RNA-binding protein DAZAP1 accelerates the advancement of pancreatic cancer by inhibiting ferroptosis

Abstract

Background

Pancreatic cancer (PC) is a highly aggressive malignancy with a poor prognosis due to its late-stage diagnosis and limited treatment options.

Objectives

This study aimed to elucidate the molecular mechanisms underlying PC progression and identify potential molecular targets for its diagnosis and treatment.

Methods

DAZAP1 expression in PC tissues, normal tissues and cell lines was assessed using immunohistochemistry (IHC), reverse transcription–quantitative polymerase chain reaction (RT–qPCR) and western blotting. DAZAP1 knockdown was achieved through plasmid transfection, and its effects on ferroptosis and PC progression were evaluated using RT–qPCR, western blotting, CCK-8 assays, EdU staining, Fe²⁺ content measurement, reactive oxygen species (ROS) detection, wound healing and Transwell migration assays.

Results

DAZAP1 expression was significantly upregulated in PC tissues and cell lines compared to normal counterparts. DAZAP1 knockdown suppressed PC cell proliferation and induced ferroptosis, while ferroptosis inhibition reversed these effects, enhancing PC cell proliferation and metastasis.

Conclusions

DAZAP1 suppression promotes ferroptosis, thereby inhibiting PC cell proliferation and metastasis. These findings suggest that DAZAP1 is a potential therapeutic target for PC.

Highlights

• 1. DAZAP1 facilitates the proliferation and metastasis of pancreatic cancer;

• 2. Downregulation of DAZAP1 in pancreatic cancer promotes ferroptosis;

• 3. DAZAP1 regulates pancreatic cancer proliferation and metastasis through ferroptosis;

• 4. DAZAP1 has therapeutic potential in pancreatic cancer treatment.

Introduction

Pancreatic cancer (PC) is a highly aggressive solid tumour characterised by poor prognosis, with a 5-year survival rate of less than 10% [1]. According to the National Cancer Center of China, PC ranked 10th in incidence but 6th in mortality among all malignant tumours in 2022, with the number of deaths nearly equalling new cases [2]. Currently, radical resection combined with systemic chemotherapy offers the only potential for long-term survival. However, due to challenges in early diagnosis, over 80% of patients present with advanced or metastatic disease at diagnosis, rendering them ineligible for surgery [3]. Even among those diagnosed early and successfully operated on, the risk of postoperative recurrence and metastasis remains high, contributing to poor prognosis [4]. While systemic chemotherapy is the primary treatment for patients with advanced PC, resistance to chemotherapy drugs frequently develops, limiting therapeutic efficacy [4]. Therefore, identifying effective treatment strategies remains crucial to improving the survival of patients with PC.

Deleted in azoospermia-associated protein 1 (DAZAP1), an RNA-binding protein, is ubiquitously expressed in various human and murine tissues, with particularly high expression in the testes [5, 6]. Early research on DAZAP1 focused on infertility disorders, such as its roles in germ cell development and spermatogenesis through interactions with Deleted in Azoospermia (DAZ) and Deleted in Azoospermia Like (DAZL) [7]. Subsequent molecular structure studies of DAZAP1 revealed its role as an RNA-binding protein, wherein it plays a key role in various cancers through its alternative splicing function. Moreover, DAZAP1 promotes the proliferation of multiple myeloma cells by regulating the alternative splicing of KIT proto-oncogene ligand (KITLG) mRNA, which activates the ERK pathway [8]. Conversely, in oesophageal squamous cell carcinoma, DAZAP1 acts as a tumour suppressor by modulating oncogenic autophagy via mTOR pathway-regulated alternative splicing of the TSC2 gene [9]. In addition, DAZAP1 has been implicated in ferroptosis [10]. For instance, DAZAP1 reduces the sensitivity of hepatocellular carcinoma (HCC) cells to ferroptosis inducers and significantly inhibits ferroptosis [11]. While DAZAP1’s role in cancer appears context-dependent, its precise function varies across different types of cancer.

Currently, the role of DAZAP1 in the initiation and progression of PC remains unclear. Elucidating the molecular mechanisms by which DAZAP1 regulates PC progression could facilitate the development of molecular targets for early diagnosis and precision treatment. Therefore, this study aims to assess DAZAP1 expression in clinical PC samples and cell lines and to explore its regulatory effects on PC cell proliferation and metastasis through in vitro experiments.

Materials and methods

Collection of clinical samples

Patients diagnosed with PC who underwent tumour resection at the General Hospital of Ningxia Medical University between January 2022 and June 2024 were recruited. Inclusion criteria required no prior chemotherapy or radiotherapy. Ten pairs of PC tissues and adjacent normal tissues were collected. Informed consent was obtained from all participants, and the study was approved by the Ethics Committee of the General Hospital of Ningxia Medical University (Approval No.: KYLL-2022-0670), adhering to the principles of the Helsinki Declaration.

Cell culture and treatment

Human normal pancreatic ductal epithelial cells (HPDE6-C7; HTX1979, OTWO, Shenzhen) and PC cell lines AsPC-1 (CL-0027), PANC-1 (CL-0184), SW1990 (CL-0448) and PaCa-2 (CL-0627) (Procell, Wuhan) were cultured in RPMI-1640 (PM150110), DMEM (PM150210) and Leibovitz’s L-15 (PM151010) media, supplemented with 1% streptomycin/penicillin and 10% fetal bovine serum (FBS), at 37 °C in a humidified atmosphere containing 5% CO₂ and 95% O₂.

Cells in the exponential growth phase were seeded into 6-well plates at an optimal density. When cell confluence reached 70–80%, si-NC, si-DAZAP1-1, si-DAZAP1-2, NC, or DAZAP1 plasmids were transfected using Lipofectamine 3000 (L3000008, Invitrogen, USA), following the manufacturer’s protocol. SiRNA sequences are detailed in Table 1. For the apoptosis inhibitor intervention, cells were transfected with si-DAZAP1 plasmids for 48 h. Subsequently, cells were treated with Ferrostatin-1 (2 μM, HY-100579, MCE, Shanghai), Liproxstatin-1 (100 nM, HY-12726, MCE, Shanghai), Necrostatin-1 (30 µM, HY-15760, MCE, Shanghai) or Z-VAD-FMK (10 μM, HY-16658B, MCE, Shanghai) for 24 h. Cells were then harvested for subsequent experiments.

Table 1 Primer sequences of RT–qPCR and siRNA sequences

Immunohistochemistry (IHC)

Clinical tissue samples were fixed in 4% paraformaldehyde, followed by paraffin embedding, sectioning, dewaxing, antigen retrieval and blocking. The sections were then incubated with a primary antibody against DAZAP1 (ab237519, 1:200, Rabbit, Abcam, UK) and a secondary antibody (goat anti-rabbit IgG, ab150077, 1:1000, Goat, Abcam, UK). After antibody incubation, DAB and hematoxylin staining were performed, and stained sections were photographed. Criteria for positive staining: five representative high-magnification fields were selected for observation. Positive staining was defined as brown or yellow cytoplasmic staining. The integrated optical density (IOD) of immunostained samples was measured using image analysis software. The IHC score was calculated as IHC score = A × B (A: 0 for no positive cells, 1 for 10% positive cells, 2 for 11–50% positive cells, 3 for 51–80% positive cells and 4 for more than 80% positive cells; B: 0 for no staining, 1 for light staining intensity, 2 for moderate staining intensity and 3 for strong staining intensity).

Real-time quantitative reverse transcription PCR (RT–qPCR)

Total RNA from tissues and cells was extracted using TRIzol reagent (Thermo Fisher, New York) according to the manufacturer’s instructions. The RNA was reverse-transcribed into cDNA using a complementary synthesis kit. Target gene amplification was performed in a 25-µL reaction mixture under the following conditions: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 54.5 °C for 30 s and extension at 72 °C for 30 s. Primer sequences are listed in Table 1. Relative gene expression was calculated using the 2^−ΔΔCt.

Western blot

Total protein was extracted from tissues and cells using RIPA lysis buffer (P0013, Beyotime, Shanghai) supplemented with protease inhibitors. Protein concentrations were determined using a BCA protein assay kit (P00125, Beyotime, Shanghai). Equal amounts of protein were separated by 10% SDS–PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA at 25℃ for 1 h and incubated overnight at 4 °C with primary antibodies: DAZAP1 (ab237519, 1:2000), GPX4 (ab125066, 1:2000), ACSL4 (ab155282, 1:20,000) and GAPDH (ab181602, 1:10,000), with GAPDH serving as the internal control. Following three washes with TBST, membranes were incubated with HRP-conjugated goat anti-rabbit IgG (ab205718, 1:10,000, Abcam, UK) for 1 h at room temperature. Protein bands were visualised using the ECL chemiluminescence method, and band intensities were quantified using ImageJ software.

Cell counting kit-8 (CCK-8)

Cells were seeded into 96-well plates and subjected to plasmid transfection and inhibitor treatment. Subsequently, 10 μL of CCK-8 solution (KGA9310-500, KeyGen Biotech, Jiangsu) was added to each well. After a 2-h incubation, the absorbance at 450 nm was measured using a microplate reader (Multiskan FC, Thermo Fisher, Waltham, USA).

5-Ethynyl-2’-deoxyuridine (EdU) staining assay

Treated cells were seeded into 96-well plates at a density of 5 × 10³ cells per well. After 6 h of incubation, EdU solution (CA1170, Solarbio, Beijing) was added, and the cells were incubated for an additional 2 h. Cells were washed and fixed with 4% paraformaldehyde, followed by glycine incubation and membrane permeabilisation using 0.5% Triton X-100. Cells were then stained sequentially with 1 × Apollo reaction solution and 1 × Hoechst 33,342 reaction solution. An anti-fade reagent was applied before fluorescence imaging using a fluorescence microscope (MSD-S820, Murzider, Guangdong, China). The number of EdU-positive cells was recorded and statistically analysed.

Fe²⁺ content detection

Intervened cells were collected, and intracellular Fe²⁺ levels were measured using the Iron Assay Kit (ab83366, Abcam, UK) according to the manufacturer’s instructions.

Reactive oxygen species (ROS) detection

Cellular ROS levels were assessed using the ROS Assay Kit (S0033S, Beyotime, Shanghai). Following the intervention, cells were incubated with serum-free DCFH–DA at a final concentration of 10 μmol/L for 20 min in the dark. Excess dye was removed by washing with serum-free culture medium, followed by the addition of 500 μL of PBS. Fluorescence images were captured using a fluorescence microscope (MSD-S820, Murzider, Guangdong, China), and fluorescence intensity was quantified using Image J software.

Wound healing assay

Cells were seeded in 6-well plates and cultured overnight until 100% confluence was achieved. A straight scratch was created using a 20 μL capillary pipette along or perpendicular to the well's diameter. Detached cells were washed away with PBS, and images were captured at 0, 6, 12 and 24 h to monitor the wound healing process.

Transwell assay

Cell invasion was assessed using Transwell chambers with an 8 µm pore size pre-coated with Matrigel in a 24-well plate. The lower chamber was filled with medium containing 20% FBS. Intervened cells were resuspended in a serum-free medium and seeded into the upper chamber at a density of 1 × 10⁶ cells/mL. After 24 h of incubation, the chambers were removed and invaded cells were stained, photographed and counted.

Statistical analysis

Data analysis was conducted using SPSS 21.0 statistical software (SPSS, Inc., Chicago, IL, USA). Measurement data were expressed as mean ± SD. For normally distributed data, the Student’s t test was used for comparisons between two groups, and one-way ANOVA was applied for comparisons among multiple groups. Non-parametric tests were performed for non-normally distributed data, using the Mann–Whitney U test for two-group comparisons and the Kruskal–Wallis test for multiple-group comparisons. A P value < 0.05 was considered statistically significant.

Results

DAZAP1 was upregulated in PC

DAZAP1, an RNA-binding protein associated with the prognosis of PC [12], was investigated to assess its role in the development of PC. IHC analysis of DAZAP1 protein levels in 10 PC tissue samples and adjacent normal tissues showed significantly higher DAZAP1 expression in PC tissues compared to the control group (Fig. 1A, B). Consistently, RT–qPCR and western blot assays also confirmed the upregulation of DAZAP1 in PC tissues (Fig. 1C, D). These findings suggest that DAZAP1 overexpression may contribute to the progression of PC.

Fig. 1

DAZAP1 was upregulated in PC. A DAZAP1 protein levels were detected in 10 PC tissue samples and adjacent normal tissues (control) using IHC; B Bar chart of IHC scores for each group; C RT–qPCR was performed to determine the mRNA level of DAZAP1 in 10 PC tissue samples and adjacent normal tissues (Control); D western blot analysis was conducted to assess the protein level of DAZAP1 in 10 PC tissue samples and adjacent normal tissues (Control). ^*, ^**, ^*** P < 0.05, 0.01, 0.001

The effect of DAZAP1 on the proliferation of PC cells

The role of DAZAP1 in PC cell proliferation was explored through in vitro experiments. RT–qPCR and western blot analyses revealed significantly higher DAZAP1 expression in PC cell lines than in HPDE6-C7 cells, with PANC-1 cells showing the highest expression and AsPC-1 cells the lowest (Fig. 2A, B). Transfection with two DAZAP1-silencing plasmids effectively reduced DAZAP1 expression in PANC-1 and AsPC-1 cells, with si-DAZAP1-1 showing the strongest silencing effect, as confirmed by RT–qPCR and western blot (Figs. 2C, D, 3A, B). CCK-8 and EdU staining assays demonstrated that DAZAP1 silencing notably suppressed PANC-1 and AsPC-1 cell proliferation, with si-DAZAP1-1 exhibiting the greatest inhibitory effect (Figs. 2E, F, 3C, D). In contrast, DAZAP1 overexpression markedly promoted cell proliferation in both cell lines (Figs. 2E, F, 3C, D). These results indicate that DAZAP1 functions as an oncogene in PC.

Fig. 2

Effect of DAZAP1 on the proliferation of PANC-1 cells. A RT–qPCR and B western blot were performed to detect the mRNA and protein levels of DAZAP1 in human normal pancreatic ductal epithelial cells (HPDE6-C7) and PC cells (AsPC-1, PANC-1, SW1990, PaCa-2); (C) RT–qPCR and D western blot evaluated the transfection efficiency of si-DAZAP1-1, si-DAZAP1-2 and DAZAP1 overexpression plasmids in PANC-1 cells; E cell viability was assessed using the CCK-8 assay; F cell proliferation was detected using the EdU staining assay. ^*, ^**, ^*** P < 0.05, 0.01, 0.001

Fig. 3

Effect of DAZAP1 on the proliferation of AsPC-1 cells. A RT–qPCR and B western blot evaluated the transfection efficiency of si-DAZAP1-1, si-DAZAP1-2 and DAZAP1 overexpression plasmids in AsPC-1 cells; C cell viability was measured using the CCK-8 assay; D cell proliferation was detected using the EdU staining assay. ^*, ^**, ^*** P < 0.05, 0.01, 0.001

Downregulation of DAZAP1 promoted ferroptosis in PC cells

Given that DAZAP1 has been reported to promote HCC progression by inhibiting ferroptosis [11], its role in ferroptosis regulation in PC cells was further investigated. After downregulating DAZAP1 expression in PC cells, western blot revealed that DAZAP1 silencing significantly reduced GPX4 expression while increasing ACSL4 levels in PANC-1 cells treated with si-DAZAP1 compared to the si-NC group (Fig. 4A). In addition, intracellular Fe²⁺ content was significantly elevated following DAZAP1 knockdown (Fig. 4B). ROS detection assays further revealed increased intracellular ROS levels upon DAZAP1 inhibition (Fig. 4C). These findings suggest that the downregulation of DAZAP1 promotes ferroptosis in PC cells.

Fig. 4

Downregulation of DAZAP1 promoted ferroptosis in PC cells. A Western blot was used to detect the expression of ferroptosis marker proteins GPX4 and ACSL4 in cells after si-DAZAP1 treatment; B concentration of Fe²⁺ within cells was quantified using an iron assay kit; C ROS levels in cells were determined using the DCFH–DA method. ^*, ^**, ^*** P < 0.05, 0.01, 0.001

Downregulation of DAZAP1 inhibited proliferation of PC cells through ferroptosis

To elucidate the mechanism by which DAZAP1 regulates PC cell proliferation, ferroptosis inhibitors (Ferrostatin-1 and Liproxstatin-1), a necroptosis inhibitor (Necrostatin-1), and a pan-caspase apoptosis inhibitor (Z-VAD–FMK) were applied to DAZAP1-silenced PC cells. The proliferation-suppressing effect of si-DAZAP1 on PC cells was alleviated by all four apoptosis inhibitors, with ferroptosis inhibitors showing the most pronounced rescue effect (Fig. 5A–C). These findings indicate that DAZAP1 downregulation inhibits PC cell proliferation primarily through the ferroptosis pathway, although other apoptotic mechanisms may also be involved.

Fig. 5

Downregulation of DAZAP1 inhibited the proliferation of PC cells through ferroptosis. Following the transfection of PANC-1 cells with si-NC and si-DAZAP1 plasmids, ferroptosis inhibitors (Ferrostatin-1 or Liproxstatin-1), necroptosis inhibitor (Necrostatin-1), and pan caspase apoptosis inhibitor (Z-VAD–FMK) were individually added for intervention. A Cell viability was assessed using the CCK-8 assay; B statistical graph of cell proliferation levels across the groups; C cell proliferation was detected using the EdU staining assay. ^*, ^**, ^*** P < 0.05, 0.01, 0.001

Downregulation of DAZAP1 inhibited migration and invasion of PC cells through ferroptosis

The effect of DAZAP1 on PC cell migration and invasion was further explored. Wound healing assays demonstrated that DAZAP1 knockdown significantly suppressed cell migration, an effect partially reversed by the addition of apoptosis inhibitors. Among these, ferroptosis inhibitors exerted the strongest migration-promoting effects (Fig. 6A, B). Similarly, Transwell invasion assays showed that DAZAP1 knockdown reduced PC cell invasion, which was markedly reversed by ferroptosis inhibitor treatment, promoting cell invasion (Fig. 6C, D). These results suggest that DAZAP1 regulates PC cell migration and invasion through the ferroptosis pathway.

Fig. 6

Downregulation of DAZAP1 inhibited the migration and invasion of PC cells through ferroptosis. After transfecting the si-NC and si-DAZAP1 plasmids into PANC-1 cells, ferroptosis inhibitors (Ferrostatin-1 or Liproxstatin-1), necroptosis inhibitor (Necrostatin-1), and pan caspase apoptosis inhibitor (Z-VAD–FMK) were added for intervention, followed by the detection of cell migration and invasion abilities. A Migration ability of cells in each group was detected using the wound healing assay; B statistical graph of wound healing area in each group; C invasion ability of cells were detected using the transwell assay; D statistical graph of cell invasion ability in each group. ^*, ^**, ^*** P < 0.05, 0.01, 0.001

Discussion

PC is a highly fatal malignancy [2], with a poor prognosis due to its late-stage diagnosis and aggressive clinical progression. Patients with PC typically present with non-specific symptoms, such as weight loss, fatigue, nausea, anorexia and pain in the lower back and waist. By the time of diagnosis, many patients already have locally advanced disease or distant metastases, limiting treatment options and resulting in poor survival rates [13]. Therefore, identifying novel therapeutic targets is crucial for improving PC outcomes. This study demonstrated that DAZAP1 is significantly overexpressed in PC, suggesting its role as an oncogene. DAZAP1 downregulation was found to promote ferroptosis, inhibiting PC cell proliferation and metastasis, highlighting its potential as a therapeutic target for PC.

DAZAP1 is a highly conserved RNA-binding protein expressed predominantly in the testes, with lower expression in tissues, such as the heart, liver, spleen, lung and brain [5, 7]. Despite limited research on DAZAP1, studies have primarily focused on its structure, function and involvement in non-neoplastic diseases. For instance, DAZAP1 knockout mice exhibit severe defects in spermatogenesis and cell growth, underscoring its essential role in reproduction and somatic cell development [14]. In addition, Jing Mao et al. reported that DAZAP1 is implicated in osteoarthritis, where miR-320a inhibits DAZAP1 expression and the MAPK pathway, alleviating IL-1β-induced osteoarthritis [15]. These findings emphasise the critical role of DAZAP1 in human physiology, where its dysregulation can lead to abnormal cell growth and even cancer development. While limited research has investigated DAZAP1's role in cancer, existing studies have linked it to HCC, leukaemia, clear cell renal cell carcinoma and multiple myeloma [8, 16,17,18]. However, its functional mechanism in PC remains unexplored. This study revealed that DAZAP1 is significantly upregulated in PC tissues and cell lines. Silencing DAZAP1 significantly inhibited PC cell proliferation, migration and invasion, primarily through the ferroptosis pathway. These findings suggest that DAZAP1 acts as an oncogene in PC and may represent a promising therapeutic target for future PC treatment strategies.

Cell death is an inevitable cellular fate, with necroptosis representing a form of regulated necrosis that occurs when apoptosis is inhibited [19]. Necrostatin-1, a statin drug that prevents cell necrosis, is currently the most widely used necroptosis inhibitor [20]. Apoptosis is an autonomous and orderly form of cell death that occurs under genetic regulation to maintain internal environmental stability [21]. The Caspase protein family plays a central role in apoptotic signalling pathways. Z-VAD–FMK, a broad-spectrum caspase inhibitor, is irreversible and cell permeable, enabling it to suppress inflammation and apoptosis [22]. In this study, both Necrostatin-1 and Z-VAD–FMK reversed the apoptosis-promoting effects of si-DAZAP1 in PC cells, though their efficacy was notably lower than that of Ferrostatin-1 or Liproxstatin-1, which are ferroptosis inhibitors. Ferroptosis, an iron-dependent form of programmed cell death, involves iron-catalysed lipid peroxidation of polyunsaturated fatty acid-containing phospholipids, leading to membrane damage and cell death [23]. Ferrostatin-1 scavenges peroxyl radicals, interrupting lipid peroxidation chain reactions and effectively suppressing ferroptosis [24]. Similarly, Liproxstatin-1, a spiroquinoxaline amine derivative, inhibits ferroptosis through a comparable mechanism but with superior absorption and distribution properties, allowing for efficacy at lower doses [25]. Our findings showed that Ferrostatin-1 and Liproxstatin-1 significantly counteracted the pro-apoptotic effects of si-DAZAP1 in PC cells, far outperforming inhibitors targeting other cell death pathways. This result underscored ferroptosis as the primary mechanism through which DAZAP1 regulates PC cell progression. Research on DAZAP1’s role in ferroptosis regulation remains limited, especially in cancer contexts. Only one previous study reported that DAZAP1 suppresses ferroptosis in HCC by binding to SLC7A11 mRNA, stabilising its expression, and thereby promoting cancer cell proliferation, migration and invasion [11]. This finding aligns with our study, supporting DAZAP1's oncogenic role in PC. Given that Ferrostatin-1 and Liproxstatin-1 inhibit lipid peroxidation, it is plausible that DAZAP1 modulates ferroptosis in PC cells by influencing the stability of mRNAs involved in lipid peroxidation processes. However, the precise regulatory mechanism requires further investigation.

Targeting cell death pathways, particularly ferroptosis, represents a promising therapeutic strategy for cancer treatment. This study demonstrated that modulating DAZAP1 expression and ferroptosis significantly impacts PC cell progression, offering a potential direction for developing novel therapeutic approaches for PC.

Conclusion

This study, through clinical tissue analysis and in vitro experiments, identified that DAZAP1 promotes the proliferation and metastasis of PC cells by inhibiting ferroptosis. Conversely, the downregulation of DAZAP1 expression significantly impedes the progression of PC cells. Future studies will explore the potential regulatory role of DAZAP1 in PC progression through animal models. In addition, the molecular mechanisms underlying DAZAP1’s regulation of ferroptosis will be investigated in greater detail. These findings highlight new potential therapeutic targets and open avenues for further research in the clinical treatment of pancreatic cancer.