 from RNase degradation.

Based on the functional relevance of TRIB3 in NSCLC pathogenesis, we synthesized a TRIB3-silencing siRNA and packaged it into CaP nanoparticles via biomineralization, yielding CaP/siRNA nanoparticles (designated NPCaP/siTrib3) (Figure 1H). As shown in Figure 1I, transmission electron microscopy (TEM) images indicated that the NPCaP/siTrib3 nanoparticles exhibited a spherical morphology with uniform distribution, distinguishing them from the CaP control nanoparticles prepared in the absence of siTrib3 (Figure S1). Meanwhile, the presence of a phosphorus (P) signal in the elemental mapping results indicates the successful encapsulation of siTrib3 within the CaP nanoparticles. Subsequently, dynamic light scattering (DLS) measurements indicated that NPCaP/siTrib3 had a hydrodynamic diameter of approximately 159.4 nm (Figure 1J), a size range favorable for exploiting the enhanced permeability and retention (EPR) effect in tumor targeting,46,47 whereas unloaded CaP nanoparticles exhibited an average diameter of approximately 108.7 nm. In addition, compared with unloaded CaP nanoparticles, NPCaP/siTrib3 displayed a more negative zeta potential after siTrib3 loading (Figure 1K), which may be attributed to the negatively charged phosphate backbone of siRNA and is generally associated with improved colloidal dispersion. Crucially, the gel retardation assay confirmed the protective effect of the CaP mineral shell: while free siTrib3 was degraded by RNase, the siTrib3 encapsulated within the CaP nanoparticles remained intact (Figure 1L).

NPCaP/siTrib3 Induces TRIB3 Knockdown and Apoptosis in NSCLC

Next, KP-1 cells were transfected with three synthesized siTrib3 variants (siTrib3-1, siTrib3-2, and siTrib3-3) and a negative control (siNC) using Lipo8000. Western blot analysis revealed that siTrib3-3 induced the most pronounced reduction in TRIB3 protein levels (Figure 2A) and was therefore selected for subsequent experiments. To evaluate the delivery efficacy of our CaP-based nanoparticle system, KP-1 cells were treated with CaP, NPCaP/siNC, or NPCaP/siTrib3 for three days. Western blot analysis showed that cells treated with NPCaP/siTrib3 exhibited a significant decrease in TRIB3 protein expression compared to the CaP and NPCaP/siNC groups (Figure 2B). We further compared the transfection efficiency of NPCaP/siTrib3 with that of the commercial reagent Lipo8000. KP-1 cells were treated with an equal amount of siTrib3 delivered via either method. Western blot results demonstrated that both systems significantly reduced TRIB3 protein levels relative to the control (Figure 2C). Importantly, no significant difference in knockdown efficiency was observed between the two delivery systems, indicating that CaP nanoparticles are as effective as Lipo8000 in delivering functional siTrib3 into cells. Importantly, biocompatibility evaluation in normal murine lung epithelial TC-1 cells demonstrated that the CaP-based nanoparticle delivery system exhibited substantially lower cytotoxicity than the commercial transfection reagent Lipo8000 (Figure S2), supporting its safety for biological applications. Flow cytometry analysis with Annexin V/7-AAD staining (Figure 2D) showed that the proportion of late apoptotic KP-1 cells was highest in the NPCaP/siTrib3 group (13.1%), compared to the PBS (0.24%), free siTrib3 (3.33%), and NPCaP/siNC (2.5%) groups. These results demonstrate that silencing Trib3 via NPCaP/siTrib3 effectively induces apoptosis in NSCLC cells.

Figure 2.

Western blot and flow cytometry analysis of TRIB3 expression and apoptosis in KP-1 cells after various treatments. Image A: Western blot of TRIB3 in KP-1 cells with siNC, siTrib3-1, siTrib3-2, siTrib3-3. Relative expression P values: siNC vs siTrib3-1 (P = 0.8097), siNC vs siTrib3-2 (P = 0.0498), siNC vs siTrib3-3 (P = 0.001). Image B: TRIB3 in KP-1 cells with CaP, NP_CaP/siNC, NP_CaP/siTrib3. P values: CaP vs NP_CaP/siNC (P = 0.3439), NP_CaP/siNC vs NP_CaP/siTrib3 (P < 0.0001). Image C: TRIB3 in KP-1 cells with Ctrl, Lipo8000+siTrib3, NP_CaP/siTrib3. P values: Ctrl vs Lipo8000+siTrib3 (P < 0.0001), Ctrl vs NP_CaP/siTrib3 (P < 0.0001). Image D: Flow cytometry of KP-1 cells with PBS, Free siTrib3, NP_CaP/siNC, NP_CaP/siTrib3. Highest late apoptotic cells in NP_CaP/siTrib3 (13.1%).

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NPCaP/siTrib3 induces apoptosis in KP-1 NSCLC cells. (A) Western blot analysis and corresponding quantitative analysis of TRIB3 protein expression in KP-1 cells following transfection with siTrib3-1, siTrib3-2, and siTrib3-3. (B) Western blot analysis and corresponding quantitative analysis of TRIB3 protein expression in KP-1 cells following transfection with CaP, NPCaP/siNC, and NPCaP/siTrib3. (C) Western blot analysis and corresponding quantitative analysis of TRIB3 protein expression in KP-1 cells following transfection with Lipo8000/siTrib3 or treatment with NPCaP/siTrib3. (D) Representative flow cytometry dot plots of KP-1 cells stained with Annexin V‑FITC and 7-AAD after different treatments. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs control group.

NPCaP/siTrib3 Inhibits Migration and Invasion of KP-1 NSCLC Cells in vitro

To evaluate the anti-metastatic effect of NPCaP/siTrib3 on KP-1 cells, we first conducted a wound-healing assay. The results showed that while the siTrib3 and NPCaP/siNC groups did not differ significantly from the control in wound closure, NPCaP/siTrib3 treatment markedly inhibited cell migration at both 12  and 24 hours post-scratching (Figure 3A and B). We further performed a Transwell invasion assay, which demonstrated a significant decrease in the number of KP-1 cells invading through the Matrigel-coated membrane upon NPCaP/siTrib3 treatment (Figure 3C and D). Collectively, these data indicate that NPCaP/siTrib3 effectively suppresses both the migration and invasion of NSCLC cells.

Figure 3.

Four-part image showing wound-healing and invasion assays of KP-1 NSCLC cells under different treatments. The image A shows wound-healing assay results at 0, 12 and 24 hours for KP-1 NSCLC cells treated with PBS, siTrib3, NP subscript CaP/siNC and NP subscript CaP/siTrib3. The image B shows a bar graph of relative migration rates at 12 and 24 hours, with significant differences noted between treatments, especially NP subscript CaP/siTrib3 showing reduced migration. The image C shows Transwell invasion assay results for KP-1 cells treated with PBS, siTrib3, NP subscript CaP/siNC and NP subscript CaP/siTrib3. The image D shows a bar graph of the number of invasion cells, highlighting significant reduction in invasion for NP subscript CaP/siTrib3 treatment. P-values indicate statistical significance between groups.

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NPCaP/siTrib3 suppresses the migration and invasion of KP-1 NSCLC cells. (A) Representative images of wound-healing assay at 0, 12, and 24 hours after scratch in KP-1 cells treated as indicated. (B) Quantitative analysis of the wound closure rate from (A). (C) Representative images of Transwell invasion assay showing KP-1 cells that penetrated through the Matrigel-coated membrane. (D) Quantitative analysis of the number of invaded cells per field from (C). Data are presented as mean ± SD (n = 3).

NPCaP/siTrib3 Inhibits Migration and Invasion of NSCLC KP-1 Cells

We then evaluated the antitumor activity of NPCaP/siTrib3 in C57 mice bearing KP-1 xenografts. When tumor volumes reached approximately 80 mm3, the mice were randomly divided into four treatment groups: (1) PBS, (2) free siTrib3, (3) NPCaP/siNC, and (4) NPCaP/siTrib3. Treatments were administered via tail-vein injection every other day for 10 days. On day 11, the mice were euthanized and tumors were excised. Analysis of tumor volume changes during treatment and the final excised tumor volumes (Figure 4A and B) revealed that, compared to the PBS group, neither the free siTrib3 nor the NPCaP/siNC group showed a significant difference in tumor size during progression or after excision. In contrast, the NPCaP/siTrib3 treatment group exhibited a marked reduction in tumor volume. Representative tumor images (Figure 4C) and quantitative tumor weight data (Figure 4D) further confirmed that only NPCaP/siTrib3 effectively inhibited NSCLC tumor growth in vivo. Moreover, body weights of mice in all treatment groups remained comparable to those of the control group throughout the study (Figure S3), indicating that NPCaP/siTrib3 exhibits a favorable safety profile. Intravenous administration was selected to better mimic clinically relevant systemic treatment conditions for NSCLC, particularly metastatic disease. Compared with intratumoral injection, systemic delivery enables evaluation of nanoparticle circulation stability, tumor accumulation via the EPR effect, and in vivo biosafety. Furthermore, biosafety evaluation demonstrated excellent biocompatibility of the NPCaP/siTrib3 delivery system. Hemolysis assay showed negligible hemolytic activity (<5%) (Figure S4). Blood biochemistry analysis (liver and kidney function) and routine hematological parameters in treated mice remained within normal physiological ranges (Figure S5). Furthermore, H&E staining of major organs (heart, liver, spleen, lungs, and kidneys) revealed no obvious pathological alterations or signs of tissue damage (Fig. S6), collectively confirming the high biosafety profile of the delivery system. Due to the intrinsic biodegradability and biocompatibility of calcium phosphate, NPCaP/siTrib3 nanoparticles can undergo intracellular acid-triggered dissolution, yielding calcium and phosphate ions that are readily processed through physiological metabolic pathways. Although direct biodistribution analysis was not performed in the current study, the significant antitumor efficacy and molecular changes observed in tumor tissues indirectly support successful tumor delivery of NPCaP/siTrib3 following intravenous administration.

Figure 4.

Image: tumor volume/weight in mice treated with PBS, siTrib3, NPCaP/siNC, NPCaP/siTrib3. The image A showing a line graph of tumor volume in mm superscript 3 over days for four groups: PBS, siTrib3, NPCaP/siNC and NPCaP/siTrib3. The graph indicates a significant reduction in tumor volume for the NPCaP/siTrib3 group with a p-value of 0.0032. The image B showing a bar graph comparing final tumor volumes in mm superscript 3 for the same groups, highlighting significant differences with p-values: PBS vs NPCaP/siTrib3 (p=0.0147), PBS vssiTrib3 (p=0.5248), PBS vs NPCaP/siNC (p = 0.7500). The image C showing photographs of excised tumors from each treatment group labeled as PBS, siTrib3, NPCaP/siNC and NPCaP/siTrib3. The image D showing a bar graph of tumor weight in mg for the groups, with p-values: PBS vs NPCaP/siTrib3 (p=0.0025), siTrib3 vs NPCaP/siTrib3 (p=0.8495), NPCaP/siNC vs NPCaP/siTrib3 (p=0.9774), indicating significant weight reduction in the NPCaP/siTrib3 group.

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In vivo antitumor efficacy of NPCaP/siTrib3. (A) Tumor growth curves of KP-1 xenograft-bearing mice treated with PBS, free siTrib3, NPCaP/siNC, or NPCaP/siTrib3. (B) Final tumor volumes measured at the endpoint of the study. (C) Representative photographs of excised tumors from each treatment group.(D) Tumor weights at the endpoint. Data are presented as mean ± SD (n = 4).

Related Markers in NSCLC

To further investigate the impact of TRIB3 knockdown on NSCLC tumorigenesis and progression, we performed KEGG pathway enrichment analysis on TCGA data comparing NSCLC tissues with high versus low TRIB3 expression. The results indicated that TRIB3 significantly influences pathways related to cell adhesion molecules (CAMs) in NSCLC (Figure 5A). Cell adhesion molecules, which are widely present in multicellular organisms, play crucial roles in tissue development, immune cell trafficking, neural connectivity, and tumor metastasis by regulating intercellular interactions.48,49 Among CAMs, cadherins are key components of EMT pathway.41 During EMT, epithelial cells acquire mesenchymal characteristics, leading to enhanced motility and invasiveness—a critical process in lung cancer metastasis.42

Figure 5.

Eight-panel plots and blots of TRIB3, EMT-related proteins and stemness markers across treatments. The image A showing a bubble scatter plot titled KEGG Enrichment Dotplot. The x-axis is labeled GeneRatio, ranging from 0.02 to 0.08. The y-axis lists 10 pathways: Neuroactive ligand–receptor interaction; Cytokine–cytokine receptor interaction; Cell adhesion molecules; Hematopoietic cell lineage; Bile secretion; Complement and coagulation cascades; Metabolism of xenobiotics by cytochrome P450; Arachidonic acid metabolism; Retinol metabolism; Primary immunodeficiency. Ten bubbles lie near GeneRatio about 0.02 to 0.08, with the largest near 0.08 at the top pathway. The image B showing Western blots for KP-1 with two lanes labeled NPCap siNC and NPCap siTrib3. Rows: TRIB3 40 kilodalton, E-cadherin 125 kilodalton, N-cadherin 140 kilodalton, Snail 29 kilodalton, beta-actin 42 kilodalton. The image C showing a grouped bar chart of Relative Expression Levels from 0.0 to 2.0. Categories: TRIB3, E-cadherin, N-cadherin, Snail. Two groups: NPCap siNC and NPCap siTrib3. Approximate bar heights: TRIB3 about 1.0 vs 0.7 (P equals 0.0006); E-cadherin about 1.0 vs 1.7 (P less than 0.0001); N-cadherin about 1.0 vs 0.5 (P less than 0.0001); Snail about 1.0 vs 0.7 (P equals 0.0008). The image D showing Western blots with four groups labeled PBS, siTrib3, NPCap siNC, NPCap siTrib3, each with lanes 1, 2, 3. Rows: TRIB3 40 kilodalton, E-cadherin 125 kilodalton, N-cadherin 140 kilodalton, Vimentin 57 kilodalton, beta-actin 42 kilodalton. The image E showing Western blots for KP-1 with two lanes labeled NPCap siNC and NPCap siTrib3. Rows: Sox2 34 kilodalton, Nanog 40 kilodalton, Pou5f1 45 kilodalton, beta-actin 42 kilodalton. The image F showing a grouped bar chart of Relative Expression Levels from 0.0 to 1.5 for Sox2, Nanog, Pou5f1, comparing NPCap siNC vs NPCap siTrib3. Approximate heights: Sox2 about 1.0 vs 0.6 (P less than 0.0001); Nanog about 1.0 vs 0.55 (P less than 0.0001); Pou5f1 about 1.0 vs 0.95 (P equals 0.0973). The image G showing a grouped bar chart of Relative messenger ribonucleic acid levels from 0.0 to 1.5 for Sox2, Nanog, Pou5f1, Klf4, c-Myc, comparing NPCap siNC vs NPCap siTrib3. Approximate heights: Sox2 about 1.0 vs 0.3 (P equals 0.0027); Nanog about 1.0 vs 0.5 (P equals 0.0239); Pou5f1 about 1.0 vs 0.4 (P equals 0.0036); Klf4 about 1.0 vs 0.7 (P equals 0.0127); c-Myc about 1.0 vs 0.2 (P equals 0.0032). The image H showing a grouped bar chart of Relative messenger ribonucleic acid levels from 0.0 to 2.0 for Sox2, Nanog, Pou5f1, Klf4, c-Myc with four groups: PBS, siTrib3, NPCap siNC, NPCap siTrib3. Approximate heights: Sox2 about 1.0, 1.3, 0.5, 1.4 (P equals 0.0313); Nanog about 1.0, 1.2, 0.6, 1.1 (P equals 0.0425); Pou5f1 about 1.0, 0.9, 0.7, 0.8 (P equals 0.0877); Klf4 about 1.0, 1.2, 0.3, 1.0 (P equals 0.0088); c-Myc about 1.0, 1.2, 1.0, 1.3 (P equals 0.9843).

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Molecular mechanisms underlying the anti‑metastatic effect of NPCaP/siTrib3: suppression of EMT and stemness. (A) KEGG pathway enrichment analysis highlighting the significant association of TRIB3 with cell adhesion molecule pathways in NSCLC. (B) Representative Western blots showing the expression of EMT-related proteins (E-cadherin, N-cadherin, Snail, Vimentin) in KP-1 cells following indicated treatments. (C) Quantitative densitometric analysis of the protein levels shown in (B). (D) Western blot analysis of EMT-related protein expression in tumor tissues from different treatment groups. (E) Representative Western blots showing the expression of stemness-related proteins (Sox2, Nanog) in KP-1 cells. (F) Quantitative analysis of the protein levels shown in (E) and (G) qRT-PCR analysis of stemness-associated gene expression (Sox2, Nanog, Pou5f1, Klf4, c-Myc) in KP-1 cells. (H) qRT-PCR analysis of stemness-associated gene expression in tumor tissues from different treatment groups. Data in quantitative panels are presented as mean ± SD (n = 3). *p < 0.05, *p < 0.01 vs control group.

To investigate whether NPCaP/siTrib3 suppresses NSCLC metastasis by modulating EMT, we first analyzed the expression of EMT-related proteins by Western blot (Figure 5B and C). In KP-1 cells, NPCaP/siTrib3 treatment significantly upregulated the epithelial marker E-cadherin while downregulating the mesenchymal markers N-cadherin and Snail compared with the control. These results indicate that NPCaP/siTrib3 attenuates EMT in NSCLC cells, thereby reducing their invasive and metastatic capacity. To validate the in vivo mechanism of NPCaP/siTrib3-mediated tumor suppression, we extracted proteins from tumor tissues of mice in four experimental groups and examined EMT-related markers by Western blot (Figure 5D). Compared with the PBS, free siTrib3, and NPCaP/siNC groups, tumors from the NPCaP/siTrib3 group exhibited significantly decreased expression of TRIB3, N-cadherin, and Snail, accompanied by increased E-cadherin levels. These in vivo data confirm that NPCaP/siTrib3 inhibits EMT activation in NSCLC tumors.

Cancer stem cells (CSCs), which possess self-renewal capacity, generate heterogeneous tumor cell populations, and drive tumor initiation, exhibit enhanced migratory and invasive potential.44,50 By secreting cytokines, CSCs remodel the tumor microenvironment to facilitate metastatic colonization.43 Based on our previous findings that TRIB3 promotes NSCLC cell migration and invasion (scratch assay, Transwell, and Western blot), we next investigated whether NPCaP/siTrib3 treatment modulates CSC-associated markers in KP-1 cells. Western blot analysis (Figure 5E and F) showed that Sox2 and Nanog protein levels were significantly downregulated in the NPCaP/siTrib3‑treated group compared to the NPCaP/siNC group, while Pou5f1 expression remained unchanged. Correspondingly, qRT-PCR analysis (Figure 5G) revealed a marked reduction in the mRNA levels of Sox2, Nanog, Pou5f1, Klf4, and c-Myc following NPCaP/siTrib3 treatment relative to the NPCaP/siNC control. To extend these observations in vivo, we extracted RNA from tumor tissues of the four experimental mouse groups and performed qRT-PCR (Figure 5H). Compared with the PBS, free siTrib3, and NPCaP/siNC groups, tumors from the NPCaP/siTrib3-treated group exhibited significantly lower mRNA expression of Sox2, Nanog, and Klf4. Collectively, these in vitro and in vivo results demonstrate that NPCaP/siTrib3 downregulates key stemness-related genes and proteins, thereby suppressing CSC-associated traits and inhibiting NSCLC progression.

Conclusion

In summary, this study establishes TRIB3 as a therapeutically relevant target in NSCLC and develops a biocompatible CaP-based nanoplatform, NPCaP/siTrib3, for its effective silencing. Bioinformatics and experimental validation confirmed that TRIB3 is overexpressed in NSCLC and associated with poor prognosis, justifying its therapeutic targeting. We engineered an optimized siRNA against TRIB3 and encapsulated it into CaP nanoparticles via biomineralization. In KP-1 NSCLC cells, NPCaP/siTrib3 achieved TRIB3 knockdown comparable to a commercial reagent (Lipo8000) but with lower cytotoxicity. This silencing robustly induced apoptosis and suppressed migration and invasion in vitro. In a murine xenograft model, NPCaP/siTrib3 significantly inhibited tumor growth relative to controls, while comprehensive biosafety assessments confirmed its excellent biocompatibility and minimal systemic toxicity. Mechanistically, NPCaP/siTrib3 attenuated EMT, as shown by increased E-cadherin, decreased N-cadherin and Snail, and downregulated key CSC markers (eg, Sox2, Nanog), thereby suppressing pro-metastatic and stemness-related traits. Thus, NPCaP/siTrib3 represents a novel and safe nanotherapeutic that inhibits NSCLC progression by targeting TRIB3-driven EMT and CSC pathways, offering a promising siRNA-based strategy for NSCLC treatment. Moreover, the favorable biosafety profile and biomineralization-based preparation of NPCaP/siTrib3 may provide potential advantages over conventional lipid nanoparticles for long-term administration and future translational manufacturing.

Acknowledgments

This work was supported by the Major Project of Natural Science Research in Higher Educational Institutions of Jiangsu Province (H.D. 20KJA320005), Open Project from Guangxi Key Laboratory of Reproductive Health and Birth Defects Prevention (H.D. GXWCH-ZDKF-2023-16), the National Natural Science Foundation of China (J.X. 22472146), the Fund for Graduate Student Research and Practice and Innovation Program of Jiangsu Province (Y.W. KYCX22_3571).

Ethics Statement

The use of the gifted, non-commercial KP-1 murine lung adenocarcinoma cell line in this study was approved by the Institutional Research Ethics Committee of Yangzhou University Medical School (approval no. 202403081; March 5, 2024). KP-1 cells were used only for in vitro experiments and were handled in compliance with institutional biosafety regulations. All experiments were conducted in accordance with institutional guidelines for animal welfare and biosafety. KP-1 cells were used for in vitro TRIB3 knockdown and functional assays, including proliferation, migration, invasion, and apoptosis analyses.

Disclosure

The authors declare no competing interests in this work.

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