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Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors of the digestive system. Due to its deep anatomical location and occult clinical manifestations, there are no effective methods for early diagnosis, resulting in a high mortality rate (1). Despite advances in the diagnosis and treatment of pancreatic cancer, with a 5-year survival rate of 9%, PDAC is predicted to become the second leading cause of cancer-related mortality worldwide by 2030 (2).

KRAS mutations are considered to initiate PDAC and the frequency of KRAS mutations in PDAC ranges from 90–95% (3). The dominance of KRAS mutations suggests that targeted therapy against the Ras signaling network may be an effective treatment modality for PDAC. To date, several targeted therapies for PDAC have been approved (4). The first approved was EGFR inhibitor erlotinib, which in combination with gemcitabine had a survival benefit compared to gemcitabine alone, but was effectively abandoned by the community after negative data emerged on EGFR-targeted therapy for KRAS mutant colorectal cancer (5). Then, the TRK kinase inhibitors larotrectinib and entrectinib were approved for solid tumors containing the NTRK-fusion gene. However, the NTRK-fusion gene occurs in only 0.5% of PDAC and the majority of PDAC without the NTRK-fusion gene has not been systematically evaluated (6). Finally, the PARP inhibitor olaparib can extend progression free survival but not overall survival in patients of late-stage PDAC with germline mutations in BRCA1 or BRCA2 (~7.5%) (7). In conclusion, their clinical activity has been disappointing so far. Therefore, there is an urgent need to identify new therapeutic modalities for pancreatic cancer.

It is well known that the vast majority of mutations in Ras are missense mutations in three hotspot residues, G12, G13 and Q61. The order of frequency observed at the G12 is G12D, G12V, G12C, G12A, G12S and G12R and G12C mutation prevalent in lung cancer and G12D being the most common in PDAC. In fact, KRAS G12D (KRASG12D) is one of the most important tumor therapeutic targets (8).

A previous study demonstrated that wild-type KRAS (KRASWT) has a survival advantage in PDAC and patients with KRASWT have a longer overall survival time (9). Patients with mutated KRAS have been shown to have worse survival and a shorter overall survival following gemcitabine-based first-line chemotherapy, regardless of age, sex, tumor stage, tumor morphology, or chemotherapy regimen. KRASWT can antagonize the effects of mutated KRASG12D, resulting in inefficient cellular transformation (10). In addition, the increased dose of mutated KRASG12D is accompanied by the deletion of KRASWT in PDAC (11). This suggests that KRASWT may exert a potential tumor suppressive effect through certain pathways, but the mechanism has not yet been elucidated.

In view of the characteristics of KRAS wild-type and mutant genes in cancer, the present study overexpressed these two genes. It focused on the differences in biological behavior between KRASWT and KRASG12D and explored the pathway and mechanism in pancreatic cancer and provides theoretical basis for the study of the KRAS gene.

Materials and methods

Cells and lentiviruses

A PANC-1 human pancreatic ductal adenocarcinoma cell line was provided by the Sichuan Institute of Medical Imaging, the lentivirus was purchased from Shanghai GeneChem Co., Ltd., and KRASWT and KRASG12D cells were constructed by the authors.

Lentiviral infection

Lentiviruses expressing KRASWT and KRASG12D were generated and purchased from Shanghai GeneChem Co., Ltd. Lentiviruses were infected into cells at a multiplicity of infection (MOI) of 200 for KRASG12D and 50 for KRASWT. Briefly, cell suspensions of 3–5×104/ml were prepared in 96-well plates (100 µl per well) and culture was performed for 18–24 h. According to cell MOI and virus titer, KRASWT and KRASG12D-overexpressing viruses were added to the wells. Culture was performed at 37°C for 12–16 h, after which the medium was replaced with conventional medium. Additional culture was performed for a further 3–4 days and the infection efficiency was observed in >10 fields of view under a fluorescence microscope (magnification, ×20; Leica DMIL LED; Leica Microsystems, Inc.). The medium was then replaced with medium containing 2 µg/ml purinomycin for further culture (37°C, 5% CO2) for over a week and follow-up experiments were carried out.

Cell culture

Cells were cultured in DMEM (Hyclone; Cytiva) containing 10% fetal bovine serum(Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, 100 µg/ml streptomycin in a humidified incubator at 37.5°C with 5% CO2. Trypsin (0.25%) was used for digestion and subculture at a rate of 1:3. KRASWT and KRASG12D cells were cultured and subcultured with DMEM containing 2 µg/ml purinomycin, which was replaced with conventional DMEM during the subsequent experiments.

Reverse transcription-quantitative (RT-q) PCR

The expression of the KRAS gene in transfected KRASWT and KRASG12D cells was detected using RT-qPCR. When cell density was ~80%, total RNA was extracted by TRIzol® (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.), cDNA was synthesized (RevertAid First Strand cDNA Synthesis Kit; cat. no. K1622; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and qPCR (Power Up™ SYBR™ Green; cat. no. A25742; Applied Biosystems; Thermo Fisher Scientific, Inc.) was performed. All steps were carried out according to the manufacturers' protocols. qPCR was conducted using the Light Cycler 96 (Roche Diagnostics) under the following conditions: 2 min at 50°C, 2 min at 95°C, followed by 40 cycles at 95°C for 15 sec, 57°C for 15 sec and 72°C for 1 min. Fold change in expression was calculated using the standard 2-ΔΔCq formula (12). GAPDH was used as an internal loading control and the experiment was repeated three times. The primer sequences were as follows, GAPDH: 5′-ACTAGGCGCTCACTGTTCTCT-3′ forward and 5′-GGAATTTGCCATGGGTGGAA-3′ reverse; KRASWT: 5′-GCCTGCTGAAAATGACTGA-3′ forward and 5′-CTCCTCTTGACCTGCTGTG-3′ reverse; KRASG12D: 5′-ACACAAAACAGGCTCAGGA-3′ forward and 5′-GTCGGATCTCTCTCACCAA-3′ reverse.

CCK-8 assay

Cell suspensions of 2×104/ml were prepared in 96-well plates (100 µl per well) and five repeats in each group were detected at 24, 48, 72, 96 and 120 h, respectively. CCK-8 reagent (10 µl) was added to each well and incubated at 37°C for 1 h. Absorbance was then determined at 450 nm by SpectraMax Paradigm microplate reader (Molecular Devices. Inc.).

Colony formation assay

A total of 200 cells/well were inoculated in a 6-well plate and cultured at 37°C with 5% CO2 for 2 weeks. When there were visible colonies (>50 cells), cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature and stained with 0.1% crystal violet for 15 min at room temperature. The dying solution was washed with clear water and dried naturally. The colonies were scanned and their number was counted.

Wound healing assay

Cell suspensions of 1×105/well were prepared in a 6-well plate and cultured in a medium containing 10% FBS. The cells were scratched when the monolayer fusion was ~90%. Horizontal lines were drawn at the 6-well plate, cells were washed with PBS and then a position selected where the scratches were clear, followed by further culture with serum-free medium (37°C, 5% CO2). Images of the same positions were captured at 0, 24, 48, 72, 96 and 120 h respectively.

Transwell assay

Cell suspensions of 2×105/ml were prepared in serum-free medium. A total of 500 µl medium with 10% FBS was added to the lower Transwell chamber and 100 µl cell suspension was added to the upper chamber, followed by culture for 48 h (37°C, 5% CO2). The Transwell chamber was cleaned with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature and stained with 0.1% crystal violet for 15 min at room temperature.

Tumor formation in nude mice

A total of 30 male nude mice (BALB-/c-nu, specific pathogen-free; Beijing Hufukang Biotechnology Co., Ltd.) aged 4–6 weeks (16–18 g) were randomly divided into three groups, termed PANC-1, KRASWT and KRASG12D and raised under the same conditions (room temperature ~26–28°C; relative humidity ~40–60% with ventilation at ~10–15 times/h and a 10/14 h light/dark cycle). Cell suspensions of 3–5×106/ml were prepared in each group, a total of 100 µl of cell suspension was collected and inoculated into the underarm skin of nude mice. A subcutaneous mass was observed ~1 week after inoculation, the long (a) and short diameter (b) of the tumor were measured (every other day, a total of 12 times) and the tumor volume was calculated according to the formula ab2/2. Following sacrifice by cervical dislocation (mortality ascertained by the observation of respiratory, heartbeat, pupil and nervous reflex), the tumors were removed and stored in liquid nitrogen. The present study was approved by the Ethics Committee of North Sichuan Medical College (approval no. 2022035) and all procedures were carried out in accordance with relevant guidelines and regulations.

Western blotting

Cells were lysed with lysate (1 ml protein lysate mixed with 10 µl protease inhibitors) and the lysates were centrifuged at 1,3500 × g at 4°C for 10 min. The protein concentration was detected using a BCA protein kit, according to the manufacturer's instructions. Protein lysates (30 g) were separated via 10% SDS-PAGE and transferred to PVDF membranes, which were blocked with 5% skimmed milk for 1 h at room temperature. The membranes were subsequently incubated overnight at 4°C with the following primary antibodies: Anti-e-cadherin (1:5,000; cat. no. 60335-1; ProteinTech Group, Inc.), anti-α-E-catenin (1:3,000; cat. no. 66221-1; ProteinTech Group, Inc.), anti-MMP-9 (1:1,000; cat. no. 13667; CST), anti-MMP-3 (1:1,000; cat. no. 14351; CST), anti-STAT3 (1:1,000; cat. no. 22785, ZenBio) and anti-phosphorylated (p-) STAT3 (1:2,000; cat. no. 9145; CST). Following the primary antibody incubation, the membranes were washed three times with PBS for 15 min and incubated with HRP-conjugated goat anti-rabbit (1:5,000; cat. no. BA1039; Wuhan Boster Biological Technology, Ltd.) or anti-mouse (1:5,000; cat. no. BA1038; Wuhan Boster Biological Technology, Ltd.) secondary antibodies for 2 h at room temperature, then washed with PBS 3 times for 15 min. Protein bands were visualized with an ECL development kit (MilliporeSigma) using an enhanced chemiluminescence detection system (FUSION Fx; Vilber Lourmat).

Immunohistochemistry

Tumor tissues were fixed in 10% paraformaldehyde for >12 h at room temperature, were paraffin-embedded and sectioned (3–5 µm), and the sections were then placed into xylene for dewaxing. A descending alcohol series was added for dehydration. Sections were placed in citrate buffer (pH 6.0) for antigen retrieval (95°C for 15 min) and the endogenous peroxidase was blocked using an endogenous peroxidase blocker (cat. no. SP-9000; OriGene Technologies, Inc.) at 25°C for 10 min. Sections were blocked with goat serum (cat. no. SP-9000; OriGene Technologies, Inc.) at 25°C for 10 min and then incubated with a primary antibody (α-E-catenin; 1:500; cat. no. 66221-1; ProteinTech Group, Inc.; e-cadherin; 1:2,000; cat. no. 60335-1; ProteinTech Group, Inc.; MMP-9; 1:300, cat. no. 13667; CST) for 1 h at 37°C, washed with PBS three times and incubated with goat anti-rabbit (1:5,000; cat. no. BA1039; Wuhan Boster Biological Technology, Ltd.) or anti-mouse (1:5,000; cat. no. BA1038; Wuhan Boster Biological Technology, Ltd.) secondary antibodies at 25°C for 15 min. Subsequently, sections were incubated with 3,3′-diaminobenzidine substrate for 5–10 min at room temperature, stained for 30 sec with hematoxylin and differentiated for 2 sec with 1% hydrochloric acid alcohol at room temperature followed by gradient alcohol series for dehydration and xylene clearing before being sealed with neutral gum and >10 fields of view were observed under a light microscope (magnification, ×5; ECLIPSE 80i; Nikon Corp.).

Statistical analysis

All statistical analysis was performed by SPSS 19.0 (IBM Corp.) and GraphPad Prism 7 (GraphPad Software, Inc.) and the data are expressed as the mean ± standard deviation. One-way analysis of variance was used for multiple group comparisons and the post-hoc test was performed by the LSD method. P<0.05 was considered to indicate a statistically significant difference.

Results

KRASWT gene inhibits the proliferation of continuous tumor-cell line (PANC-1)

The transfection effect is shown in Fig. 1A and the verification of transfection effect was performed using RT-qPCR assay (Fig. 1B). CCK-8 and clone formation assays were used to detect the proliferation ability of pancreatic cancer following the overexpression of the KRAS gene. The CCK-8 assay results showed that, compared with the PANC-1, the proliferation of KRASWT was reduced and the proliferation of KRASG12D was not significantly changed (Fig. 2), which was consistent with the results of the colony formation assay (Fig. 3). These results indicated that the KRASWT gene could inhibit the proliferation of pancreatic cancer cells.

Figure 1. Efficiency of lentiviral transfection after a week of culture in medium containing 2 µg/ml purinomycin and (A) detection of infection efficiency under a microscope. (B) Reverse transcription-quantitative PCR was used to measure the expression of the KRAS gene. **P<0.01. Scale bar=50 µm. KRAS, Kirsten rat sarcoma virus; WT, wild-type.

Figure 2. CCK-8 assay was used to detect cell proliferation. As compared with PANC-1 group, the proliferation of KRASWT group was decreased and no statistically significant differences in the proliferation of KRASG12D group were observed (**P<0.01). KRAS, Kirsten rat sarcoma virus; WT, wild-type.

Figure 3. Colony formation assay was used to detect cell proliferation. Compared with PANC-1 group, the proliferation of KRASWT group was decreased (P<0.01) and the changes in the proliferation of KRASG12D group were not statistically significant. (A) Representative images from one of the experiments. (B) Statistical data from three experiments presented as the mean ± standard deviation (**P<0.01). KRAS, Kirsten rat sarcoma virus; WT, wild-type.

KRASWT gene inhibits the migration and invasion of PANC-1

Next, the effect of the KRAS gene on the invasion and migration of pancreatic cancer cells was investigated. Transwell and wound healing assays were performed to detect cell invasion and migration capacity. As shown in Fig. 4, the migration of KRASG12D cells was enhanced and that of KRASWT cells was weakened, as compared with those of PANC-1 cells after 48 h and the difference was more pronounced after 120 h. Similarly, a Transwell assay revealed an enhanced invasion of KRASG12D cells and decreased invasion of KRASWT cells (Fig. 5). The results suggested that the KRASWT gene can inhibit the migration and invasion of pancreatic cancer.

Figure 4. Wound healing assay was used to detected cell migration. As compared with PANC-1 group, the migration of KRASWT group was decreased and that of KRASG12D group was increased. Scale bar=400 µm. KRAS, Kirsten rat sarcoma virus; WT, wild-type.

Figure 5. Transwell assay was used to detected cell invasion. Compared with PANC-1 group, the invasion of KRASWT group was weakened and the invasion of KRASG12D group was enhanced. (A) Representative images from one of the experiments. (B) Statistical data from three experiments presented as the mean ± standard deviation (n=3; **P<0.01). Scale bar=400 µm. KRAS, Kirsten rat sarcoma virus; WT, wild-type.

KRASWT gene may inhibit the migration and invasion of PANC-1 through the Wnt/β-catenin pathway

In order to explore the mechanism through which KRAS regulates pancreatic cancer cell invasion and migration, some signaling pathway molecules that serve key roles in cell invasion and migration were examined. E-cadherin and α-E-catenin, which are key regulators of the Wnt/β-catenin pathway, as well as MMP-3, MMP-9, STAT3 and p-STAT3 were selected for western blotting. As shown in Fig. 6, compared with PANC-1, all the expressions of proteins of KRASWT were upregulated and that of KRASG12D was not significantly changed. These data suggested that the inhibition of migration and invasion of PANC-1 cells by the KRASWT gene may be mediated by the Wnt/β-catenin pathway.

Figure 6. Protein expression detected by western blotting. As compared with PANC-1 group, the protein expression of KRASWT group was increased and no significant difference was observed in KRASG12D group. KRAS, Kirsten rat sarcoma virus; WT, wild-type; p-, phosphorylated.

KRASWT gene inhibits tumor growth in nude mice

In order to explore the different effects of KRAS on pancreatic cancer proliferation, migration and invasion in vivo and in vitro, tumor growth curves were drawn according to the average tumor volume measured at each monitoring point. The results showed that, compared with PANC-1, the average tumor volume in the KRASWT was decreased, while that of KRASG12D was increased, indicating that the KRASWT gene can inhibit the proliferation of pancreatic cancer in vivo, while the mutant KRAS gene can promote the proliferation of pancreatic cancer (Fig. 7).

Figure 7. Tumor growth in nude mice. (A) Each group comprised 10 nude mice and the average tumor volume was calculated. Compared with PANC-1 group, the average tumor volume of KRASWT group decreased and that of KRASG12D group increased (**P<0.01). (B) Images of tumors in nude mice. (C) Changes in body weight of nude mice. KRAS, Kirsten rat sarcoma virus; WT, wild-type.

KRASWT gene inhibits protein expression in nude mouse tumors

Immunohistochemistry was performed on tumors from nude mice and the results showed that, compared with PANC-1, the expression of E-cadherin, α-E-catenin and MMP-9 of KRASG12D exhibited no significant changes. However, the expression of E-cadherin, α-E-catenin and MMP-9 in KRASWT was significantly upregulated (Fig. 8), which suggested that the inhibition of the migration and invasion of PANC-1 cells by KRASWT gene may be mediated by the Wnt/β-catenin pathway.

Figure 8. Immunohistochemistry was performed to detect the protein expression of tumors in nude mice. Compared with PANC-1 group, the proteins of KRASWT group were all upregulated, while no significant differences were observed in KRASG12D group). Scale bar=400 µm. KRAS, Kirsten rat sarcoma virus; WT, wild-type.

Discussion

KRAS mutations are considered to be the driving event in the development of pancreatic cancer. Studies (13,14) have shown that KRAS mutations can be detected in 90% of pancreatic ductal intraepithelial neoplasia (PANIN), 45–60% of pancreatic ductal papillary myxomas and >90% of pancreatic adenocarcinoma, with 84% of the mutations leading to the substitution of a single amino acid at the G12 site, among which G12D is the most common (42%).

The G12D mutant subtype is associated with a decreased overall survival. Windon et al (9) suggested that KRASWT may have a survival advantage in PDAC, as patients with KRASG12D exhibited a worse survival rate and shorter overall survival time following gemcitabine-based first-line chemotherapy (11.6 vs. 5.6 months, P=0.03). Ambrogio et al (10) suggested that KRASWT can antagonize the effect of KRASG12D and the loss of KRASWT can accelerate cell proliferation and tumor progression through the increase of the GTPase level of KRASG12D. KRASWT can also disrupt the inhibitory response of KRASG12D to MEK. According to Mueller et al (15), KRASWT is absent to varying degrees during tumor development and is associated with a high mRNA expression of the KRASG12D gene. Mueller et al also performed a microdissection of 19 patients with low-grade pancreatic intraepithelial neoplasia and deep sequencing of KRAS exon 2, confirming that KRASG12D significantly increases metastatic potential, which explains the high incidence of early progression and metastasis in PDAC. The above studies show that KRASWT exerts different degrees of tumor suppression. Therefore, the KRASWT gene was directly overexpressed in the present study to investigate the differences in biological behaviors and the mechanism of action.

The results of CCK-8 and clone formation assay were consistent. As compared with PANC-1, the proliferation of KRASWT cells was weakened, while no significant difference was observed in KRASG12D, indicating that the KRASWT gene can reduce the proliferation of PANC-1 (Figs. 2 and 3). At the same time, a tumor formation model of pancreatic cancer was established in nude mice (Fig. 7). In vivo, the average tumor volume in the KRASWT group was smaller than PANC-1 group and KRASG12D group, which was consistent with the results of the in vitro CCK-8 and clone formation assays, indicating that KRASWT gene can reduce the proliferation of pancreatic cancer cells in vivo and in vitro. In addition, it was found that the tumor volume of the KRASG12D group was larger than that of the PANC-1 group, indicating that the mutant KRAS gene can promote the proliferation of pancreatic cancer in vivo. However, the promotion effect was not obvious in vitro.

Wound healing and Transwell assays were then performed to detect migration and invasion. As shown in Figs. 4 and 5, as compared with PANC-1 group, the migration and invasion of KRASG12D group was enhanced, while that of KRASWT group was significantly weakened, suggesting that the KRASWT gene could inhibit the migration and invasion of pancreatic cancer.

Epithelial-to-mesenchymal transition (EMT) is a progressive process of phenotype conversion from epithelial to mesenchymal (16,17). Among them, E-cadherin ensures the integrity of epithelial phenotypes by influencing cell polarity and tissue integrity to form stable adhesion (18), which is a key event in the EMT process (19,20). α-E-catenin also plays an important role in the regulation and coordination of intracellular adhesion and is a key regulator of the Wnt/β-catenin pathway (21). α-E-catenin is a major sensor of mechanical force in adherens junctions and its cytoplasmic domain is connected to the actin cytoskeleton by β-catenin and α-E-catenin. α-E-catenin can bind to E-cadherin to form intercellular adhesion and mediate the invasion and migration of tumor cells (22–24). E-cadherin and α-E-catenin were therefore selected to verify whether the Wnt/β-catenin pathway plays a role in inhibiting migration and invasion. According to western blotting (Fig. 6) and immunohistochemistry (Fig. 8), as compared with PANC-1, there was no significant difference in the expression of E-cadherin and α-E-catenin in KRASG12D. The upregulated expression of E-cadherin and α-E-catenin in KRASWT indicated that the KRASWT gene may play a potential role in inhibiting the invasion and migration of pancreatic cancer through the Wnt/β-catenin pathway. A previous study revealed that the expression levels of β-catenin and RAS are increased in gemcitabine-resistant pancreatic cancer cells (25); inhibition of Wnt/β-catenin and RAS/ERK pathways may provide a therapeutic strategy for gemcitabine-resistant pancreatic cancer. In fact, Wnt/β-catenin signaling and RAS/ERK pathways not only dominate gemcitabine resistance, it is possible that Wnt/β-catenin signaling directly responds to mutations in the RAS gene, which the present study confirmed.

In the process of tumor development, the destruction of basement membrane is an important step for tumor invasion and metastasis (26). In addition to degrading various protein components of extracellular matrix, some MMPs, MMP-3, MMP-9, MMP-14 and MMP-2 can also induce EMT or its related processes (27). A previous study demonstrated that MMP-9 is highly expressed in non-small cell lung, cervical and ovarian cancer, among others, and has become one of the important factors related to cancer metastasis and invasion (28). MMP-3 protein breaks down a variety of extracellular matrix molecules and cleaves various adhesion molecules, growth factors and other MMPs (29). MMP-3 can activate MMP-1 and other family members and its overexpression is associated with the growth and metastasis of various cancers, including breast cancer (30). When stimulated by external signals, STAT3 migrates to the nucleus in the form of activated p-STAT3, which stimulates cell growth and angiogenesis, activates the transcription of target genes and regulates cell proliferation, differentiation and metastasis (31,32). Studies have shown that STAT3/p-STAT3 is highly expressed in a variety of tumors and its activation product, p-STAT3, is associated with tumor grade and patient prognosis (33,34). As a major mediator of the JAK/STAT pathway, STAT3 may be an important regulator of tumor progression by enhancing aggressiveness or promoting EMT (35,36).

These studies indicate that the high expression of MMP-3, MMP-9, STAT3 and p-STAT3 is an important manifestation of tumor invasion and metastasis. The present results showed that, as compared with PANC-1 group, the migration and invasion of KRASWT group was weakened. However, western blotting and immunohistochemistry results showed that the expression of MMP-3, MMP-9, STAT3 and p-STAT3 in KRASWT group were upregulated, which was inconsistent with previous reports. The present study hypothesized that the inhibition of pancreatic cancer cell invasion and migration by the KRASWT gene may not be due to the degradation of extracellular matrix or the inhibition of the function of the JAK/STAT pathway, it is also possible that the inhibitory effect of Wnt/β-catenin pathway on the invasion and migration of pancreatic cancer cells was greater compared with that of extracellular matrix or JAK/STAT pathway and the reasons need to be further studied.

In conclusion, the present study investigated the biological effects of KRASWT on the proliferation and migration of pancreatic cancer cells, it was found that KRASWT could inhibit the proliferation of pancreatic cancer in vivo and in vitro and mutated KRAS could enhance the proliferation of pancreatic cancer in vivo, but this promotion was not obvious in vitro. Meanwhile, KRASWT can inhibit the migration and invasion of pancreatic cancer, which may be achieved through the Wnt/β-catenin pathway. The present study provided a theoretical and experimental basis for the strategy of KRAS gene therapy for pancreatic cancer.

Acknowledgements

Not applicable.

Funding

Funding was provided by the Science and Technology Innovation project of Sichuan Province (grant no. 2021033) and the Nanchong City School cooperation project (grant nos. 22SXCXTD0002 and 22SXQT0100).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

In the planning and execution of this paper, the authors XH and CZ designed the study, XH completed the main writing and most of the experiments, CZ and RZ completed the feeding, data collection and statistical analysis of the body weight and tumor data of the nude mice., BM and JY participated in the data statistics. XH and BM confirm the authenticity of all raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of North Sichuan Medical College (approval no. 2022035) and all procedures were carried out in accordance with relevant guidelines and regulations.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References