IL20RB promotes pancreatic cancer progression by activating NF- kB signalling and promoting EMT

IL20RB promotes pancreatic cancer progression by activating NF- kB signalling and promoting EMT | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article IL20RB promotes pancreatic cancer progression by activating NF- kB signalling and promoting EMT Wanbai Ruan, Junfen Li, Yanmei Yin, Lei Peng, Kexiang Zhu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5929013/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Currently, the pathogenesis and biological features of pancreatic cancer are not fully understood. Interleukin-20 receptor subunit beta (IL20RB) is a risk factor for poor prognosis in a variety of solid tumours, including breast cancer. However, the biological characteristics of IL20RB in pancreatic cancer and its impact on patient prognosis remain unclear. The aim of this study was to investigate the effect of IL20RB on the biological characteristics of pancreatic cancer and to explore the underlying mechanisms. Methods The expression of IL20RB in pancreatic cancer and its effect on the prognosis of pancreatic cancer patients were analysed by The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Immunohistochemistry and Western blot were used to detect IL20RB expression in pancreatic cancer tissues. Pancreatic cancer cell lines PANC-1 and BxPC-3 with stable knockdown of IL20RB were constructed.The effects of IL20RB on the proliferation, migration, apoptosis and cell cycle of pancreatic cancer cells were analysed by CCK-8 assay, plate clone formation assay, wound healing assay, Transwell assay and flow cytometry. Transcriptome sequencing analysis and Western blot were used to detect changes in NF-κB signalling and key molecules during EMT after IL20RB knockdown. Results IL20RB is highly expressed in pancreatic cancer and that patients with high expression have a poor prognosis. Knockdown of IL20RB significantly inhibited the proliferation and migration of pancreatic cancer cells, induced cell apoptosis, and resulted in cell arrest in the S phase. Gene set enrichment analysis (GSEA) and transcriptome analysis showed that IL20RB regulated related signalling pathways in pancreatic cancer cells, including Nuclear factor-κb (NF-κB) and Epithelial-mesenchymal transition(EMT). After the knockdown of IL20RB, the expression of EMT-related protein E-cadherin was increased, and the expression of N-cadherin, Vimentin and Snail was decreased. At the same time, the expression levels of IKKα/β and P-NF-κB P65 were significantly reduced after IL20RB knockdown. The expression level of NF-κB P65 was not changed considerably, and the ratio of P-NF-κB P65/NF-κB P65 was significantly decreased. Conclusion IL20RB is highly expressed in pancreatic cancer and is associated with poor prognosis in patients. IL20RB may promote the proliferation, migration and apoptosis of pancreatic cancer cells by activating the IKKα/β/NF-κB P65 signalling pathway to promote the EMT process. interleukin-20 receptor subunit beta pancreatic cancer biological role nuclear factor-κb epithelial-mesenchymal transition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Globally, pancreatic cancer (PC) is the seventh most common cause of cancer-related mortality [1] . The incidence of pancreatic cancer in developed countries is approximately four to five times higher than in developing countries [1, 2] . The peak age of onset is 65–69 years for men and 75–79 years for women [3] . In recent years, there has been a notable shift in the age at which pancreatic cancer is diagnosed, with a higher incidence observed in younger individuals, particularly women [4–6] . Surgery remains the primary treatment for pancreatic cancer, with a high success rate. However, only 15–20% of patients are eligible for surgery at diagnosis [7] . Nearly three-quarters of patients who undergo surgical resection will experience recurrence within two years, suggesting the presence of micrometastatic lesions [8] . Preclinical studies have also demonstrated that circulating disseminated pancreatic cancer cells can be observed prior to limited invasion of the primary tumour [9] . Currently, the poor prognosis of pancreatic cancer patients is primarily attributed to a lack of comprehensive understanding of the biological characteristics and mechanisms of pancreatic cancer cells, particularly in the transformation of normal pancreatic tissues to cancerous processes. There are still numerous undefined aspects of this process. Therefore, it is significant to study pancreatic cancer's biological characteristics and mechanisms. IL20RB is located at position 3q22.3 and has a molecular weight of approximately 35,076 Da. As a subunit of the IL-20 subfamily of receptors, it forms a complete heterodimeric receptor with IL20RA or IL22RA1, and its ligands are mainly IL-19, IL-20 and IL-24 [10] . In recent years, IL20RB has been found to play an essential role in the development and progression of a variety of cancers, such as lung cancer [11, 12] 、nasopharyngeal carcinoma [13] 、renal cancer [14, 15] and breast cancer [16] . Cui et al. [15] showed that high expression of IL20RB in renal papillary cell carcinoma was associated with poorer patient prognosis. He et al. [11] found that IL20RB showed abnormally high expression in lung cancer tissues but not normal lung tissues. Osteoclasts activate JAK1 by secreting IL-19 ligands, which act on IL20RB in lung cancer cells, activating the JAK1 /STAT3 signalling pathway and promoting the proliferation and intraosseous colonisation of disseminated tumour cells. These findings suggest that IL20RB may be involved as an oncogenic in tumour cells' proliferation, migration and invasion process and is closely associated with EMT. In addition, many bioinformatics analyses have pointed to the high expression of IL20RB in pancreatic cancer as a marker of poor prognosis [17–20] . However, research into the function and mechanism of action of IL20RB in pancreatic cancer is still in its infancy, and relevant experiments are needed to elucidate better its role in the development, progression and treatment of pancreatic cancer. This study aimed to analyse the expression of IL20RB in pancreatic cancer tissues and cells, explore its effect on the proliferation and migration ability of pancreatic cancer cells, and preliminarily investigate the mechanisms involved. Materials and methods 1. Clinical specimen collection This study collected 12 surgical specimens of pancreatic cancer definitively diagnosed between November 2022 and October 2023 at the First Hospital of Lanzhou University (Lanzhou, China), including pancreatic cancer tissues and corresponding normal tissue samples. All samples were collected and used with the patient's informed consent and approved by the Ethics Committee of the First Hospital of Lanzhou University(approval number: LDYY-2024-330). 2. Bioinformatics analysis Combined data from the TCGA database (https://cancergenome.nih.gov) and the GTEx database (https://www.gtexportal.org/) were used to analyse IL20RB expression in pancreatic cancer and corresponding normal pancreatic tissue. Pairwise comparisons of tumour and normal tissues were performed using the GSE15471 dataset (containing a tumour sample size of 39 and a normal sample size of 39), which was used to validate the expression status of IL20RB. Survival data and related clinical information of 178 pancreatic cancer patients from the TCGA database were extracted and grouped by median IL20RB expression, and Kaplan-Meier survival curves were plotted using the R package (Survival and Survminer) to compare the differences between the high- and low-expression groups in terms of overall survival time and disease-specific survival time, respectively. A COX regression model was developed to analyse the association between IL20RB expression and overall survival time in pancreatic cancer patients and to assess its prognostic value. Transcriptome profiles of TCGA PAAD were obtained using the R package TCGA Biolinks, data were transformed using DESeq2, and samples were stratified according to median IL20RB expression. GSEA was used to identify overexpressed pathways or gene modules. p<0.05 indicates statistically significant enrichment. 3. Antibodies For IHC, rabbit polyclonal antibody IL20RB (PU595351, dilution 1/200) was purchased from Abmart Co (Shanghai, China). For Western blot, mouse polyclonal antibody IL20RB (PU595351) was purchased from Proteintech Co (Wuhan, China); mouse polyclonal antibodies β-actin (T40104) and rabbit polyclonal antibodies E-cadherin (TA0131), N-cadherin (T55015), vimentin (T55134), snail (TA6032), IKK α/β (T55660), NF-κB P65 (T55034) and P-NF-κB P65 (Ser529) (TP56371) were purchased from Abmart Co (Shanghai, China) and all antibody dilutions were 1/1000. 4. Immunohistochemical assay For IHC, paraffin-embedded tissue was selected for HE staining, sectioned to a thickness of 3 μm, deparaffinised in xylene and rehydrated in a series of dilute alcohol solutions. Thermal epitope retrieval was performed for 20 min in a target retrieval solution at pH 7.5. Sections were incubated with rabbit polyclonal human IL20RB antibody (Catalogue No. PU595351, Abmart, Shanghai, China) at a dilution of 1:200 at 4°C overnight. The sections were then incubated with horseradish peroxidase (HRP) for 30 min at room temperature and observed microscopically for 5-10 min using DAB as a colour developer. IHC photographs were obtained by photographing 12 pairs of IHC sections stained by inverted fluorescence microscopy at 100x magnification. These IHC photographs were automatically analysed using the IHC Profiler [21] , which combines the mean grey value of positive cells (staining intensity) and the percentage of positive area (stained area) as IHC measurements to produce four final scores: High Positive (3+), Positive (2+), Low Positive (1+) and Negative (0). Quick scores (Quick score = staining intensity score × percentage of corresponding stained area) were calculated for each section for statistical analysis. 5. Western Blot Pancreatic cancer tissues and cells were lysed and sonicated using RIPA lysis buffer from Beyotime Biotechnology Co (Shanghai, China). Lysates containing soluble proteins were collected and stored at -80°C. Protein concentration was determined using the BCA method from BOSTER Biological Technology Co (Wuhan, China). An equal amount (30 μg) of protein was separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane with a transfer time of 1 h. A single wash was performed with TBS/0.1% Tween-20 (TBST) buffer, and the membrane was incubated with a 1:1000 dilution of primary antibody solution at 4°C overnight. The membrane was washed thrice with TBST and incubated with a 1:1000 dilution of HRP-conjugated secondary antibody for 1 h at 20-25°C. After incubation, the membrane was washed three more times with TBST. Finally, the immunoreactive bands were detected using Enhanced Chemiluminescent Liquid (ECL) from BOSTER Co (Wuhan, China). 6. Cell Culture Four human pancreatic cancer cell lines (MIA PaCa-2, SW1990, PANC-1 and BxPC-3) and one normal human pancreatic ductal epithelial (HPDE) cell line were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These cells were cultured according to the culture protocol provided by the supplier; BxPC-3 was cultured in RPMI-1640 complete medium (Gibco, America) containing 10% fetal bovine serum (FBS, Uruguay), MIA PaCa-2, SW1990, PANC-1 and HPDE were cultured in RPMI-1640 complete medium (Gibco, America) containing 10% fetal bovine serum (FBS, Uruguay) in DMEM complete medium (Gibco, America) and all cells were incubated at 37°C, 5% CO2. 7. Construction of cell lines PANC-1 and BxPC-3 cells were transfected with GV493 lentiviral vector (SHANGHAI GENECHEM Co, China) to obtain IL20RB knockdown experimental groups (IL20RB-sh1, IL20RB-sh2 and IL20RB-sh3); transfections were performed using empty vector viruses as a negative control group. Lentiviruses were transfected according to the manufacturer's instructions. Stable transfected cell lines were screened with 2-3 µg/ml puromycin. The lentiviral vector sequences are detailed in Appendix 1: Table S1. 8. RT-PCR Total RNA was extracted using the M5 Universal RNA Mini Kit (Mei5bio Co.), and the quality and concentration of RNA were determined using the Nanodrop2000 (Thermo Co.). Subsequently, cDNA was synthesised using a quantitative reverse transcription kit containing de-gDNA (Takara Co.). Finally, real-time quantitative fluorescence RT-PCR analysis was performed using the TB Green dye method standardised quantification kit (Takara Co.). All steps were performed according to the kit instructions. Each sample was repeated three times. The RT-PCR results were analysed by the 2 -ΔΔCT method using GAPDH as an internal reference. The sequence list of the gene-specific primers is detailed in Appendix 1: Table S2. 9. Cell Proliferation Assay Cell Counting Kit-8 (CCK-8, APExBIO Technology LLC, USA) was used for the cell proliferation assay. Cells at 2×10 3 /well were inoculated into 96-well plates and incubated in a thermostat for 1-4 h. The original medium in the wells to be tested was removed after the cells had attached to the wall, and 100 ul of CCK-8 reaction solution was added (the corresponding medium was mixed with the original CCK-8 solution in a 10:1 configuration). The wells were incubated again for 2 hours at 37°C under 5% CO2, and the absorbance (OD) at 450 nm was measured using an enzyme-linked immunosorbent assay (BioTek Instruments Inc., USA). After 24, 48, 72, 96 and 120 h, the medium in the corresponding wells to be tested was replaced with CCK-8 reaction solution, and the OD values were recorded. Differences in the proliferation of cells in different groups were assessed by comparing their OD values at the same time points. For plate cloning experiments, 2000/well cells were inoculated into 6-well plates, and the medium was changed every four days in a humidified incubator at 37°C. After two weeks, the fixed cells were washed with PBS and stained with 0.1% crystal violet (Solarbio Co, Beijing, China) for 10-15 min. After staining, the cells were washed three times with PBS and allowed to dry naturally before being photographed. The number of cell colonies formed in each group was counted using ImageJ software to compare proliferation differences between groups. 10. Cell migration assay For wound healing experiments, 1×10 5 /well cells were inoculated into 6-well plates and 1ml of sterile lance tip was used to scratch the plates to create trauma. After washing with PBS, the cells were incubated in a serum-free medium, photographed, and preserved at 0 and 24 h for each cell group under a 200× lens. The area between the two edges of the wound after the specified time was measured using ImageJ software, and the difference in the migration ability of the cells in each group was compared according to the cell migration rate = (0 h wound area – 24 h wound area)/0h wound area × 100%. Transwell migration assays were performed using Corning (3422, 8 μm) migration chambers from the USA. The upper chamber was seeded with 1×105 PANC-1 or BxPC-3 cells in a serum-free medium, approximately 200 μl. The corresponding RPMI-1640, or DMEM medium containing 30% fetal bovine serum, was added to the lower chamber, approximately 800 ul. Migration experiments were performed by incubation at 37°C, 5% CO2 for 24-48 h. Cells on the lower membrane were stained with 0.1% crystal violet staining solution (Solarbio Co, China). Global perforation images were taken at 40×, and five randomly selected fields of view were photographed and stored at 200×. The number of cells crossing the chambers was counted using ImageJ software to compare differences in migration ability between groups. 11. Flow Cytometry According to the kit instructions, Apoptosis was detected using the Annexin V-APC/7-AAD Apoptosis Kit from MULTI SCIENCES Co (Hangzhou, China) (Catalogue No. AP104-30). Apoptosis was induced according to the experimental protocol. Cells were washed by centrifugation with pre-cooled PBS, and 1-10×10 5 cells (including cells in the culture supernatant) were collected. Dilute 5× binding buffer to 1× working solution and resuspend cells with 500ul of 1× binding buffer. 5ul of Annexin V-APC and 10ul of 7-AAD were added to each tube, and after mixing by gentle vortexing, the cells were incubated for 5 min at room temperature in the dark. Apoptosis was detected using an Agilent flow cytometer (Agilent Technologies Co), and data were analysed using Novoexpress software. A cell cycle staining kit (catalogue number CCS012) from MULTI SCIENCES Co was used according to the kit instructions. Approximately 2×10 5 -1×10 6 cells were collected from each negative control and IL20RB knockdown group, and the supernatant was discarded by centrifugation. The cells were resuspended and washed once with PBS. Next, 1 ml DNA staining solution and 10 μl permeabilization solution were added to each sample and incubated for 30 min at room temperature in the dark. The lowest sampling rate was selected, and the cell cycle was detected by Agilent flow cytometry. The data was also analyzed using Novoexpress software. 12. Cellular transcriptome sequencing The stably transfected PANC-1 cell line, including the negative control group and the knockdown experimental group (IL20RB-sh1, IL20RB-sh2), were provided with two samples each to be sent to BMK Co (China) for eukaryotic reference transcriptome sequencing, and the data were further analysed using BMKCloud (www.biocloud.net). 13. Statistical analysis SPSS 27.0, R4.3.1 and GraphPad Prism 9.0 software were used for statistical analysis and presentation. Multiple comparisons between groups were analysed using ANOVA, and post-hoc tests were performed using the Student-Newman-Keuls test. The two-tailed t-test was used for comparisons between the two groups. The Kaplan-Meier method was used to assess overall survival. Data are expressed as mean ± standard deviation. p<0.05 was considered statistically significant. Results 1. Bioinformatic analysis of IL20RB expression in pancreatic cancer and its association with prognosis Combined analysis of the TCGA and GTEx databases revealed that IL20RB expression was significantly upregulated in pancreatic cancer tissue compared to normal pancreatic tissue (171 vs 179, P<0.001) (Figure 1 a). Re-validation of 39 paired normal pancreatic tissues and pancreatic cancer tissues using the GSE15471 dataset showed that IL20RB expression was significantly increased in pancreatic cancer tissues (P<0.01) (Figure 1 b). Furthermore, the area under the ROC curve (AUC) was 0.949, indicating that IL20RB has a high diagnostic value and can be used as a unique diagnostic marker to discriminate between the normal population and pancreatic cancer patients (Figure 1 c). The time-dependent ROC curves showed that the AUC values at 1, 3 and 5 years were greater than 0.6, suggesting that IL20RB has a specific reference value in prognostic assessment (Figure 1 d). Survival analysis showed that the OS time of patients in the IL20RB high-expression group was significantly shorter than that of the low-expression group (P = 0.0024) (Figure 1 e), and the DSS time was also significantly shorter than that of the low-expression group (P = 0.0079) (Figure 1 f). The results of univariate and multivariate COX regression analyses showed whether the surgery was R0 resection and IL20RB expression level were independent predictors affecting OS in pancreatic cancer patients (Table 1). In conclusion, high expression of IL20RB in pancreatic cancer is strongly associated with poor prognosis and is an independent risk factor for it. Table 1 COX regression analysis of IL20RB clinical data Characteristics Total（N） Univariate analysis Multivariate analysis HR（95%CI） P value HR（95%CI） P value Pathologic T stage 177 T1&T2 31 Reference Reference T3 143 2.056(1.090-3.878) 0.026 1.410(0.716-2.777) 0.320 T4 3 1.091(0.140-8.489) 0.934 0.909(0.117-7.092) 0.928 Pathologic N stage 174 N0 50 Reference Reference N1 124 2.161(1.287-3.627) 0.004 1.609(0.901-2.872) 0.108 Pathologic M stage 85 M0 80 Reference M1 5 0.773(0.185-3.227) 0.724 Gender 179 Female 80 Reference Male 99 0.813(0.541-1.222) 0.319 Age 179 65 85 1.285(0.853-1.937) 0.230 Histologic grade 177 G1&G2&G3 175 Reference Characteristics Total（N） Univariate analysis Multivariate analysis HR（95%CI） P value HR（95%CI） P value G4 2 0.864(0.120-6.217) 0.885 Smoker 145 No 66 Reference Yes 79 1.075(0.682-1.694) 0.755 Residual tumor 165 R0 107 Reference Reference R1&R2 58 1.650(1.064-2.558) 0.025 1.602(1.021-2.516) 0.040 IL20RB 179 Low 89 Reference Reference High 90 2.025（1.325-3.095) 0.001 1.757(1.124-2.747) 0.013 Interleukin-20 receptor subunit beta; TNM, tumor, node and metastasis 2. Expression analysis of IL20RB in human pancreatic cancer tissue Twelve pancreatic cancer surgical specimens, including matched normal pancreatic tissue, were collected. Immunohistochemistry (IHC) results showed that in pancreatic cancer cells, IL20RB was positively stained mainly at the cell membrane and was positively expressed in pancreatic cancer tissues. In contrast, the corresponding normal pancreatic tissues were negatively expressed (Fig. 2 a). In 8 of the 12 cases, pancreatic cancer tissues expressed IL20RB positively, with a positive rate of 66.7% (8/12), higher than the corresponding normal pancreatic tissues with a positive rate of 16.7% (8/12). 66.7% (8/12), which was higher than the positive rate of IL20RB expression in its corresponding normal pancreatic tissues of 16.7% (2/12); at the same time, the expression level of IL20RB in pancreatic cancer tissues was significantly higher than that in the corresponding normal pancreatic tissues (P=0.036), as shown in Table 2. In addition, IL20RB in pancreatic cancer tissues showed a higher IHC staining score (P<0.001) compared with the corresponding normal pancreatic tissues (Figure 2 b). Western blot experiments showed that the expression level of IL20RB was significantly up-regulated in tumour samples compared with normal pancreatic tissues (P<0.05) (Figure 2 c, d). Table 2 Expression of IL20RB in pancreatic cancer and corresponding normal pancreatic tissue Type of organisation Number of examples Positive Negative Positivity rate P Normal pancreatic tissue 12 8 4 66.7% 0.036* Pancreatic cancer tissue 12 2 10 16.7% Note: * is Fisher's exact probability method; Differences are statistically significant. 3. Knockdown of IL20RB in pancreatic cancer cell lines The expression of IL20RB mRNA and protein in normal pancreatic epithelial cell lines (HPDE) and pancreatic cancer cell lines (MIA PaCa-2, SW1990, PANC-1 and BxPC-3) was investigated by RT-PCR and Western blot assays. The results of the RT-PCR assay showed that IL20RB mRNA expression was significantly increased in the MIA PaCa-2, SW1990, PANC-1 and BxPC-3 cancer cell groups compared with the HPDE group, and the difference was statistically significant (P<0.05, P<0.05, P<0.05). The MIA PaCa-2, SW1990, PANC-1, and BxPC-3 cancer cell groups showed a significant increase in IL20RB mRNA expression, and the difference was statistically significant (P<0.05, P<0.001, P<0.0001, P<0.0001) (Figure 2 e). The results of Western blot experiments also confirmed this trend, and the MIA PaCa-2, SW1990, PANC-1, and BxPC-3 cells showed a significant increase in IL20RB protein expression. Among them, in MIA PaCa-2 cells, IL20RB protein expression was increased compared to HPDE cells. However, the difference was insignificant (P<0. 1780). In contrast, the protein level of IL20RB was also significantly increased in the other three pancreatic cancer cell lines (SW1990, PANC-1, and BxPC-3), and the difference was statistically significant (P<0.05, P<0.05, and P<0.) 0.05, P<0.001, P<0.0001) (Figure 2 f, g). These results indicated that both IL20RB mRNA and protein levels were significantly up-regulated in pancreatic cancer, suggesting that IL20RB may be involved in pancreatic carcinogenesis and development as an oncogenic factor. In PANC-1 and BxPC-3 cells, IL20RB was the most highly expressed at both mRNA and protein levels. Therefore, in this study, the expression of IL20RB was stably knocked down in these two pancreatic cancer cell lines by lentiviral transfection. The knockdown effect of IL20RB at the mRNA and protein levels was then detected using RT-PCR and Western blot experiments. The results showed that in the three groups of cells transfected with knockdown IL20RB lentivirus (i.e. IL20RB-sh1, IL20RB-sh2 and IL20RB-sh3 groups), the expression of IL20RB was significantly reduced at both the mRNA and protein levels. The most significant knockdown effect was observed in the IL20RB-sh1 and IL20RB-sh2 groups (all P values: P<0.05) (Figure 2 h, i and j). Therefore, in the follow-up experiments in the present study, IL20RB-sh1 and IL20RB-sh2 were selected as the knockdown experimental groups and further studies were conducted with the negative control group. 4. Analysis of the effect of knocking down IL20RB expression on the biological properties of pancreatic cancer cells 4.1 IL20RB knockdown inhibits pancreatic cancer cell proliferation and migration capacity in vitro In this study, the effect of IL20RB knockdown on the proliferative capacity of pancreatic cancer cells was jointly verified by performing two parallel experiments, CCK-8 assay and cell clone formation assay. In cells stably transfected with the virus, the differences in OD levels and the number of clonal cell clusters were compared between groups of cells at different time points (0h, 24h, 48h, 72h, 96h and 120h). The results showed that in PANC-1 and BxPC-3 cells, the OD values of cells in the negative control group were significantly higher than those in the IL20RB knockdown group, and the difference gradually increased (P<0.0001) (Figure 3 a, b). Meanwhile, similar results were also observed in the cell cloning assay (P<0.01) (Figure 3 c, d). The above two validations indicated that IL20RB knockdown attenuated the proliferative ability of PANC-1 and BxPC-3 cells. Similarly, the wound healing rate of cells in each group within 24 hours and the number of cells migrating through the opening of the transwell within a given period were compared under the condition of stable viral transfection. The results showed that the wound healing rate of cells in the IL20RB knockdown group was significantly lower than that of the negative control group (both P values: P<0.01) (Figure 3 e, f). Meanwhile, the results of the transwell migration assay also showed that the number of cells migrating through the aperture of the chamber was significantly reduced in the IL20RB knockdown group compared to the negative control group (P<0.0001) (Figure 3 g, h). The above results indicated that the migration ability of PANC-1 and BxPC-3 cells was also significantly reduced after IL20RB knockdown treatment. 4.2 IL20RB knockdown induces apoptosis and promotes cell cycle arrest in pancreatic cancer cells in vitro The effects of IL20RB knockdown on apoptosis and cell cycle of PANC-1 and BxPC-3 cells were investigated by flow cytometry. The apoptosis ratio (including early and late apoptosis) and the distribution of cells in each group throughout the cell cycle were determined. The experimental results showed that the number of apoptotic cells in the IL20RB knockdown group was significantly increased (P<0.05) compared to the negative control group (Figure 4 a, b). In addition, there was no significant change in the distribution of cells in the G1 and G2 phases in the knockdown group. However, their distribution in the S phase was significantly increased (P<0.05) (Figure 4 c, d). The above flow cytometry experimental results suggested that the knockdown of IL20RB gene expression promoted pancreatic cancer cells PANC-1 and BxPC-3 to undergo apoptosis and resulted in these cells being blocked from remaining in the S phase. 5. Analysis of IL20RB-related downstream signalling pathways in pancreatic cancer cells 5.1 Effect of IL20RB knockdown on the EMT process in pancreatic cancer cells Bioinformatic analysis of GSEA revealed that the EMT process was significantly enriched in pancreatic cancer (Figure 5 b). In addition, cell function assays showed that knockdown of IL20RB significantly inhibited the migration ability of pancreatic cancer cells, further suggesting that IL20RB is associated with the EMT process in pancreatic cancer. Therefore, the present study confirmed by Western blot that the PANC-1 cells stably transfected with IL20RB-sh1 and IL20RB-sh2 groups showed increased E-cadherin expression (P<0.0001, P<0.0001) and down-regulation of EMT-related protein expression (P<0.0001, P<0.0001) compared to the negative control group in EMT-related protein expression. N-cadherin (P=0.0001, P<0.0001), vimentin (P<0.0001, P=0.0013) and snail (P=0.0079, P<0.0001) expression levels (Figure 6 a, c). These results were also confirmed in BxPC-3 cells (Figure 6 b, d). The above experimental results further confirmed that the knockdown of IL20RB expression in pancreatic cancer cells could inhibit the EMT process. 5.2 Inhibition of the IKK α/β/NF-κB P65 signalling pathway by IL20RB knockdown According to the cellular transcriptome sequencing results (Figure 5 c, d) and GSEA enrichment analysis (Figure 5 a), a large number of genes related to IL20RB function were found to be enriched in the NF-κB signalling pathway, suggesting that the functional realisation of IL20RB is closely related to NF-κB signalling. Moreover, IKK/NF-κB, as a classical signalling pathway, plays a vital role in cancer cell proliferation, metastasis and apoptosis. Therefore, the present study hypothesised that IL20RB may regulate pancreatic cancer cell proliferation, metastasis and apoptosis through the IKK/NF-κB pathway. To test this hypothesis, the changes in key protein molecules in this pathway after IL20RB knockdown were detected at the protein level using Western blot experiments. The results showed that in PANC-1 cells, IL20RB knockdown significantly decreased IKKα/β expression compared to the negative control group (P<0.0001, P<0.0001), whereas there was no significant change in NF-κB P65 expression; however, P-NF-κB P65 expression was significantly decreased (P<0.0001, P<0.0001). P-NF-κB P65/NF-κB P65 was also significantly reduced (P<0.0001, P=0.0001) (Figure 6 e, g). Consistent results were obtained in BxPC-3 cells (Figure 6 f, h). The above results confirm our hypothesis that IL20RB may promote pancreatic cancer progression through the IKK/NF-κB pathway. Discussion In pancreatic cancer, IL20RB was found to be highly expressed, to have some diagnostic predictive value and to be an independent risk factor for poor prognosis. This finding is consistent with previous studies [17–20] . Functional experiments showed that knockdown of IL20RB significantly inhibited the proliferation and migration ability of pancreatic cancer cells, induced apoptosis and led to S-phase blockade. In addition, GSEA enrichment analysis and cellular transcriptome sequencing showed that IL20RB was closely associated with EMT, apoptosis and proliferation and closely correlated with NF-κB signalling. This result stimulated our interest to investigate further the relationship between IL20RB and EMT and NF-κB signalling in pancreatic cancer. NF-κB acts as a nuclear transcription factor and regulates tumour cells' proliferative, anti-apoptotic, and invasive metastatic properties [22–24] . It is activated through classical and non-classical pathways and interacts with I-κBs proteins to control gene transcription [25] . The frequent activation of NF-κB in pancreatic cancer suggests its potential as a therapeutic target. In addition, most studies of the relationship between NF-κB and tumourigenesis have focused primarily on the p65 subunit. In particular, nuclear translocation of NF-κB P65 has been shown to induce the EMT process in various tumour cells [25] . Conversely, the involvement of the NF-κB pathway in promoting tumour cell progression by inducing EMT is closely linked to the upstream mediators of EMT, such as ZEB1, Slug, Twist and Snail. For example, in hepatocellular carcinoma, Wu et al. [26] found that Snail acted as a direct target gene downstream of NF-κB signalling, and the IKKβ/NF-κB P65 signalling pathway was activated, upregulating Snail expression and inducing the EMT process. On the other hand, Zuo et al. [27] found that the natural product daehyenoside induced EMT in cervical cancer cells by inhibiting the NF-κB signalling pathway induced by stimulation of the hypoxic tumour microenvironment activation, decreased the expression levels of Slug and Snail in cervical cancer cells, thereby decreasing the expression of N-cadherin and Vimentin and increasing the expression of E-cadherin, which served to inhibit EMT and ultimately inhibited the migration and invasion of cervical cancer cells. The study by Qu et al. [28] showed that AKR1B10 was effective in breast cancer through the induction of the PI3K/Akt signalling pathway to stimulate the activation of the NF-κB pathway and upregulate the expression levels of ZEB1, Slug, and Twist to promote EMT, which in turn promoted the migration of breast cancer cells. Meanwhile, in a variety of tumours, including pancreatic cancer, it has been shown to have a tumour-initiating capacity after induction of EMT [29–31] . At the same time, the expression levels of N-cadherin, vimentin, E-cadherin and Snail are often used to detect the presence of EMT processes [32–36] . In this study, we found increased expression of E-cadherin protein and decreased expression of N-cadherin, vimentin and Snail protein after knockdown of IL20RB in both pancreatic cancer cell lines, PANC-1 and BxPC-3. This result suggests that IL20RB may promote pancreatic carcinogenesis, proliferation, migration and invasion by activating the EMT process. This finding is consistent with the results obtained in this study using CCK8, clone formation assay, and scratch and transwell assay. In addition, protein levels of IKKα/β, critical kinases involved in the NF-κB signalling pathway, as well as NF-κB-P65 and P-NF-κB P65, were detected by Western bolt, and it was found that knockdown of IL20RB resulted in decreased IKKα/β expression. However, there was no significant change in NF-κB-P65 expression, but P-NF-κB P65 expression was decreased, and the ratio of P-NF-κB P65/NF-κB-P65 was reduced. These results suggest that the knockdown of IL20RB inhibits the activation of the IKKα/β/NF-κB-P65 pathway. IL20RB, which is highly expressed in pancreatic cancer, may regulate the expression of multiple genes by activating the IKKα/β/NF-κB-P65 signalling pathway, thereby promoting the development and progression of pancreatic cancer. In addition, cell proliferation must depend on a balanced cell cycle process, and abnormalities in any component of the cell cycle can trigger tumours [37] . Han et al. [38] found that berberine interfered with bladder cancer cells' survival, proliferation and metastasis by inhibiting the NF-κB pathway. In addition, berberine-induced bladder cancer cells arrest in the S phase, resulting in anti-tumour effects. The present study obtained similar results, i.e., knockdown of IL20RB expression could effectively inhibit the proliferative ability of pancreatic cancer cells, increasing the number of cells remaining in the S phase. At the same time, there was no significant change in the distribution of cells in the G1 and G2 phases. This result suggests that IL20RB may regulate the cell cycle process of pancreatic cancer cells, and considering that the S phase is a critical period for DNA synthesis, it can be inferred after combining the results of in vitro experiments of IL20RB, it can be concluded that high expression of IL20RB may be involved in promoting DNA synthesis in the occurrence and development of pancreatic cancer. Moreover, targeting therapy to IL20RB expression in pancreatic cancer may be similar to the findings of Han et al. [38] , which is of great significance in tumour therapy. Upon comprehensive analysis, the results of this study showed that IL20RB was highly expressed in both pancreatic cancer tissues and cell lines and correlated with poor patient prognosis. Knockdown of IL20RB inhibited the proliferation and migration of pancreatic cancer cells and induced apoptosis, contributing to the arrest of cells in the S phase. In contrast, IL20RB may promote the proliferation and migration of pancreatic cancer cells by activating the IKKα/β/NF-κB P65 signalling pathway and facilitating the EMT process, ultimately promoting the proliferation and migration of pancreatic cancer cells. In conclusion, IL20RB acts as an oncogenic factor in pancreatic cancer, and a more comprehensive understanding of its effects on the biological properties of pancreatic cancer and more in-depth studies on the related mechanisms will provide an essential theoretical basis for revealing the mechanisms of pancreatic cancer development and progression.IL20RB is a highly promising therapeutic target for pancreatic cancer. Declarations Acknowledgements The author would like to thank Dr Kexiang Zhu for his techinal assistanse Funding This study was supported by the National Natural Science Foundation of China (grant no. 81960516). Natural Science Foundation of Gansu Province (grant no.22JR11RA023) Availability of data and materials The analyzed datasets generated during the study are available from the corresponding author on reasonable request. Authors’ contributions declaration Wanbai Ruan and Kexiang Zhu designed the study. Wanbai Ruan, Junfen Li, Yanmei Yin and Lei Peng performed the experiments. Wanbai Ruan and Junfen Li analyzed the results. All authors have read and approved the final manuscript Ethics approval and consent to participate In accordance with the Declaration of Helsinki. For human specimens, all samples were collected and used with the patient's informed consent and approved by the Ethics Committee of the First Hospital of Lanzhou University (Lanzhou, China) (approval number: LDYY-2024-330). Consent for publication Not applicable Competing interests The author declare that they have no competing interests References Sung H, Ferlay J, Siegel R L, Laversanne M, Soerjomataram I, Jemal A, Bray F Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries [J]. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5929013","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":409568683,"identity":"387429ce-e4ce-4a98-8288-a0eac5db6171","order_by":0,"name":"Wanbai Ruan","email":"","orcid":"","institution":"The 940th Hospital of the Joint Logistic Support Force of the PLA","correspondingAuthor":false,"prefix":"","firstName":"Wanbai","middleName":"","lastName":"Ruan","suffix":""},{"id":409568684,"identity":"fd0c36d9-2144-4a09-ad02-fb7dd0340d7f","order_by":1,"name":"Junfen Li","email":"","orcid":"","institution":"The First Clinical Medical College, Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Junfen","middleName":"","lastName":"Li","suffix":""},{"id":409568687,"identity":"c4217741-f771-464c-93de-6d906fa845fd","order_by":2,"name":"Yanmei Yin","email":"","orcid":"","institution":"The First Clinical Medical College, Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yanmei","middleName":"","lastName":"Yin","suffix":""},{"id":409568688,"identity":"c79c53dd-c258-4e80-8550-91d90228db7d","order_by":3,"name":"Lei Peng","email":"","orcid":"","institution":"The First Clinical Medical College, Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Peng","suffix":""},{"id":409568689,"identity":"b0ffb48d-99b4-4795-92a8-6d500b711835","order_by":4,"name":"Kexiang Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIie3PIQvCQBTA8RsPznJ4dQeyz3BjYDH4VXYImhTBsmDYUG5BBD+KcVF5sHRiNc5i1qbNoVHZZjPcP7z0fg8eITbbH9ZKy+HExOPgJEUYzesJwzcJRAooC5M3J0oe6VCcl9CAAOTikWEokXUjFVPC01VYQ+hACoMTUZKTyjrENYdtJekD8wtf46z9IoYS6Y6rCQN+3SmNKi7JVGloQphT7EuyQTokDQkN/ESPArEAdEOTs9pfWGtxEQ/d8zjfJ7d7NPd4uq4mnzd+W7fZbDbb1554HUc9xt3legAAAABJRU5ErkJggg==","orcid":"","institution":"The First Hospital of Lanzhou University","correspondingAuthor":true,"prefix":"","firstName":"Kexiang","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2025-01-30 09:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5929013/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5929013/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75410006,"identity":"c7cddcc7-a777-49fd-bb25-9414669f60b4","added_by":"auto","created_at":"2025-02-04 09:06:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":310349,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of IL20RB expression differences in pancreatic cancer and its association with patient diagnosis and survival prognosis. Figure a: TCGA combined with GTEx database to analyse IL20RB expression in normal and cancerous pancreatic tissue; Figure b: GSEA15471 dataset to analyse IL20RB expression in normal and cancerous pancreatic tissues; Figure c: Categorical ROC curves of IL20RB with pancreatic cancer patients; Figure d: Time-dependent ROC curves of IL20RB with pancreatic cancer patients; Figure e、 f: Kaplan-Meier survival curves analysing the relationship between IL20RB expression and patients' overall and disease-specific survival.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5929013/v1/136c6902cd3120de2dac7f63.png"},{"id":75411679,"identity":"c6818542-7ce2-400c-bbd4-01e1b0b0ef3f","added_by":"auto","created_at":"2025-02-04 09:14:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":329170,"visible":true,"origin":"","legend":"\u003cp\u003eIL20RB expression in human pancreatic cancer and its knockdown in pancreatic cancer cell lines. Figure a、 b: Representative graph of immunohistochemical results for IL20RB in pancreatic cancer tissues and score statistics; Figure c、 d: Graphical representation of IL20RB protein expression and quantitative statistics in pancreatic cancer tissues; Figure e: Expression statistical graphs of IL20RB mRNA in normal pancreatic epithelial cell lines and several pancreatic cancer cell lines by RT-PCR assay; Figures f、 g: Protein expression graph of IL20RB in multiple pancreatic cancer cell lines and quantification of results; Figure h: Results of IL20RB knockdown assay of mRNA levels in PANC-1 and BxPC-3 cell lines; Figure i, j: Results and quantification of knockdown of protein levels by IL20RB in PANC-1 and BxPC-3 cell lines (****.P\u0026lt;0.0001, ***.P\u0026lt;0.001, **.P\u0026lt;0.01, *.P\u0026lt;0.05, ns.P\u0026gt;0.05)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5929013/v1/019ea0e8c1ed0d468cd370e8.png"},{"id":75411680,"identity":"42b5bfd4-91e7-46e3-9ab5-9e36b0f48256","added_by":"auto","created_at":"2025-02-04 09:14:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":414672,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of IL20RB knockdown on proliferation and migration ability of PANC-1 and BxPC-3 cells. Figure a: Statistical graph of the results of CCK-8 experiments for PANC-1 cells; Figure b: Statistical graph of the results of CCK-8 experiments for BxPC-3 cells; Figure c: Results of cloning experiments for PANC-1 and BxPC-3 cells; Figure d: Statistical graph of the results of clone formation experiments for PANC-1 and BxPC-3 cells; Figure e, f: Results and statistical graphs of scratch experiments for PANC-1 and BxPC-3 cells; Figure g, h: Results and statistical graphs of transwell experiments for PANC-1 and BxPC-3 cells. (****.P\u0026lt;0.0001,***.P\u0026lt;0.001,**.P\u0026lt;0.01, compared to negative control group cells)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5929013/v1/22b5691307d78531d397323e.png"},{"id":75410010,"identity":"e6f16a16-ce81-4a6f-8567-748da06d55fd","added_by":"auto","created_at":"2025-02-04 09:06:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":435328,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of IL20RB knockdown on apoptosis and cell cycle of PANC-1 and BxPC-3 cells. Figure a、 b: Results and statistics of apoptosis experiment of PANC-1 and BxPC-3 cells; Figure c、 d: Results and statistics of cell cycle experiment of PANC-1 and BxPC-3 cells. (***.P\u0026lt;0.001, **.P\u0026lt;0.01, *.P\u0026lt;0.05, ns.P\u0026gt;0.05, compared with cells in Negative Control group)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5929013/v1/eaa1d3ad72e7ba31426477fa.png"},{"id":75410028,"identity":"4ddd96b3-77e0-4b29-80ff-f93a8ea3a3ca","added_by":"auto","created_at":"2025-02-04 09:06:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":378649,"visible":true,"origin":"","legend":"\u003cp\u003eBioinformatics and transcriptome sequencing analysis of IL20RB-regulated downstream molecules. Figure a、 b: Results of GSEA enrichment analysis based on IL20RB in pancreatic cancer; Figure c: Volcano plot of differentially expressed genes in transcriptome sequencing data; Figure d: KEGG enrichment analysis of differentially expressed genes in transcriptome sequencing data.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5929013/v1/1c3b56a278c1cc58f7cb9c62.png"},{"id":75410031,"identity":"4994572b-c921-40b6-85e3-6552f99418d5","added_by":"auto","created_at":"2025-02-04 09:06:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":297960,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of IL20RB knockdown on the expression of EMT and NF-κB signalling proteins in PANC-1 and BxPC-3 cells. Figure a、 b: Results of Western blot detection of N-cadherin, E-cadherin, vimentin and snail protein expression in PANC-1, BxPC-3 cells; Figure c、 d: Results of quantitative plots of N-cadherin, E-cadherin, vimentin and snail protein expression in PANC-1, BxPC-3 cells; Figure e、 f: Western blot detection of IKK α/β, NF-κB P65 and P-NF-κB P65 protein expression in PANC-1, BxPC-3 cells; Figure g、 h: Quantitative plots of Wb detection of IKK α/β, NF-κB P65 and P-NF-κB P65 protein expression in PANC-1, BxPC-3 cells. (****.P\u0026lt;0.0001, ***.P\u0026lt;0.001, **.P\u0026lt;0.01, *.P\u0026lt;0.05, ns.P\u0026gt;0.05, compared with negative Control group)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5929013/v1/81310a0429ed4fc450bd701d.png"},{"id":75433408,"identity":"cec86215-138b-44e4-8005-8267be70f2c8","added_by":"auto","created_at":"2025-02-04 13:54:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2799586,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5929013/v1/9487c87a-9d09-40ed-b1c3-1205346c7f57.pdf"},{"id":75410004,"identity":"6e1f5fda-6fca-4894-8418-fd18e06f9847","added_by":"auto","created_at":"2025-02-04 09:06:03","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16189,"visible":true,"origin":"","legend":"","description":"","filename":"Appendix1TableS1andS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5929013/v1/c2e282dfa76d6d869319b21d.docx"},{"id":75410012,"identity":"7e724521-4c03-4a50-8f84-41a5f287fd8e","added_by":"auto","created_at":"2025-02-04 09:06:04","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3351468,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.zip","url":"https://assets-eu.researchsquare.com/files/rs-5929013/v1/9b215a8b45e4edffe3660320.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"IL20RB promotes pancreatic cancer progression by activating NF- kB signalling and promoting EMT","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlobally, pancreatic cancer (PC) is the seventh most common cause of cancer-related mortality\u003csup\u003e[1]\u003c/sup\u003e. The incidence of pancreatic cancer in developed countries is approximately four to five times higher than in developing countries\u003csup\u003e[1, 2]\u003c/sup\u003e. The peak age of onset is 65\u0026ndash;69 years for men and 75\u0026ndash;79 years for women\u003csup\u003e[3]\u003c/sup\u003e. In recent years, there has been a notable shift in the age at which pancreatic cancer is diagnosed, with a higher incidence observed in younger individuals, particularly women\u003csup\u003e[4\u0026ndash;6]\u003c/sup\u003e. Surgery remains the primary treatment for pancreatic cancer, with a high success rate. However, only 15\u0026ndash;20% of patients are eligible for surgery at diagnosis\u003csup\u003e[7]\u003c/sup\u003e. Nearly three-quarters of patients who undergo surgical resection will experience recurrence within two years, suggesting the presence of micrometastatic lesions\u003csup\u003e[8]\u003c/sup\u003e. Preclinical studies have also demonstrated that circulating disseminated pancreatic cancer cells can be observed prior to limited invasion of the primary tumour\u003csup\u003e[9]\u003c/sup\u003e. Currently, the poor prognosis of pancreatic cancer patients is primarily attributed to a lack of comprehensive understanding of the biological characteristics and mechanisms of pancreatic cancer cells, particularly in the transformation of normal pancreatic tissues to cancerous processes. There are still numerous undefined aspects of this process. Therefore, it is significant to study pancreatic cancer's biological characteristics and mechanisms.\u003c/p\u003e \u003cp\u003eIL20RB is located at position 3q22.3 and has a molecular weight of approximately 35,076 Da. As a subunit of the IL-20 subfamily of receptors, it forms a complete heterodimeric receptor with IL20RA or IL22RA1, and its ligands are mainly IL-19, IL-20 and IL-24\u003csup\u003e[10]\u003c/sup\u003e. In recent years, IL20RB has been found to play an essential role in the development and progression of a variety of cancers, such as lung cancer\u003csup\u003e[11, 12]\u003c/sup\u003e、nasopharyngeal carcinoma\u003csup\u003e[13]\u003c/sup\u003e、renal cancer\u003csup\u003e[14, 15]\u003c/sup\u003eand breast cancer\u003csup\u003e[16]\u003c/sup\u003e. Cui et al.\u003csup\u003e[15]\u003c/sup\u003e showed that high expression of IL20RB in renal papillary cell carcinoma was associated with poorer patient prognosis. He et al.\u003csup\u003e[11]\u003c/sup\u003e found that IL20RB showed abnormally high expression in lung cancer tissues but not normal lung tissues. Osteoclasts activate JAK1 by secreting IL-19 ligands, which act on IL20RB in lung cancer cells, activating the JAK1 /STAT3 signalling pathway and promoting the proliferation and intraosseous colonisation of disseminated tumour cells. These findings suggest that IL20RB may be involved as an oncogenic in tumour cells' proliferation, migration and invasion process and is closely associated with EMT. In addition, many bioinformatics analyses have pointed to the high expression of IL20RB in pancreatic cancer as a marker of poor prognosis\u003csup\u003e[17\u0026ndash;20]\u003c/sup\u003e. However, research into the function and mechanism of action of IL20RB in pancreatic cancer is still in its infancy, and relevant experiments are needed to elucidate better its role in the development, progression and treatment of pancreatic cancer.\u003c/p\u003e \u003cp\u003eThis study aimed to analyse the expression of IL20RB in pancreatic cancer tissues and cells, explore its effect on the proliferation and migration ability of pancreatic cancer cells, and preliminarily investigate the mechanisms involved.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e1. Clinical specimen collection\u003c/p\u003e\n\u003cp\u003eThis study collected 12 surgical specimens of pancreatic cancer definitively diagnosed between November 2022 and October 2023 at the First Hospital of Lanzhou University (Lanzhou, China), including pancreatic cancer tissues and corresponding normal tissue samples. All samples were collected and used with the patient\u0026apos;s informed consent and approved by the Ethics Committee of the First Hospital of Lanzhou University(approval number: LDYY-2024-330).\u003c/p\u003e\n\u003cp\u003e2. Bioinformatics analysis\u003c/p\u003e\n\u003cp\u003eCombined data from the TCGA database (https://cancergenome.nih.gov) and the GTEx database (https://www.gtexportal.org/) were used to analyse IL20RB expression in pancreatic cancer and corresponding normal pancreatic tissue. Pairwise comparisons of tumour and normal tissues were performed using the GSE15471 dataset (containing a tumour sample size of 39 and a normal sample size of 39), which was used to validate the expression status of IL20RB. Survival data and related clinical information of 178 pancreatic cancer patients from the TCGA database were extracted and grouped by median IL20RB expression, and Kaplan-Meier survival curves were plotted using the R package (Survival and Survminer) to compare the differences between the high- and low-expression groups in terms of overall survival time and disease-specific survival time, respectively. A COX regression model was developed to analyse the association between IL20RB expression and overall survival time in pancreatic cancer patients and to assess its prognostic value. Transcriptome profiles of TCGA PAAD were obtained using the R package TCGA Biolinks, data were transformed using DESeq2, and samples were stratified according to median IL20RB expression. GSEA was used to identify overexpressed pathways or gene modules. p\u0026lt;0.05 indicates statistically significant enrichment.\u003c/p\u003e\n\u003cp\u003e3. Antibodies\u003c/p\u003e\n\u003cp\u003eFor IHC, rabbit polyclonal antibody IL20RB (PU595351, dilution 1/200) was purchased from Abmart Co (Shanghai, China). For Western blot, mouse polyclonal antibody IL20RB (PU595351) was purchased from Proteintech Co (Wuhan, China); mouse polyclonal antibodies \u0026beta;-actin (T40104) and rabbit polyclonal antibodies E-cadherin (TA0131), N-cadherin (T55015), vimentin (T55134), snail (TA6032), IKK \u0026alpha;/\u0026beta; (T55660), NF-\u0026kappa;B P65 (T55034) and P-NF-\u0026kappa;B P65 (Ser529) (TP56371) were purchased from Abmart Co (Shanghai, China) and all antibody dilutions were 1/1000.\u003c/p\u003e\n\u003cp\u003e4. Immunohistochemical assay\u003c/p\u003e\n\u003cp\u003eFor IHC, paraffin-embedded tissue was selected for HE staining, sectioned to a thickness of 3 \u0026mu;m, deparaffinised in xylene and rehydrated in a series of dilute alcohol solutions. Thermal epitope retrieval was performed for 20 min in a target retrieval solution at pH 7.5. Sections were incubated with rabbit polyclonal human IL20RB antibody (Catalogue No. PU595351, Abmart, Shanghai, China) at a dilution of 1:200 at 4\u0026deg;C overnight. The sections were then incubated with horseradish peroxidase (HRP) for 30 min at room temperature and observed microscopically for 5-10 min using DAB as a colour developer.\u003c/p\u003e\n\u003cp\u003eIHC photographs were obtained by photographing 12 pairs of IHC sections stained by inverted fluorescence microscopy at 100x magnification. These IHC photographs were automatically analysed using the IHC Profiler\u003csup\u003e[21]\u003c/sup\u003e, which combines the mean grey value of positive cells (staining intensity) and the percentage of positive area (stained area) as IHC measurements to produce four final scores: High Positive (3+), Positive (2+), Low Positive (1+) and Negative (0). Quick scores (Quick score = staining intensity score \u0026times; percentage of corresponding stained area) were calculated for each section for statistical analysis.\u003c/p\u003e\n\u003cp\u003e5. Western Blot\u003c/p\u003e\n\u003cp\u003ePancreatic cancer tissues and cells were lysed and sonicated using RIPA lysis buffer from Beyotime Biotechnology Co (Shanghai, China). Lysates containing soluble proteins were collected and stored at -80\u0026deg;C. Protein concentration was determined using the BCA method from BOSTER Biological Technology Co (Wuhan, China). An equal amount (30 \u0026mu;g) of protein was separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane with a transfer time of 1 h. A single wash was performed with TBS/0.1% Tween-20 (TBST) buffer, and the membrane was incubated with a 1:1000 dilution of primary antibody solution at 4\u0026deg;C overnight. The membrane was washed thrice with TBST and incubated with a 1:1000 dilution of HRP-conjugated secondary antibody for 1 h at 20-25\u0026deg;C. After incubation, the membrane was washed three more times with TBST. Finally, the immunoreactive bands were detected using Enhanced Chemiluminescent Liquid (ECL) from BOSTER Co (Wuhan, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e6. Cell Culture\u003c/p\u003e\n\u003cp\u003eFour human pancreatic cancer cell lines (MIA PaCa-2, SW1990, PANC-1 and BxPC-3) and one normal human pancreatic ductal epithelial (HPDE) cell line were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These cells were cultured according to the culture protocol provided by the supplier; BxPC-3 was cultured in RPMI-1640 complete medium (Gibco, America) containing 10% fetal bovine serum (FBS, Uruguay), MIA PaCa-2, SW1990, PANC-1 and HPDE were cultured in RPMI-1640 complete medium (Gibco, America) containing 10% fetal bovine serum (FBS, Uruguay) in DMEM complete medium (Gibco, America) and all cells were incubated at 37\u0026deg;C, 5% CO2.\u003c/p\u003e\n\u003cp\u003e7. Construction of cell lines\u003c/p\u003e\n\u003cp\u003ePANC-1 and BxPC-3 cells were transfected with GV493 lentiviral vector (SHANGHAI GENECHEM Co, China) to obtain IL20RB knockdown experimental groups (IL20RB-sh1, IL20RB-sh2 and IL20RB-sh3); transfections were performed using empty vector viruses as a negative control group. Lentiviruses were transfected according to the manufacturer\u0026apos;s instructions. Stable transfected cell lines were screened with 2-3 \u0026micro;g/ml puromycin. The lentiviral vector sequences are detailed in Appendix 1: Table S1.\u003c/p\u003e\n\u003cp\u003e8. RT-PCR\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using the M5 Universal RNA Mini Kit (Mei5bio Co.), and the quality and concentration of RNA were determined using the Nanodrop2000 (Thermo Co.). Subsequently, cDNA was synthesised using a quantitative reverse transcription kit containing de-gDNA (Takara Co.). Finally, real-time quantitative fluorescence RT-PCR analysis was performed using the TB Green dye method standardised quantification kit (Takara Co.). All steps were performed according to the kit instructions. Each sample was repeated three times. The RT-PCR results were analysed by the 2\u003csup\u003e-\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method using GAPDH as an internal reference. The sequence list of the gene-specific primers is detailed in Appendix 1: Table S2.\u003c/p\u003e\n\u003cp\u003e9. Cell Proliferation Assay\u003c/p\u003e\n\u003cp\u003eCell Counting Kit-8 (CCK-8, APExBIO Technology LLC, USA) was used for the cell proliferation assay. Cells at 2\u0026times;10\u003csup\u003e3\u003c/sup\u003e/well were inoculated into 96-well plates and incubated in a thermostat for 1-4 h. The original medium in the wells to be tested was removed after the cells had attached to the wall, and 100 ul of CCK-8 reaction solution was added (the corresponding medium was mixed with the original CCK-8 solution in a 10:1 configuration). The wells were incubated again for 2 hours at 37\u0026deg;C under 5% CO2, and the absorbance (OD) at 450 nm was measured using an enzyme-linked immunosorbent assay (BioTek Instruments Inc., USA). After 24, 48, 72, 96 and 120 h, the medium in the corresponding wells to be tested was replaced with CCK-8 reaction solution, and the OD values were recorded. Differences in the proliferation of cells in different groups were assessed by comparing their OD values at the same time points.\u003c/p\u003e\n\u003cp\u003eFor plate cloning experiments, 2000/well cells were inoculated into 6-well plates, and the medium was changed every four days in a humidified incubator at 37\u0026deg;C. After two weeks, the fixed cells were washed with PBS and stained with 0.1% crystal violet (Solarbio Co, Beijing, China) for 10-15 min. After staining, the cells were washed three times with PBS and allowed to dry naturally before being photographed. The number of cell colonies formed in each group was counted using ImageJ software to compare proliferation differences between groups.\u003c/p\u003e\n\u003cp\u003e10. Cell migration assay\u003c/p\u003e\n\u003cp\u003eFor wound healing experiments, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e/well cells were inoculated into 6-well plates and 1ml of sterile lance tip was used to scratch the plates to create trauma. After washing with PBS, the cells were incubated in a serum-free medium, photographed, and preserved at 0 and 24 h for each cell group under a 200\u0026times; lens. The area between the two edges of the wound after the specified time was measured using ImageJ software, and the difference in the migration ability of the cells in each group was compared according to the cell migration rate = (0 h wound area \u0026ndash; 24 h wound area)/0h wound area \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003eTranswell migration assays were performed using Corning (3422, 8 \u0026mu;m) migration chambers from the USA. The upper chamber was seeded with 1\u0026times;105 PANC-1 or BxPC-3 cells in a serum-free medium, approximately 200 \u0026mu;l. The corresponding RPMI-1640, or DMEM medium containing 30% fetal bovine serum, was added to the lower chamber, approximately 800 ul. Migration experiments were performed by incubation at 37\u0026deg;C, 5% CO2 for 24-48 h. Cells on the lower membrane were stained with 0.1% crystal violet staining solution (Solarbio Co, China). Global perforation images were taken at 40\u0026times;, and five randomly selected fields of view were photographed and stored at 200\u0026times;. The number of cells crossing the chambers was counted using ImageJ software to compare differences in migration ability between groups.\u003c/p\u003e\n\u003cp\u003e11. Flow Cytometry\u003c/p\u003e\n\u003cp\u003eAccording to the kit instructions, Apoptosis was detected using the Annexin V-APC/7-AAD Apoptosis Kit from MULTI SCIENCES Co (Hangzhou, China) (Catalogue No. AP104-30). Apoptosis was induced according to the experimental protocol. Cells were washed by centrifugation with pre-cooled PBS, and 1-10\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells (including cells in the culture supernatant) were collected. Dilute 5\u0026times; binding buffer to 1\u0026times; working solution and resuspend cells with 500ul of 1\u0026times; binding buffer. 5ul of Annexin V-APC and 10ul of 7-AAD were added to each tube, and after mixing by gentle vortexing, the cells were incubated for 5 min at room temperature in the dark. Apoptosis was detected using an Agilent flow cytometer (Agilent Technologies Co), and data were analysed using Novoexpress software.\u003c/p\u003e\n\u003cp\u003eA cell cycle staining kit (catalogue number CCS012) from MULTI SCIENCES Co was used according to the kit instructions. Approximately 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e-1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells were collected from each negative control and IL20RB knockdown group, and the supernatant was discarded by centrifugation. The cells were resuspended and washed once with PBS. Next, 1 ml DNA staining solution and 10 \u0026mu;l permeabilization solution were added to each sample and incubated for 30 min at room temperature in the dark. The lowest sampling rate was selected, and the cell cycle was detected by Agilent flow cytometry. The data was also analyzed using Novoexpress software.\u003c/p\u003e\n\u003cp\u003e12. Cellular transcriptome sequencing\u003c/p\u003e\n\u003cp\u003eThe stably transfected PANC-1 cell line, including the negative control group and the knockdown experimental group (IL20RB-sh1, IL20RB-sh2), were provided with two samples each to be sent to BMK Co (China) for eukaryotic reference transcriptome sequencing, and the data were further analysed using BMKCloud (www.biocloud.net).\u003c/p\u003e\n\u003cp\u003e13. Statistical analysis\u003c/p\u003e\n\u003cp\u003eSPSS 27.0, R4.3.1 and GraphPad Prism 9.0 software were used for statistical analysis and presentation. Multiple comparisons between groups were analysed using ANOVA, and post-hoc tests were performed using the Student-Newman-Keuls test. The two-tailed t-test was used for comparisons between the two groups. The Kaplan-Meier method was used to assess overall survival. Data are expressed as mean \u0026plusmn; standard deviation. p\u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e1. Bioinformatic analysis of IL20RB expression in pancreatic cancer and its association with prognosis\u003c/p\u003e\n\u003cp\u003eCombined analysis of the TCGA and GTEx databases revealed that IL20RB expression was significantly upregulated in pancreatic cancer tissue compared to normal pancreatic tissue (171 vs 179, P\u0026lt;0.001) (Figure 1 a). Re-validation of 39 paired normal pancreatic tissues and pancreatic cancer tissues using the GSE15471 dataset showed that IL20RB expression was significantly increased in pancreatic cancer tissues (P\u0026lt;0.01) (Figure 1 b). Furthermore, the area under the ROC curve (AUC) was 0.949, indicating that IL20RB has a high diagnostic value and can be used as a unique diagnostic marker to discriminate between the normal population and pancreatic cancer patients (Figure 1 c). The time-dependent ROC curves showed that the AUC values at 1, 3 and 5 years were greater than 0.6, suggesting that IL20RB has a specific reference value in prognostic assessment (Figure 1 d). Survival analysis showed that the OS time of patients in the IL20RB high-expression group was significantly shorter than that of the low-expression group (P = 0.0024) (Figure 1 e), and the DSS time was also significantly shorter than that of the low-expression group (P = 0.0079) (Figure 1 f). The results of univariate and multivariate COX regression analyses showed whether the surgery was R0 resection and IL20RB expression level were independent predictors affecting OS in pancreatic cancer patients (Table 1). In conclusion, high expression of IL20RB in pancreatic cancer is strongly associated with poor prognosis and is an independent risk factor for it.\u003c/p\u003e\n\u003cp\u003eTable 1 COX regression analysis of IL20RB clinical data\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"563\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 110px;\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 53px;\"\u003e\n \u003cp\u003eTotal（N）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 202px;\"\u003e\n \u003cp\u003eUnivariate analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 183px;\"\u003e\n \u003cp\u003eMultivariate analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003eHR（95%CI）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u0026nbsp;\u003c/em\u003evalue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eHR（95%CI）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u0026nbsp;\u003c/em\u003evalue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003ePathologic T stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e177\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eT1\u0026amp;T2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e2.056(1.090-3.878)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.026\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e1.410(0.716-2.777)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e0.320\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e1.091(0.140-8.489)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.934\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e0.909(0.117-7.092)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e0.928\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003ePathologic N stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eN1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e2.161(1.287-3.627)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e1.609(0.901-2.872)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e0.108\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003ePathologic M stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eM0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e0.773(0.185-3.227)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.724\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e0.813(0.541-1.222)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.319\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e\u0026lt;=65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e\u0026gt;65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e1.285(0.853-1.937)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.230\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eHistologic grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e177\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eG1\u0026amp;G2\u0026amp;G3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"563\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 110px;\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 53px;\"\u003e\n \u003cp\u003eTotal（N）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 202px;\"\u003e\n \u003cp\u003eUnivariate analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 183px;\"\u003e\n \u003cp\u003eMultivariate analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003eHR（95%CI）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u0026nbsp;\u003c/em\u003evalue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eHR（95%CI）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u0026nbsp;\u003c/em\u003evalue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eG4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e0.864(0.120-6.217)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.885\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eSmoker\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e1.075(0.682-1.694)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.755\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eResidual tumor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eR0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eR1\u0026amp;R2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e1.650(1.064-2.558)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e1.602(1.021-2.516)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e0.040\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eIL20RB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 134px;\"\u003e\n \u003cp\u003e2.025（1.325-3.095)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e1.757(1.124-2.747)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eInterleukin-20 receptor subunit beta; TNM, tumor, node and metastasis\u003c/p\u003e\n\u003cp\u003e2. Expression analysis of IL20RB in human pancreatic cancer tissue\u003c/p\u003e\n\u003cp\u003eTwelve pancreatic cancer surgical specimens, including matched normal pancreatic tissue, were collected. Immunohistochemistry (IHC) results showed that in pancreatic cancer cells, IL20RB was positively stained mainly at the cell membrane and was positively expressed in pancreatic cancer tissues. In contrast, the corresponding normal pancreatic tissues were negatively expressed (Fig. 2 a). In 8 of the 12 cases, pancreatic cancer tissues expressed IL20RB positively, with a positive rate of 66.7% (8/12), higher than the corresponding normal pancreatic tissues with a positive rate of 16.7% (8/12). 66.7% (8/12), which was higher than the positive rate of IL20RB expression in its corresponding normal pancreatic tissues of 16.7% (2/12); at the same time, the expression level of IL20RB in pancreatic cancer tissues was significantly higher than that in the corresponding normal pancreatic tissues (P=0.036), as shown in Table 2. In addition, IL20RB in pancreatic cancer tissues showed a higher IHC staining score (P\u0026lt;0.001) compared with the corresponding normal pancreatic tissues (Figure 2 b). Western blot experiments showed that the expression level of IL20RB was significantly up-regulated in tumour samples compared with normal pancreatic tissues (P\u0026lt;0.05) (Figure 2 c, d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2 Expression of IL20RB in pancreatic cancer and corresponding normal pancreatic tissue\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 180px;\"\u003e\n \u003cp\u003eType of organisation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eNumber of examples\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003ePositivity rate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 180px;\"\u003e\n \u003cp\u003eNormal pancreatic tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e66.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e0.036*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 180px;\"\u003e\n \u003cp\u003ePancreatic cancer tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e16.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNote: * is Fisher\u0026apos;s exact probability method; Differences are statistically significant.\u003c/p\u003e\n\u003cp\u003e3. Knockdown of IL20RB in pancreatic cancer cell lines\u003c/p\u003e\n\u003cp\u003eThe expression of IL20RB mRNA and protein in normal pancreatic epithelial cell lines (HPDE) and pancreatic cancer cell lines (MIA PaCa-2, SW1990, PANC-1 and BxPC-3) was investigated by RT-PCR and Western blot assays. The results of the RT-PCR assay showed that IL20RB mRNA expression was significantly increased in the MIA PaCa-2, SW1990, PANC-1 and BxPC-3 cancer cell groups compared with the HPDE group, and the difference was statistically significant (P\u0026lt;0.05, P\u0026lt;0.05, P\u0026lt;0.05). The MIA PaCa-2, SW1990, PANC-1, and BxPC-3 cancer cell groups showed a significant increase in IL20RB mRNA expression, and the difference was statistically significant (P\u0026lt;0.05, P\u0026lt;0.001, P\u0026lt;0.0001, P\u0026lt;0.0001) (Figure 2 e). The results of Western blot experiments also confirmed this trend, and the MIA PaCa-2, SW1990, PANC-1, and BxPC-3 cells showed a significant increase in IL20RB protein expression. Among them, in MIA PaCa-2 cells, IL20RB protein expression was increased compared to HPDE cells. However, the difference was insignificant (P\u0026lt;0. 1780). In contrast, the protein level of IL20RB was also significantly increased in the other three pancreatic cancer cell lines (SW1990, PANC-1, and BxPC-3), and the difference was statistically significant (P\u0026lt;0.05, P\u0026lt;0.05, and P\u0026lt;0.) 0.05, P\u0026lt;0.001, P\u0026lt;0.0001) (Figure 2 f, g). These results indicated that both IL20RB mRNA and protein levels were significantly up-regulated in pancreatic cancer, suggesting that IL20RB may be involved in pancreatic carcinogenesis and development as an oncogenic factor.\u003c/p\u003e\n\u003cp\u003eIn PANC-1 and BxPC-3 cells, IL20RB was the most highly expressed at both mRNA and protein levels. Therefore, in this study, the expression of IL20RB was stably knocked down in these two pancreatic cancer cell lines by lentiviral transfection. The knockdown effect of IL20RB at the mRNA and protein levels was then detected using RT-PCR and Western blot experiments. The results showed that in the three groups of cells transfected with knockdown IL20RB lentivirus (i.e. IL20RB-sh1, IL20RB-sh2 and IL20RB-sh3 groups), the expression of IL20RB was significantly reduced at both the mRNA and protein levels. The most significant knockdown effect was observed in the IL20RB-sh1 and IL20RB-sh2 groups (all P values: P\u0026lt;0.05) (Figure 2 h, i and j). Therefore, in the follow-up experiments in the present study, IL20RB-sh1 and IL20RB-sh2 were selected as the knockdown experimental groups and further studies were conducted with the negative control group.\u003c/p\u003e\n\u003cp\u003e4. Analysis of the effect of knocking down IL20RB expression on the biological properties of pancreatic cancer cells\u003c/p\u003e\n\u003cp\u003e4.1 IL20RB knockdown inhibits pancreatic cancer cell proliferation and migration capacity in vitro\u003c/p\u003e\n\u003cp\u003eIn this study, the effect of IL20RB knockdown on the proliferative capacity of pancreatic cancer cells was jointly verified by performing two parallel experiments, CCK-8 assay and cell clone formation assay. In cells stably transfected with the virus, the differences in OD levels and the number of clonal cell clusters were compared between groups of cells at different time points (0h, 24h, 48h, 72h, 96h and 120h). The results showed that in PANC-1 and BxPC-3 cells, the OD values of cells in the negative control group were significantly higher than those in the IL20RB knockdown group, and the difference gradually increased (P\u0026lt;0.0001) (Figure 3 a, b). Meanwhile, similar results were also observed in the cell cloning assay (P\u0026lt;0.01) (Figure 3 c, d). The above two validations indicated that IL20RB knockdown attenuated the proliferative ability of PANC-1 and BxPC-3 cells.\u003c/p\u003e\n\u003cp\u003eSimilarly, the wound healing rate of cells in each group within 24 hours and the number of cells migrating through the opening of the transwell within a given period were compared under the condition of stable viral transfection. The results showed that the wound healing rate of cells in the IL20RB knockdown group was significantly lower than that of the negative control group (both P values: P\u0026lt;0.01) (Figure 3 e, f). Meanwhile, the results of the transwell migration assay also showed that the number of cells migrating through the aperture of the chamber was significantly reduced in the IL20RB knockdown group compared to the negative control group (P\u0026lt;0.0001) (Figure 3 g, h). The above results indicated that the migration ability of PANC-1 and BxPC-3 cells was also significantly reduced after IL20RB knockdown treatment.\u003c/p\u003e\n\u003cp\u003e4.2 IL20RB knockdown induces apoptosis and promotes cell cycle arrest in pancreatic cancer cells in vitro\u003c/p\u003e\n\u003cp\u003eThe effects of IL20RB knockdown on apoptosis and cell cycle of PANC-1 and BxPC-3 cells were investigated by flow cytometry. The apoptosis ratio (including early and late apoptosis) and the distribution of cells in each group throughout the cell cycle were determined. The experimental results showed that the number of apoptotic cells in the IL20RB knockdown group was significantly increased (P\u0026lt;0.05) compared to the negative control group (Figure 4 a, b). In addition, there was no significant change in the distribution of cells in the G1 and G2 phases in the knockdown group. However, their distribution in the S phase was significantly increased (P\u0026lt;0.05) (Figure 4 c, d). The above flow cytometry experimental results suggested that the knockdown of IL20RB gene expression promoted pancreatic cancer cells PANC-1 and BxPC-3 to undergo apoptosis and resulted in these cells being blocked from remaining in the S phase.\u003c/p\u003e\n\u003cp\u003e5. Analysis of IL20RB-related downstream signalling pathways in pancreatic cancer cells\u003c/p\u003e\n\u003cp\u003e5.1 Effect of IL20RB knockdown on the EMT process in pancreatic cancer cells\u003c/p\u003e\n\u003cp\u003eBioinformatic analysis of GSEA revealed that the EMT process was significantly enriched in pancreatic cancer (Figure 5 b). In addition, cell function assays showed that knockdown of IL20RB significantly inhibited the migration ability of pancreatic cancer cells, further suggesting that IL20RB is associated with the EMT process in pancreatic cancer. Therefore, the present study confirmed by Western blot that the PANC-1 cells stably transfected with IL20RB-sh1 and IL20RB-sh2 groups showed increased E-cadherin expression (P\u0026lt;0.0001, P\u0026lt;0.0001) and down-regulation of EMT-related protein expression (P\u0026lt;0.0001, P\u0026lt;0.0001) compared to the negative control group in EMT-related protein expression. N-cadherin (P=0.0001, P\u0026lt;0.0001), vimentin (P\u0026lt;0.0001, P=0.0013) and snail (P=0.0079, P\u0026lt;0.0001) expression levels (Figure 6 a, c). These results were also confirmed in BxPC-3 cells (Figure 6 b, d). The above experimental results further confirmed that the knockdown of IL20RB expression in pancreatic cancer cells could inhibit the EMT process.\u003c/p\u003e\n\u003cp\u003e5.2 Inhibition of the IKK \u0026alpha;/\u0026beta;/NF-\u0026kappa;B P65 signalling pathway by IL20RB knockdown\u003c/p\u003e\n\u003cp\u003eAccording to the cellular transcriptome sequencing results (Figure 5 c, d) and GSEA enrichment analysis (Figure 5 a), a large number of genes related to IL20RB function were found to be enriched in the NF-\u0026kappa;B signalling pathway, suggesting that the functional realisation of IL20RB is closely related to NF-\u0026kappa;B signalling. Moreover, IKK/NF-\u0026kappa;B, as a classical signalling pathway, plays a vital role in cancer cell proliferation, metastasis and apoptosis. Therefore, the present study hypothesised that IL20RB may regulate pancreatic cancer cell proliferation, metastasis and apoptosis through the IKK/NF-\u0026kappa;B pathway. To test this hypothesis, the changes in key protein molecules in this pathway after IL20RB knockdown were detected at the protein level using Western blot experiments. The results showed that in PANC-1 cells, IL20RB knockdown significantly decreased IKK\u0026alpha;/\u0026beta; expression compared to the negative control group (P\u0026lt;0.0001, P\u0026lt;0.0001), whereas there was no significant change in NF-\u0026kappa;B P65 expression; however, P-NF-\u0026kappa;B P65 expression was significantly decreased (P\u0026lt;0.0001, P\u0026lt;0.0001). P-NF-\u0026kappa;B P65/NF-\u0026kappa;B P65 was also significantly reduced (P\u0026lt;0.0001, P=0.0001) (Figure 6 e, g). Consistent results were obtained in BxPC-3 cells (Figure 6 f, h). The above results confirm our hypothesis that IL20RB may promote pancreatic cancer progression through the IKK/NF-\u0026kappa;B pathway.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn pancreatic cancer, IL20RB was found to be highly expressed, to have some diagnostic predictive value and to be an independent risk factor for poor prognosis. This finding is consistent with previous studies\u003csup\u003e[17\u0026ndash;20]\u003c/sup\u003e. Functional experiments showed that knockdown of IL20RB significantly inhibited the proliferation and migration ability of pancreatic cancer cells, induced apoptosis and led to S-phase blockade. In addition, GSEA enrichment analysis and cellular transcriptome sequencing showed that IL20RB was closely associated with EMT, apoptosis and proliferation and closely correlated with NF-κB signalling. This result stimulated our interest to investigate further the relationship between IL20RB and EMT and NF-κB signalling in pancreatic cancer.\u003c/p\u003e \u003cp\u003eNF-κB acts as a nuclear transcription factor and regulates tumour cells' proliferative, anti-apoptotic, and invasive metastatic properties\u003csup\u003e[22\u0026ndash;24]\u003c/sup\u003e. It is activated through classical and non-classical pathways and interacts with I-κBs proteins to control gene transcription\u003csup\u003e[25]\u003c/sup\u003e. The frequent activation of NF-κB in pancreatic cancer suggests its potential as a therapeutic target. In addition, most studies of the relationship between NF-κB and tumourigenesis have focused primarily on the p65 subunit. In particular, nuclear translocation of NF-κB P65 has been shown to induce the EMT process in various tumour cells\u003csup\u003e[25]\u003c/sup\u003e. Conversely, the involvement of the NF-κB pathway in promoting tumour cell progression by inducing EMT is closely linked to the upstream mediators of EMT, such as ZEB1, Slug, Twist and Snail. For example, in hepatocellular carcinoma, Wu et al.\u003csup\u003e[26]\u003c/sup\u003e found that Snail acted as a direct target gene downstream of NF-κB signalling, and the IKKβ/NF-κB P65 signalling pathway was activated, upregulating Snail expression and inducing the EMT process. On the other hand, Zuo et al.\u003csup\u003e[27]\u003c/sup\u003e found that the natural product daehyenoside induced EMT in cervical cancer cells by inhibiting the NF-κB signalling pathway induced by stimulation of the hypoxic tumour microenvironment activation, decreased the expression levels of Slug and Snail in cervical cancer cells, thereby decreasing the expression of N-cadherin and Vimentin and increasing the expression of E-cadherin, which served to inhibit EMT and ultimately inhibited the migration and invasion of cervical cancer cells. The study by Qu et al.\u003csup\u003e[28]\u003c/sup\u003e showed that AKR1B10 was effective in breast cancer through the induction of the PI3K/Akt signalling pathway to stimulate the activation of the NF-κB pathway and upregulate the expression levels of ZEB1, Slug, and Twist to promote EMT, which in turn promoted the migration of breast cancer cells. Meanwhile, in a variety of tumours, including pancreatic cancer, it has been shown to have a tumour-initiating capacity after induction of EMT\u003csup\u003e[29\u0026ndash;31]\u003c/sup\u003e. At the same time, the expression levels of N-cadherin, vimentin, E-cadherin and Snail are often used to detect the presence of EMT processes\u003csup\u003e[32\u0026ndash;36]\u003c/sup\u003e. In this study, we found increased expression of E-cadherin protein and decreased expression of N-cadherin, vimentin and Snail protein after knockdown of IL20RB in both pancreatic cancer cell lines, PANC-1 and BxPC-3. This result suggests that IL20RB may promote pancreatic carcinogenesis, proliferation, migration and invasion by activating the EMT process. This finding is consistent with the results obtained in this study using CCK8, clone formation assay, and scratch and transwell assay. In addition, protein levels of IKKα/β, critical kinases involved in the NF-κB signalling pathway, as well as NF-κB-P65 and P-NF-κB P65, were detected by Western bolt, and it was found that knockdown of IL20RB resulted in decreased IKKα/β expression. However, there was no significant change in NF-κB-P65 expression, but P-NF-κB P65 expression was decreased, and the ratio of P-NF-κB P65/NF-κB-P65 was reduced. These results suggest that the knockdown of IL20RB inhibits the activation of the IKKα/β/NF-κB-P65 pathway. IL20RB, which is highly expressed in pancreatic cancer, may regulate the expression of multiple genes by activating the IKKα/β/NF-κB-P65 signalling pathway, thereby promoting the development and progression of pancreatic cancer.\u003c/p\u003e \u003cp\u003eIn addition, cell proliferation must depend on a balanced cell cycle process, and abnormalities in any component of the cell cycle can trigger tumours\u003csup\u003e[37]\u003c/sup\u003e. Han et al.\u003csup\u003e[38]\u003c/sup\u003e found that berberine interfered with bladder cancer cells' survival, proliferation and metastasis by inhibiting the NF-κB pathway. In addition, berberine-induced bladder cancer cells arrest in the S phase, resulting in anti-tumour effects. The present study obtained similar results, i.e., knockdown of IL20RB expression could effectively inhibit the proliferative ability of pancreatic cancer cells, increasing the number of cells remaining in the S phase. At the same time, there was no significant change in the distribution of cells in the G1 and G2 phases. This result suggests that IL20RB may regulate the cell cycle process of pancreatic cancer cells, and considering that the S phase is a critical period for DNA synthesis, it can be inferred after combining the results of in vitro experiments of IL20RB, it can be concluded that high expression of IL20RB may be involved in promoting DNA synthesis in the occurrence and development of pancreatic cancer. Moreover, targeting therapy to IL20RB expression in pancreatic cancer may be similar to the findings of Han et al.\u003csup\u003e[38]\u003c/sup\u003e, which is of great significance in tumour therapy.\u003c/p\u003e \u003cp\u003eUpon comprehensive analysis, the results of this study showed that IL20RB was highly expressed in both pancreatic cancer tissues and cell lines and correlated with poor patient prognosis. Knockdown of IL20RB inhibited the proliferation and migration of pancreatic cancer cells and induced apoptosis, contributing to the arrest of cells in the S phase. In contrast, IL20RB may promote the proliferation and migration of pancreatic cancer cells by activating the IKKα/β/NF-κB P65 signalling pathway and facilitating the EMT process, ultimately promoting the proliferation and migration of pancreatic cancer cells. In conclusion, IL20RB acts as an oncogenic factor in pancreatic cancer, and a more comprehensive understanding of its effects on the biological properties of pancreatic cancer and more in-depth studies on the related mechanisms will provide an essential theoretical basis for revealing the mechanisms of pancreatic cancer development and progression.IL20RB is a highly promising therapeutic target for pancreatic cancer.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author would like to thank Dr Kexiang Zhu for his techinal assistanse\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (grant no. 81960516). Natural Science Foundation of Gansu Province (grant no.22JR11RA023)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analyzed datasets generated during the study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions declaration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWanbai Ruan and Kexiang Zhu designed the study. Wanbai Ruan, Junfen Li, Yanmei Yin and Lei Peng performed the experiments. Wanbai Ruan and Junfen Li analyzed the results. All authors have read and approved the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn accordance with the Declaration of Helsinki. For human specimens, all samples were collected and used with the patient\u0026apos;s informed consent and approved by the Ethics Committee of the First Hospital of Lanzhou University (Lanzhou, China) (approval number: LDYY-2024-330).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel R L, Laversanne M, Soerjomataram I, Jemal A, Bray F Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries [J]. 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Nature. 411, 342-348(2001).https://doi.org/10.1038/35077213\u003c/li\u003e\n\u003cli\u003eHan C, Wang Z, Chen S, Li L, Xu Y, Kang W, Wei C, Ma H, Wang M, Jin X Berbamine Suppresses the Progression of Bladder Cancer by Modulating the ROS/NF-\u0026kappa;B Axis [J]. Oxid Med Cell Longev. 2021, 8851763(2021).https://doi.org/10.1155/2021/8851763\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"interleukin-20 receptor subunit beta, pancreatic cancer, biological role, nuclear factor-κb, epithelial-mesenchymal transition","lastPublishedDoi":"10.21203/rs.3.rs-5929013/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5929013/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose \u003c/strong\u003eCurrently, the pathogenesis and biological features of pancreatic cancer are not fully understood. Interleukin-20 receptor subunit beta (IL20RB) is a risk factor for poor prognosis in a variety of solid tumours, including breast cancer. However, the biological characteristics of IL20RB in pancreatic cancer and its impact on patient prognosis remain unclear. The aim of this study was to investigate the effect of IL20RB on the biological characteristics of pancreatic cancer and to explore the underlying mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eThe expression of IL20RB in pancreatic cancer and its effect on the prognosis of pancreatic cancer patients were analysed by The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Immunohistochemistry and Western blot were used to detect IL20RB expression in pancreatic cancer tissues. Pancreatic cancer cell lines PANC-1 and BxPC-3 with stable knockdown of IL20RB were constructed.The effects of IL20RB on the proliferation, migration, apoptosis and cell cycle of pancreatic cancer cells were analysed by CCK-8 assay, plate clone formation assay, wound healing assay, Transwell assay and flow cytometry. Transcriptome sequencing analysis and Western blot were used to detect changes in NF-κB signalling and key molecules during EMT after IL20RB knockdown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eIL20RB is highly expressed in pancreatic cancer and that patients with high expression have a poor prognosis. Knockdown of IL20RB significantly inhibited the proliferation and migration of pancreatic cancer cells, induced cell apoptosis, and resulted in cell arrest in the S phase. Gene set enrichment analysis (GSEA) and transcriptome analysis showed that IL20RB regulated related signalling pathways in pancreatic cancer cells, including Nuclear factor-κb (NF-κB) and Epithelial-mesenchymal transition(EMT). After the knockdown of IL20RB, the expression of EMT-related protein E-cadherin was increased, and the expression of N-cadherin, Vimentin and Snail was decreased. At the same time, the expression levels of IKKα/β and P-NF-κB P65 were significantly reduced after IL20RB knockdown. The expression level of NF-κB P65 was not changed considerably, and the ratio of P-NF-κB P65/NF-κB P65 was significantly decreased.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIL20RB is highly expressed in pancreatic cancer and is associated with poor prognosis in patients. IL20RB may promote the proliferation, migration and apoptosis of pancreatic cancer cells by activating the IKKα/β/NF-κB P65 signalling pathway to promote the EMT process.\u003c/p\u003e","manuscriptTitle":"IL20RB promotes pancreatic cancer progression by activating NF- kB signalling and promoting EMT","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-04 09:05:59","doi":"10.21203/rs.3.rs-5929013/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5abfe911-2156-4779-8c49-f0459581b2c7","owner":[],"postedDate":"February 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-04T13:53:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-04 09:05:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5929013","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5929013","identity":"rs-5929013","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}