KIFC1 overexpression promotes pancreatic carcinoma progression via the BUB1/WNT/β- catenin pathway

KIFC1 overexpression promotes pancreatic carcinoma progression via the BUB1/WNT/β- catenin pathway | 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 KIFC1 overexpression promotes pancreatic carcinoma progression via the BUB1/WNT/β- catenin pathway Ao Cui, Ying-Xue Yu, Ji-Yang Wang, Ye-Qing Zou, Ya-Qiong Zhu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4838885/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 Background: Pancreatic cancer (PC) is a highly lethal tumor of the gastrointestinal tract. New molecular targets are urgently needed for its treatment. Kinesin family member C1 (KIFC1) is implicated in the development and progression of several types of cancer. Previously, our studies indicated that KIFC1 is overexpressed in hepatocellular carcinoma and activates the malignant behavior of hepatocellular carcinoma through the PI3K/AKT pathway. However, the molecular and functional mechanisms of KIFC1 in PC have not been investigated. Methods: In this study, high-throughput sequencing technology was utilized to characterize differential gene expression profiles in patients with PC. KIFC1 was revealed by screening up-regulated genes from our sequenced data and the Gene Expression Omnibus (GEO) database. Sixty-two PC tissues were analyzed to determine the correlation of KIFC1 expression with the clinicopathological features and prognosis of patients. The role of KIFC1 in proliferation, migration and invasion in PC was verified both in vitro and in vivo. Bioinformatics analysis, coimmunoprecipitation (CoIP), and western blotting were performed to identify proteins that interact with KIFC1and further affect the downstream pathway. Results: According to high-throughput sequencing and the GEO database, KIFC1 is highly expressed in PC. KIFC1 is highly expressed in PC tissues and cells and is positively correlated with poor patient prognosis and malignant cellular behavior. Silencing KIFC1 inhibited the proliferation, migration, and invasion of PC cells, and overexpression of KIFC1 had the opposite effect. Protein‒protein interaction (PPI) and Co-IP analyses indicated that KIFC1 interacts with and regulates BUB1. Overexpression of BUB1 can also promote the proliferation, migration, and invasion of PC cells. BUB1 acts as an intermediary in the activation of the Wnt/β-catenin pathway by KIFC1, leading to an increase in the malignant behaviors of PC cells. The reversal of Wnt/β-catenin activation and increase in cellular malignant behavior induced by KIFC1 overexpression are achieved by silencing BUB1. These biological functions of KIFC1 in PC were also confirmed in a nude mouse xenograft model. Conclusions: Our experiments demonstrated for the first time that KIFC1 can influence PC progression by regulating BUB1 to activate the Wnt/β-catenin pathway. Therefore, KIFC1 shows promise as an attractive therapeutic target for PC in the future. KIFC1 BUB1 Pancreatic cancer Protein interactions Tumorigenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Pancreatic cancer (PC) causes approximately 50,000 deaths each year in America. Unfortunately, most PC patients have a poor prognosis. Investigations have shown that the 5-year survival rate for patients with PC is only 13%[ 1 ]. Surgical removal of the lesion is currently the only viable option for eradicating PC, but early metastasis or extensive local invasion renders resection for PC patients less than 20%[ 2 , 3 ]. Although adjuvant and systemic chemotherapy are currently indispensable therapeutic methods for improving long-term outcomes, their effects are still unclear[ 4 ]. In 1985, Ronald D. Vale discovered kinesin in the squid giant axon, which induces microtubule-based movements[ 5 ]. To date, 14 distinct families of kinesin superfamily proteins (KIFs) have been identified, ranging from KIF1 to KIF14[ 6 ]. KIFs participate in a range of cellular processes, including synaptic vesicle transport, centrosome clustering, and chromosomal transport during mitosis or meiosis, due to their function as molecular motors[ 7 , 8 ]. Kinesin family member C1 (KIFC1) is a member of the kinesin-14 family of C-type kinesins. Previous research has shown that KIFC1 is linked to the development and progression of several types of tumors, including prostate carcinoma[ 9 ], breast carcinoma[ 10 ], gastric carcinoma[ 11 ], endometrial carcinoma[ 12 ], and non-small cell lung carcinoma[ 13 ]. Our previous experiments showed that KIFC1 is significantly overexpressed in hepatocellular carcinoma and contributes to the progression of the disease, resulting in a poor prognosis[ 14 ]. However, the molecular and functional mechanisms of KIFC1 in PC have not been reported. The BUB1 protein plays a crucial regulatory role in mitosis, as demonstrated by recent studies[ 15 ]. It encodes a protein kinase that activates the spindle checkpoint by phosphorylating members of the mitotic checkpoint complex[ 16 – 18 ]. Various types of cancer, including bladder[ 19 ], pancreatic[ 20 ], and breast cancers[ 21 ], have been linked to mutations in BUB1. Kenneth B. Kaplan et al. reported that BUB1 phosphorylates the adenomatosis polyposis coli (APC) protein in the WNT pathway[ 22 ]. Phosphorylated APC proteins may lose their ability to inhibit the WNT pathway. Similarly, no studies have reported on the specific molecular mechanisms and functions of BUB1 in PC. The Wnt signaling pathway was initially identified in a mouse mammary tumor virus model and was designated Int-1 by Roel Nusse[ 23 ]. The Wnt/β-catenin signaling pathway is a canonical signaling pathway in which the β-catenin protein plays a key role. In the nucleus, β-catenin binds to DNA-binding proteins of the T-cell factor/lymphoid enhancing factor (TCF/LEF) family, leading to the activation of downstream target genes. Wnt pathway activation is inhibited by the degradation of β-catenin, which is phosphorylated by a disruption complex composed of APC, Axin, and GSK3β. This phosphorylation recruits the E3 ubiquitin ligase containing β-TrCP to degrade β-catenin. [ 24 ]. However, the molecular interactions between the KIFC1, BUB1, and Wnt signaling pathways in PC have not been fully elucidated. Our study revealed that KIFC1 interacts with downstream BUB1, ultimately activating the Wnt pathway and promoting PC development. This study provides a new molecular mechanism of KIFC1 in PC development and identifies new targets for the future treatment of PC. Materials and methods Patients and tissue samples This study included sixty-two patients with PC who were pathologically diagnosed between 2016 and 2023 and who underwent pancreatectomy at the Department of Hepatobiliary Surgery of the Second Affiliated Hospital of Nanchang University. The specimens were subjected to immunohistochemical analysis after being embedded in formalin and paraffin. The patients' clinicopathological data were also collected. All patients provided informed consent. Tumor staging was determined according to the Union for International Cancer Control TNM classification guide (8th edition, 2019). The Ethics Committee of the Second Affiliated Hospital approved the study. Cell lines PANC-1, SW-1990, BXPC-3, ASPC-1, and H6C7 (human normal pancreatic ductal epithelial cells) cells were purchased from the Institutes for Life Sciences, Chinese Academy of Sciences (Beijing, China). All cells were cultured in DMEM (Thermo Fisher, 12430054, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, SH30396.02, USA), except for the ASPC-1 cells, which were cultured in RPMI-1640 medium (Thermo Fisher, 11875119, USA) supplemented with 10% FBS in a humidified incubator supplemented with 5% carbon dioxide at 37°C, and the medium was changed every 3 days. Immunohistochemistry Tissue specimens were dewaxed in xylene and rehydrated in a graded series of ethanol. Afterwards, the tissue sections were placed in a pressure cooker at 100°C for 15 minutes to repair the antigens. Subsequently, the sections were incubated with H 2 O 2 for 15 minutes at room temperature to block endogenous peroxidase activity. Next, the sections were blocked with goat serum (Thermo Fisher, 16210064, USA) for 30 minutes. Next, the sections were incubated with an anti-KIFC1 antibody (1:200, ORIGENE, TA38608) at 4°C overnight. Afterwards, the sections were incubated with the corresponding secondary antibodies. Both the staining intensity and area of positive staining for KIFC1 were evaluated by two pathologists in a mutually blinded manner. Staining was graded as 0 (negative), 1 (weakly positive), 2 (moderate) or 3 (strongly positive) based on the intensity of staining. The extent of staining was scored as 1 ( 75%). The intensity and extent of the staining were multiplied to obtain a total staining score. The KIFC1 score ranged from 0 to 12, which allowed the specimens to be categorized into a low-expression group (0–3) and a high-expression group (3–12). Data mining and bioinformatics analysis The expression profiles of KIFC1 and BUB1 in PC and their respective survival curves were obtained from the Gene Expression Profiling Interactive Analysis (GEPIA) online database ( http://gepia.cancer-pku.cn/ ). KIFC1 and BUB1 gene correlation analyses were performed with the Gene_Corr module of the TIMER2.0 database ( http://timer.cistrome.org/ ). The PPIs were first identified from the STRING database and then mapped using Cytoscape v3.9.0 software. The GSE107160, GSE16515 and GSE15471 datasets were obtained from the GEO database ( https://www.ncbi.nlm.nih.gov/geo/ ). Cell transfection Small interfering RNA (siRNA) targeting KIFC1 and lentiviruses for the knockdown or overexpression of KIFC1 and BUB1 were purchased from General Biol (Anhui, China). Small interfering RNA (siRNA) was used to grow the cells in 6-well plates to 70–80% confluence, and the cells were transfected with Lipofectamine 2000 (Invitrogen, 11668-019, USA) according to the manufacturer's protocol. After the cells reached 90% confluence, they were infected with lentivirus, followed by screening with puromycin to obtain stable overexpression and knockdown cell lines. The sequences of the siRNAs used were as follows: siRNA1: 5'-GGACUUAAAGGGUCAGUUATT-3' and siRNA2: 5'-CGGGAACGCCUUCGGGAAATT-3'. Quantitative real-time PCR (qRT‒PCR) The expression of KIFC1 was assessed by qRT‒PCR. Total RNA was isolated from PC cells using TRIzol reagent, and cDNA was obtained by reverse transcription according to the instructions of TRAN One-Step gDNA Removal and cDNA Synthesis SuperMix Kits (TransGen, AE311, China). qRT‒PCR was subsequently performed using SYBR® Premix Ex Taq II Kits (Takara, RR420B, USA). The 2 −ΔΔCt method was used to calculate KIFC1 using GAPDH as an internal reference[ 25 ] for relative expression. The sequences of the primers used were as follows: KIFC1-F: 5'-GCAGGAACTCAAGGGCAA-3', KIFC1 -R 5'-GCTAAGGCGGGGTTGGAG-3'; and GAPDH-F: 5'-GGACCTGACCTGCCGTCTAG-3', GAPDH-R 5'-GTAGCCCAGGATGCCCTTGA-3'. Western blotting Cell or tissue samples were lysed using RIPA lysis buffer supplemented with protease inhibitors. The proteins were separated by SDS‒PAGE using either an 8% or 10% gel and then transferred to 0.22 \(\:\mu\:m\) PVDF membranes (Millipore, ISEQ00010, USA). The membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4°C. The strips were then washed with TBST and incubated with secondary antibodies for one hour at room temperature. Finally, the strips were visualized using a bioimaging system and analyzed with ImageJ software. Primary antibodies against KIFC1 (cat. no. TA384608, ORIGENE), BUB1 (cat. no. HA60053, HUABIO), β-catenin (cat. no. #8480, Celling Signaling), TCF-4 (cat. no. #2569, Celling Signaling), c-Myc (cat. no. #9402, Celling Signaling), cyclin D1 (cat. no. #55506, Celling Signaling), N-cadherin (cat. no. #13116, Celling Signaling), E-cadherin (cat. no. #3195, Celling Signaling), vimentin (cat. no. #5741, Celling Signaling), and GAPDH (cat. no. #5174, Celling Signaling) were used. Cell Counting Kit-8 The transfected cells were inoculated into 96-well plates at a density of 4000 cells per well and cultured for 24, 48, or 72 hours. Then, 10 \(\:\mu\:L\:\) of CCK-8 (Abcam, ab228554, USA) reagent was added to each well at the end of each incubation cycle. After a 2-hour incubation, the absorbance of each well at 450 \(\:\:nm\:\) was measured using a microplate reader. Colony Formation Assay Cells that had been transfected and resuspended were inoculated into six-well plates at a density of 800 cells per well. The cells were cultured for 14 days, and the cells in each well were fixed with paraformaldehyde for 30 minutes at room temperature and then stained with crystal violet for 10 minutes. A microscope was used to count the number of cell clusters. Cell proliferation assay Cell proliferation was assessed by a 5-ethynyl-2ʹdeoxyuridine (EdU) proliferation assay, and an EdU kit (UElandy, C6044S, China) was purchased from UElandy Biotechnology Co. in Suzhou, China. Transfected and resuspended cells were inoculated into 96-well plates at 1 × 10 5 cells per well. Once the cells had attached to the wall, the EdU reagent was added to the medium, and the cells were incubated for two hours. After this, the cells were fixed with 4% paraformaldehyde and destained with glycine, and the cell membrane was permeabilized with 0.5% Triton X-100. YF594 or YF488 was added for 30 minutes at 25°C in the dark. After washing, Hoechst 33342 was added for another 30 minutes under the same conditions. Images were acquired using a fluorescence microscope. Wound healing The fused cells were treated with mitomycin (1 \(\:\:\mu\:g/mL\) ) for one hour after being switched to serum-free medium. Vertical scratches were made at the bottom using a 200 \(\:\mu\:l\:\) pipette gun tip. The shed cells were washed away with PBS, and the scratches were imaged at 0 h. Images were captured again 24 h after incubation, and the rate of cell migration was calculated. Transwell assay For the invasion assay, Matrigel was spread into Transwell chambers. Transfected cells were then resuspended in serum-free DMEM and added to the upper chambers, while DMEM containing 10% FBS was added to the lower chambers. The chambers were incubated for 48 hours in a 24-well plate. After incubation, the chambers were fixed with 4% paraformaldehyde for 25 minutes and stained with crystal violet for 5 minutes. Images were captured using a microscope, and the cells were counted. Cell Cycle Assay A total of 1×10 5 posttransfection cells were collected and fixed with precooled 70% ethanol in a refrigerator at 4°C overnight. The following day, the cells were washed twice with PBS, stained with 0.5 ml of PI/RNase (Abcam, ab112116, USA) and resuspended. The cells were incubated for 30 minutes at room temperature in the dark. Afterward, the cell cycle distribution was analyzed using flow cytometry. Animal Experiments Female BALB/c nude mice (6–8 weeks old) weighing 15 ± 1 g from Beijing SPF Biotechnology Co., Ltd., were used to construct a xenograft tumor model. All animal experiments were conducted at Nanchang Royo Biotech Co,. Ltd. The aim of this study was to investigate the effect of KIFC1 on tumor formation in vivo. The mice were injected subcutaneously with 1×10 6 PANC-1 cells (with stable KIFC1 knockdown) resuspended in 100 \(\:\:\mu\:L\:\) of serum-free DMEM. Tumor volumes were measured at five-day intervals, and images of the mice were captured after 30 days using a small animal live imager. After the mice were euthanized, the tumor tissue was removed for detection. All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanchang Royo Biotech Co,. Ltd (Nanchang, China, IACUC No. RYE2024033001) Statistical analysis All analyses were performed using SPSS 26.0 software. Survival curves were plotted using the Kaplan‒Meier method and compared using the log-rank test, and the association between KIFC1 and clinicopathology was tested using the \(\:\chi\:2\) test. Bivariate correlations were calculated using Pearson's correlation coefficient. Comparisons between two groups were made using two independent samples t tests, and differences were considered statistically significant at P < 0.05. The data are presented as the means ± SDs, and P < 0.05 was considered to indicate statistical significance. Results KIFC1 is highly expressed in PC tissue and is associated with advanced clinical stage and poor prognosis. Both the high-throughput sequencing and GSE107610 datasets indicated that KIFC1 is highly expressed in PC (Fig. 1 A). To explore the expression and location of KIFC1 in PC patients, we performed immunohistochemistry (IHC) analysis on 62 PC patient samples. We observed almost no KIFC1 expression in normal tissues. On the other hand, KIFC1 expression was significantly high in neoplastic tissues, and it was detected mainly in the nuclei of tumor cells (Fig. 1 B). This finding was supported by findings obtained from the online GEPIA database (Fig. 1 C). Furthermore, by analyzing the clinicopathological features of 62 patients, we found that a high KIFC1 expression level was closely correlated with T stage (P = 0.016), TNM stage (P = 0.017) and histological grade (P = 0.001) in patients with PC (Table 1 ). These findings indicate that KIFC1 expression is clearly related to poor clinical and pathological stage. Therefore, we analyzed the association between KIFC1 expression and patient survival by Kaplan‒Meier analysis, and the results demonstrated that KIFC1-high PC patients had a significantly worse overall survival (OS) than did KIFC1-low patients (log rank P = 0.008; Fig. 1 E). This result was also confirmed by the GEPIA database (log rank P = 0.0059; Fig. 1 D). Table 1 Relationships between KIFC1 expression and the clinicopathological features of 62 PC patients. Variables n KIFC1 χ 2 P value High expression Low expression Sex 0.466 0.495 Male 25 17(68.0%) 8(32.0%) Female 37 22(59.5%) 15(40.5%) Age (years) 0.726 0.394 ≥ 60 34 23(67.6%) 11(32.4%) < 60 28 16(57.1%) 12(42.9%) T staging 8.290 0.016* T1 20 8(40.0%) 12(60.0%) T2 27 18(66.7%) 9(33.3%) T3 15 13(86.7%) 2(13.3%) Nerve infiltration 0.777 0.378 Yes 47 31(66.0%) 16(34.0%) No 15 8(53.3%) 7(46.6%) Venous invasion 2.503 0.114 Yes 35 25(71.4%) 10(28.6%) No 27 14(51.9%) 13(48.1%) Histological grade 11.519 0.001* High 19 6(31.6%) 13(68.4%) Low / Medium 43 33(76.7%) 10(23.3%) Lymph node status 0.521 0.470 Negative 26 15(57.7%) 11(42.3%) Positive 36 24(66.7%) 12(33.3%) TNM staging 8.127 0.017* I 19 7(36.8%) 12(63.2%) II 29 21(72.4%) 8(27.6%) III 14 11(78.6%) 3(21.4%) KIFC1 overexpression promotes PC cell growth in vitro The KIFC1 protein and mRNA were examined by western blotting and qRT‒PCR, and the results revealed that KIFC1 was more highly expressed in PC cell lines than in normal pancreatic ductal epithelial cells (Fig. 1 F and G). To confirm that KIFC1 promotes PC cell proliferation, two independent siRNAs and one KIFC1-overexpressing lentiviral vector were used to silence or overexpress KIFC1, respectively. SiRNA transfection and lentivirus infection were detected in PANC-1 and SW-1990 cells via western blotting, and the KIFC1 protein levels decreased and increased, respectively (Fig. 2 A). Compared with control cells, SW-1990 cells overexpressing KIFC1 exhibited greater cell viability, colony formation and proliferation (Fig. 2 B, C and 3 A). However, KIFC1 knockdown in PANC-1 cells had opposite effects on cell viability, colony formation and proliferation (Fig. 2 B, C and 3 A). Furthermore, a cell cycle assay demonstrated that KIFC1 promoted progression through the cell cycle from the G1 to the S and G2 phases. (Fig. 3 B). KIFC1 promoted PC cell migration and invasion. Scratch and Transwell assays were performed in PANC-1 and SW-1990 cells to examine the impact of KIFC1 on PC cell migration and invasion. A scratch assay indicated that KIFC1 overexpression markedly enhanced migration, while the opposite effect was observed in the control group (Fig. 4 A). In addition, Transwell assays revealed that PC invasion capacity was closely related to KIFC1 expression (Fig. 4 B). It is widely known that epithelial–mesenchymal transition (EMT) is an indispensable factor that promotes cell migration and invasion[ 26 , 27 ]. Therefore, western blotting was used to verify the key role of EMT in PC cell migration and invasion. The results demonstrated that the epithelial marker E-cadherin was repressed; in contrast, the mesenchymal markers N-cadherin and vimentin were increased in KIFC1-overexpressing PC cells. However, silencing KIFC1 had the opposite effect (Fig. 4 C). Overall, the above results indicated that KIFC1 promoted PC cell migration and invasion. KIFC1 overexpression activated the Wnt/β/catenin pathway in PC cells. In a previous study, the Wnt/β/catenin pathway was verified to contribute to PC malignancy[ 28 ]. The dephosphorylation of β-catenin prevents it from being degraded by ubiquitination, after which it accumulates in the nucleus and increases the expression of TCF/LEF. TCF/LEF subsequently interacts with downstream targets, such as c-Myc and cyclin D1[ 29 ]. Western blotting revealed that KIFC1 overexpression upregulated β-catenin, TCF4, c-My and cyclin D1, while KIFC1 depletion downregulated the expression of these genes (Fig. 4 C). Thus, the results indicated that KIFC1 might affect PC malignancy by activating the Wnt/β-catenin signaling pathway. BUB1 interacts with KIFC1 at the protein level, is highly expressed in PC patients and is associated with poor prognosis. Using high-throughput sequencing, we screened 2922 upregulated genes. After genes that were not interrelated were eliminated, PPI analysis revealed that KIFC1 may interact with BUB1. (Fig. 5 A). To investigate the relationship between KIFC1 and BUB1 in PC, we analyzed transcriptomic data from The Cancer Genome Atlas (TCGA) Spearman's correlation analysis confirmed a strong positive correlation between the expression of KIFC1 and that of BUB1 in PC (r = 0.835; Fig. 5 B). Moreover, interactions between KIFC1 and BUB1 were observed at the protein level via Co-IP assays (Fig. 5 C). To confirm the relationship between BUB1 expression levels and clinical outcomes, we selected two datasets (GSE15471 and GSE16151) from the GEO database for paired-sample t tests. The results showed that BUB1 expression was significantly greater in tumor tissues than in adjacent normal tissues (Fig. 5 D and E). Additionally, the GEPIA database revealed high expression of BUB1 in patients with tumors, which adversely affected patient prognosis (Fig. 5 F and G). BUB1 mediates the effect of KIFC1 on the Wnt/β-catenin pathway and the malignancy of PC cells. Western blotting revealed that BUB1 was successfully knocked down or overexpressed (Fig. 6 A). EdU experiments revealed that BUB1 knockdown decreased the proliferation of PC cells, whereas BUB1 overexpression had the opposite effect (Fig. 6 B). Similarly, a positive correlation between BUB1 expression and cell migration and invasion ability was observed in the scratch and Transwell assays (Fig. 6 C and D). The knockdown and overexpression of KIFC1 in PANC-1 cells resulted in a similar trend for BUB1 (Fig. 7 A). A lentivirus was used to knock down BUB1, which reversed the positive effects of KIFC1 overexpression on the proliferation, migration, and invasion ability of PANC-1 cells (Fig. 7 B-D). Similarly, decreased protein levels of β-catenin, TCF-4, c-Myc, and cyclin D1 were observed in KIFC1-overexpressing cells upon BUB1 silencing (Fig. 7 E). These results demonstrated that reducing BUB1 expression inhibits the activation of the Wnt/β-catenin pathway induced by KIFC1 overexpression. KIFC1 enhances PC growth and activation of the Wnt/β-catenin pathway in vivo We established subcutaneous tumor models to explore the function of KIFC1 in regulating PC tumorigenesis. Tumors with silenced KIFC1 exhibited significantly lower growth rates, weights, and volumes than those in the control group. (Fig. 8 A-C). Moreover, a small animal imaging system was used to evaluate tumor growth, and the results also verified that KIFC1 knockdown obviously suppressed cancer cell proliferation (Fig. 8 D). Furthermore, Western blotting revealed that KIFC1 regulates BUB1 at the protein level and affects the activation of the Wnt/β-catenin pathway in vivo (Fig. 8 E). Thus, in vivo experiments confirmed that KIFC1 regulates BUB1 and the downstream Wnt/β-catenin pathway, promoting tumor growth. Discussion PC is a highly malignant tumor of the gastrointestinal tract, with an incidence rate almost equal to the mortality rate[ 30 ]. Due to its insidious early onset, most cases are already in advanced stages when it is detected. PC can only be eliminated through surgery, but the chances of recurrence after the procedure are high, leading to a poor prognosis[ 31 ]. Therefore, finding potential molecular targets for PC may lead to new directions for its treatment. Through a series of experiments, we confirmed that KIFC1 is highly expressed in PC cell lines and patients. KIFC1 overexpression activates the Wnt/β-catenin pathway through BUB1, promoting malignant behavior and functions in PC cells. These findings indicate that the KIFC1-BUB1-Wnt pathway signaling axis could be a more effective target for PC therapy. With respect to KIFC1, growing evidence suggests that KIFC1 overexpression is strongly associated with cancer development, progression, and drug resistance[ 32 , 33 ]. Cancer cells typically exhibit centrosome amplification, and these cells survive and proliferate by clustering supernumerary centrosomes for bipolar division[ 34 , 35 ]. Coincidentally, KIFC1 can aggregate centrosomes to achieve bipolar division of cancer cells, promoting their survival and proliferation[ 36 , 37 ]. These findings demonstrate that KIFC1 may promote PC progression by facilitating centrosome aggregation in PC cells. Previous studies have demonstrated that the Wnt/β-catenin signaling pathway can promote PC tumorigenesis and drug resistance[ 38 , 39 ]. Targeting this pathway has become a crucial initiative for treating PC and other gastrointestinal tumors[ 40 ]. In head and neck carcinoma, KIFC1 has been identified as an 'activator' of the Wnt/β-catenin signaling pathway, promoting the development of head and neck squamous cell carcinoma[ 41 ]. Our experimental results suggest that KIFC1 may promote PC development by activating the Wnt/β-catenin signaling pathway. Further mechanistic exploration revealed that KIFC1 binds to BUB1 at the protein level. Knocking down BUB1 reversed the increase in cell function and Wnt/β-catenin pathway activity caused by KIFC1 overexpression, suggesting that KIFC1 may activate the Wnt/β-catenin pathway via BUB1. BUB1 functions as a protein kinase that phosphorylates APC (a component of the disruption complex)[ 42 ]. We speculate that this phosphorylation may alter the activity of the disruption complex, preventing the degradation of most β-catenin. The retained β-catenin then enters the nucleus and binds to TCF/LEF proteins, transcribing downstream target genes. BUB1 is a crucial component in chromosome segregation, and BUB1 overexpression induces Aurora-B hyperactivation, resulting in chromosomal missegregation and aneuploidy. [ 43 , 44 ]. In general, cancer cells with aneuploidy-containing and dispersed centromeres tend to undergo multipolarization, preventing them from dividing and leading to cell death[ 45 ]. However, KIFC1 promotes the aggregation of dispersed centrosomes in cancer cells, facilitating polar division and ensuring cell survival[ 36 , 37 ]. The available evidence indicates that KIFC1 and BUB1 have molecular and cellular interactions that promote the proliferation, migration, and invasion of PC cells both in vitro and in vivo by activating the downstream Wnt/β-catenin pathway. Conclusion In summary, in the clinic, KIFC1 is expressed at high levels in PC tissues and cells, which significantly reduces the overall survival and results in a poor prognosis for patients. Mechanistically, KIFC1 overexpression contributed to PC cell proliferation, migration, and invasion via the BUB1/Wnt/β-catenin pathway. (Fig. 9 ). Abbreviations KIFC1 Kinesin family member C1 PC Pancreatic cancer TCGA The Cancer Genome Atlas Program GEO Gene Expression Omnibus APC Adenomatosis polyposis coli GEPIA Gene Expression Profiling Interactive Analysis PPI Protein‒Protein Interaction Networks EMT Epithelial–mesenchymal transition IHC Immunohistochemistry TCF/LEF T-cell factor/Lymphoid enhancing factor OS Overall Survival Declarations Ethics Approval and Consent to Participate The use of human tissue specimens for this study was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University. Animal ethics were reviewed and approved by the Institutional Animal Care and Use Committee of Nanchang Royo Biotech Co., Ltd. Competing interests The authors declare no competing interests. Consent for publication All authors agreed with submission of the manuscript for publication and agree to be accountable for all aspect of the manuscript. Funding This research was funded by grants from the Natural Science Foundation of Jiangxi Province, China (No. 20232BAB216076, 20224BAB216050, 20232BAB206078 and 20224ACB206037); the Research Project of Education Department of Jiangxi Province, China (No. GJJ2200227 and GJJ2200237); the National Natural Science Foundation Incubation Project of The Second Affiliated Hospital of Nanchang University (2022YNF12020 and 2022YNFY12002); and the National Natural Science Foundation of China (No. 82360220 and 82360513). Author Contribution A.C: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, and Writing—original draft. Y.X.Y: Resources, Validation, and Writing—original draft. J.Y.W and L.J.R: Resources, Data curation and Validation. Y.Q.Z: Resources, Funding acquisition and Writing—review & editing. Y.Z, R.X.L, M.Y.D and H.W: Resources, Validation, and Data curation. L.F: Resources, Supervision, Funding acquisition, Validation, and Writing—review & editing. X.W.F: Resources, Supervision, Funding acquisition and Writing—review & editing. Acknowledgement We would like to express our gratitude to the editors and reviewers for their hard work and the authors for their efforts. Data availability Data will be made available on request by contacting the corresponding author. References Siegel RL, Giaquinto AN, Jemal A (2024) Cancer statistics, 2024. CA Cancer J Clin 74:12–49 Kindler HL (2018) A Glimmer of Hope for Pancreatic Cancer. N Engl J Med 379:2463–2464 Malafa MP (2015) Defining borderline resectable pancreatic cancer: emerging consensus for an old challenge. J Natl Compr Canc Netw 13:501–504 Macchini M, Peretti U, Orsi G, Zanon S, Mazza E, Valente MM, et al (2021) Exploring chemotherapy holiday and drugs re-challenge in advanced pancreatic cancer patients. 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Chemistry & Biology 20:1399–1410 Rath O, Kozielski F (2012) Kinesins and cancer. Nat Rev Cancer 12:527–539 Tang B, Yang Y, Kang M, Wang Y, Wang Y, Bi Y, et al (2020) m6A demethylase ALKBH5 inhibits pancreatic cancer tumorigenesis by decreasing WIF-1 RNA methylation and mediating Wnt signaling. Mol Cancer 19:3 Deng J, Zhang J, Ye Y, Liu K, Zeng L, Huang J, et al (2021) N6 -methyladenosine–Mediated Upregulation of WTAPP1 Promotes WTAP Translation and Wnt Signaling to Facilitate Pancreatic Cancer Progression. Cancer Research 81:5268–5283 White BD, Chien AJ, Dawson DW (2012) Dysregulation of Wnt/β-Catenin Signaling in Gastrointestinal Cancers. Gastroenterology 142:219–232 Yu B-Y, Shi L-G, Jiang C, Wang G-K, Liu J, Wu T-Y (2023) Kinesin Family Member C1 Overexpression Exerts Tumor-Promoting Properties in Head and Neck Squamous Cell Carcinoma via the Rac1/Wnt/β-catenin Pathway. Lab Invest 103:100134 Kaplan KB, Burds AA, Swedlow JR, Bekir SS, Sorger PK, Näthke IS (2001) A role for the Adenomatous Polyposis Coli protein in chromosome segregation. Nat Cell Biol 3:429–432 Ricke RM, Jeganathan KB, Van Deursen JM (2011) Bub1 overexpression induces aneuploidy and tumor formation through Aurora B kinase hyperactivation. Journal of Cell Biology 193:1049–1064 Ricke RM, Van Deursen JM (2011) Aurora B hyperactivation by Bub1 overexpression promotes chromosome missegregation. Cell Cycle 10:3645–3651 Kwon M, Godinho SA, Chandhok NS, Ganem NJ, Azioune A, Thery M, et al (2008) Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes. Genes Dev 22:2189–2203 Additional Declarations No competing interests reported. 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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-4838885","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":346203138,"identity":"ef053d71-bb5e-4f15-ab8d-9c4ec7de17c8","order_by":0,"name":"Ao Cui","email":"","orcid":"","institution":"The Second Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Ao","middleName":"","lastName":"Cui","suffix":""},{"id":346203139,"identity":"40a89630-5ee1-48a5-97da-21a3840db267","order_by":1,"name":"Ying-Xue Yu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Ying-Xue","middleName":"","lastName":"Yu","suffix":""},{"id":346203140,"identity":"01c1cec5-fd17-4adb-adbc-a97f6a2ccf10","order_by":2,"name":"Ji-Yang Wang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Ji-Yang","middleName":"","lastName":"Wang","suffix":""},{"id":346203141,"identity":"f2dfdc8c-0ae7-4270-bbcf-b9b0a233f144","order_by":3,"name":"Ye-Qing Zou","email":"","orcid":"","institution":"The Second Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Ye-Qing","middleName":"","lastName":"Zou","suffix":""},{"id":346203142,"identity":"ec582438-bf2d-437f-9f09-fe0fd1a7e399","order_by":4,"name":"Ya-Qiong Zhu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Nanchang 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University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Fang","suffix":""},{"id":346203149,"identity":"2ad0fa06-6dec-4435-92c6-55ab7a42386a","order_by":11,"name":"Xiao-Wei Fu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIie3RMQrCMBTG8RQhXR7OTyx6hYAgDoEexCUiZKo4uHaIOHgFwVP0BsFAJw+QQZBScNbFTTGjTombYP77D97HIyQW+8Hysxb69uRAUxVqLGmaHZVZF3QwSdoR0AMfoAgUyX4jEUAD7bWVJSWfekknqyUinoD25WpCarlQPkJJIZGxiyPFGBNl/ATI8o5CGHfYMZAgijnT2hGEQMJAz5q1kkDBbREhW1iqhHkong+3prLXkvvJBw9+zRv5VsRisdh/9AIMcT1SC6yiQwAAAABJRU5ErkJggg==","orcid":"","institution":"The Second Affiliated Hospital of Nanchang University","correspondingAuthor":true,"prefix":"","firstName":"Xiao-Wei","middleName":"","lastName":"Fu","suffix":""}],"badges":[],"createdAt":"2024-08-01 03:12:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4838885/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4838885/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64145356,"identity":"b6944796-feb1-47ae-8e16-d42432601b7b","added_by":"auto","created_at":"2024-09-08 19:49:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3782894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIFC1 is expressed at high levels in both pancreatic cancer tissues and cells A.\u003c/strong\u003e The volcano plot was constructed using the fold change values and P values. Red dots represent upregulated genes, blue dots represent downregulated genes, and gray dots represent genes whose expression did not significantly change. KIFC1 was jointly screened using our high-throughput sequencing data and the GSE107160 dataset. \u003cstrong\u003eB.\u003c/strong\u003e Immunohistochemical (IHC) staining of clinical human pancreatic adenocarcinoma and corresponding paracancerous tissue samples: (a) ×40 normal pancreatic tissue sample, (b) ×400 normal pancreatic tissue sample, (c) ×40 pancreatic tumor tissue sample, and (d) ×400 pancreatic tumor tissue sample. \u003cstrong\u003eC.\u003c/strong\u003eThe GEPIA database shows significant upregulation of KIFC1 gene expression in PC tissues (n=179) compared to normal pancreatic tissues (n=171). \u003cstrong\u003eD, E.\u003c/strong\u003e Overall survival (OS) of 62 PC patients from our collected data and OS of 178 PC patients from the GEPIA database. \u003cstrong\u003eF, G.\u003c/strong\u003e RT‒qPCR and western blotting were used to measure the expression of KIFC1 mRNA and protein in H6C7, PANC-1, BXPC-3, ASPC-1, and SW-1990 cells. The values are presented as the means ± SDs. *P \u0026lt; 0.05, **P \u0026lt;0.01, ***P \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4838885/v1/1dde42f8d4a34bd75f216fa9.png"},{"id":64144630,"identity":"100fd89a-853b-4ae8-8ed9-c19dd1ca28ad","added_by":"auto","created_at":"2024-09-08 19:41:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":954671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIFC1 promotes the proliferation of PC cells A.\u003c/strong\u003e Western blotting demonstrated that SiRNAs reduced KIFC1 expression, while overexpression of KIFC1 lentivirus increased KIFC1 expression. \u003cstrong\u003eB.\u003c/strong\u003e CCK-8 assays showed that siRNA-KIFC1 inhibited PC cell viability, while KIFC1 overexpression improved cell viability. \u003cstrong\u003eC.\u003c/strong\u003e Colony formation assays revealed that siRNA-KIFC1 reduced the colony-forming ability of cells, while KIFC1 overexpression increased the colony-forming ability of cells. The values are presented as the means± SDs. *P \u0026lt; 0.05, **P \u0026lt;0.01, ***P \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4838885/v1/7abbfde592e7747ccd0835e1.png"},{"id":64143758,"identity":"d6d7d20e-2b0f-4636-b9c7-296b098504f2","added_by":"auto","created_at":"2024-09-08 19:33:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":894026,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIFC1 promotes PC cell proliferation and cell cycle progression A.\u003c/strong\u003e An EdU assay revealed that the proliferative capacity of KIFC1-knockdown cells was decreased and that of KIFC1-overexpressing cells was increased. \u003cstrong\u003eB.\u003c/strong\u003eKIFC1 knockdown caused cell cycle arrest in the G1 phase, as determined by flow cytometry, while KIFC1 overexpression promoted cell cycle progression from the G1 phase to the S and G2 phases. The values are presented as the means ± SDs. **P \u0026lt;0.05, ***P \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4838885/v1/99935d6cb28369c1d3affc28.png"},{"id":64143763,"identity":"45194df3-b3c3-4903-befb-dbd5ec0414c6","added_by":"auto","created_at":"2024-09-08 19:33:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1740932,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIFC1 promotes PC cell migration, invasion, EMT and activation of the Wnt/β-catenin pathway\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e. A scratch assay was used to detect the effect of KIFC1 on cell migration, and KIFC1 knockdown cells had decreased migration ability, while the opposite was true for the overexpression group. \u003cstrong\u003eB.\u003c/strong\u003e Transwell assays showed that KIFC1 knockdown reduced cell invasion, while KIFC1 overexpression increased cell invasion. \u003cstrong\u003eC. \u003c/strong\u003eWestern blotting demonstrated that knockdown of KIFC1 reduced the protein expression of N-cadherin, β-catenin, TCF4, vimentin, C-Myc, and cyclin D1 and increased the protein expression of E-cadherin, while overexpression of KIFC1 had the opposite effect. The values are presented as the means ± SDs. *P \u0026lt; 0.05, **P \u0026lt;0.01, ***P \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4838885/v1/8894ade0749dba364e3e12cf.png"},{"id":64144631,"identity":"3d77078f-efa1-4333-8148-c92c6212c652","added_by":"auto","created_at":"2024-09-08 19:41:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2853715,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIFC1 interacts with BUB1 A.\u003c/strong\u003e Upregulated hub genes identified by high-throughput sequencing. The PPI network revealed that KIFC1 is correlated with BUB1. \u003cstrong\u003eB.\u003c/strong\u003e TIMER 2.0 database analysis showed that KIFC1 is strongly correlated with BUB1 expression. \u003cstrong\u003eC.\u003c/strong\u003e Co-IP experiments demonstrated that KIFC1 interacts with BUB1 at the protein level. \u003cstrong\u003eD, E.\u003c/strong\u003e Both GSE15471 and GSE16515 indicate that BUB1 expression was greater in cancer tissues than in adjacent paracancerous tissues. \u003cstrong\u003eF. \u003c/strong\u003eThe GEPIA database indicated that BUB1 expression is significantly greaterin PC tissues than innormal pancreatic tissues. \u003cstrong\u003eG.\u003c/strong\u003e In the GEPIA database, high expression of BUB1 was associated with a poor prognosis. The values are presented as the means ± SDs. ****P \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4838885/v1/c4e8477a92737b3ae40c72c9.png"},{"id":64143761,"identity":"2ba79463-0f23-45ba-9cb5-5fea97b50aca","added_by":"auto","created_at":"2024-09-08 19:33:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2202015,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBUB1 affects PC cell proliferation, migration and invasion A. \u003c/strong\u003eWestern blotting confirmed that the BUB1 knockdown and overexpression lentiviruses effectively silenced and overexpressed BUB1, respectively. \u003cstrong\u003eB-D.\u003c/strong\u003e Positive effects of BUB1 on the proliferation, migration, and invasion of PANC-1 cells. The values are presented as the means ± SDs. *P \u0026lt; 0.05, **P \u0026lt;0.01, ***P \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4838885/v1/449eba9eb9ca62cb9c55a467.png"},{"id":64143765,"identity":"11491fe8-de38-4a0f-b912-53bcda42d7a5","added_by":"auto","created_at":"2024-09-08 19:33:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1981072,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIFC1 activates the WNT/β-catenin pathway through BUB1, resulting in the malignant behaviors of PC cells.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e KIFC1 positively regulates the protein expression of BUB1 in PC cells. \u003cstrong\u003eB-E.\u003c/strong\u003e The increase in PC cell proliferation, migration, invasion, and activation of the Wnt/β-catenin pathway induced by KIFC1 overexpression was reversed upon knockdown of BUB1. The values are presented as the means ± SDs. *P \u0026lt; 0.05, **P \u0026lt;0.01, ***P \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4838885/v1/8f03111d4103b4e79ca97ef9.png"},{"id":64144633,"identity":"089abc0c-95e1-4568-98d6-5c970707ff8c","added_by":"auto","created_at":"2024-09-08 19:41:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2661666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIFC1 promotes PC proliferation and activation of the BUB1/WNT/β-catenin pathway in vivo\u003c/strong\u003e \u003cstrong\u003eA. \u003c/strong\u003eRepresentative images of subcutaneous tumor nodule size in mice on day 30. \u003cstrong\u003eB, C.\u003c/strong\u003e Final tumor mass bar graphs and line graphs of subcutaneous tumor growth versus time. \u003cstrong\u003eD.\u003c/strong\u003e Representative images of subcutaneous tumors in mice on day 30 were acquired using an animal living imager. \u003cstrong\u003eE.\u003c/strong\u003e Western blotting showed that KIFC1 regulates the downstream BUB1 and Wnt/β-catenin pathways in vivo. The values are presented as the means± SDs. ***P \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-4838885/v1/3d3aa37ab0152c7b848e12eb.png"},{"id":64143764,"identity":"8bf99340-f060-43bb-9df8-7db9b81b1b1d","added_by":"auto","created_at":"2024-09-08 19:33:58","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":59406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the potential molecular mechanism of KIFC1 as a key regulator in PC progression. \u003c/strong\u003eKIFC1 overexpression promotes PC progression via the BUB1/WNT/β-catenin pathway.\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-4838885/v1/996c6dbc794ba31c8b305d1c.png"},{"id":74100682,"identity":"0d92f74b-4c9d-4fde-9f50-c05c07b064bf","added_by":"auto","created_at":"2025-01-17 18:16:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19436816,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4838885/v1/b50a0b9d-7400-483b-afb5-b40c7c35df58.pdf"},{"id":64143767,"identity":"bc2fa2e9-f66c-4549-9da6-0fc7d58e9477","added_by":"auto","created_at":"2024-09-08 19:33:58","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":1317517,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4838885/v1/c3a921f80d10207991bb2a91.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"KIFC1 overexpression promotes pancreatic carcinoma progression via the BUB1/WNT/β- catenin pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic cancer (PC) causes approximately 50,000 deaths each year in America. Unfortunately, most PC patients have a poor prognosis. Investigations have shown that the 5-year survival rate for patients with PC is only 13%[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Surgical removal of the lesion is currently the only viable option for eradicating PC, but early metastasis or extensive local invasion renders resection for PC patients less than 20%[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although adjuvant and systemic chemotherapy are currently indispensable therapeutic methods for improving long-term outcomes, their effects are still unclear[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn 1985, Ronald D. Vale discovered kinesin in the squid giant axon, which induces microtubule-based movements[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To date, 14 distinct families of kinesin superfamily proteins (KIFs) have been identified, ranging from KIF1 to KIF14[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. KIFs participate in a range of cellular processes, including synaptic vesicle transport, centrosome clustering, and chromosomal transport during mitosis or meiosis, due to their function as molecular motors[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Kinesin family member C1 (KIFC1) is a member of the kinesin-14 family of C-type kinesins. Previous research has shown that KIFC1 is linked to the development and progression of several types of tumors, including prostate carcinoma[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], breast carcinoma[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], gastric carcinoma[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], endometrial carcinoma[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and non-small cell lung carcinoma[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Our previous experiments showed that KIFC1 is significantly overexpressed in hepatocellular carcinoma and contributes to the progression of the disease, resulting in a poor prognosis[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the molecular and functional mechanisms of KIFC1 in PC have not been reported.\u003c/p\u003e \u003cp\u003eThe BUB1 protein plays a crucial regulatory role in mitosis, as demonstrated by recent studies[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It encodes a protein kinase that activates the spindle checkpoint by phosphorylating members of the mitotic checkpoint complex[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Various types of cancer, including bladder[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], pancreatic[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and breast cancers[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], have been linked to mutations in BUB1. Kenneth B. Kaplan et al. reported that BUB1 phosphorylates the adenomatosis polyposis coli (APC) protein in the WNT pathway[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Phosphorylated APC proteins may lose their ability to inhibit the WNT pathway. Similarly, no studies have reported on the specific molecular mechanisms and functions of BUB1 in PC.\u003c/p\u003e \u003cp\u003eThe Wnt signaling pathway was initially identified in a mouse mammary tumor virus model and was designated Int-1 by Roel Nusse[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The Wnt/β-catenin signaling pathway is a canonical signaling pathway in which the β-catenin protein plays a key role. In the nucleus, β-catenin binds to DNA-binding proteins of the T-cell factor/lymphoid enhancing factor (TCF/LEF) family, leading to the activation of downstream target genes. Wnt pathway activation is inhibited by the degradation of β-catenin, which is phosphorylated by a disruption complex composed of APC, Axin, and GSK3β. This phosphorylation recruits the E3 ubiquitin ligase containing β-TrCP to degrade β-catenin. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, the molecular interactions between the KIFC1, BUB1, and Wnt signaling pathways in PC have not been fully elucidated. Our study revealed that KIFC1 interacts with downstream BUB1, ultimately activating the Wnt pathway and promoting PC development. This study provides a new molecular mechanism of KIFC1 in PC development and identifies new targets for the future treatment of PC.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and tissue samples\u003c/h2\u003e \u003cp\u003eThis study included sixty-two patients with PC who were pathologically diagnosed between 2016 and 2023 and who underwent pancreatectomy at the Department of Hepatobiliary Surgery of the Second Affiliated Hospital of Nanchang University. The specimens were subjected to immunohistochemical analysis after being embedded in formalin and paraffin. The patients' clinicopathological data were also collected. All patients provided informed consent. Tumor staging was determined according to the Union for International Cancer Control TNM classification guide (8th edition, 2019). The Ethics Committee of the Second Affiliated Hospital approved the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003ePANC-1, SW-1990, BXPC-3, ASPC-1, and H6C7 (human normal pancreatic ductal epithelial cells) cells were purchased from the Institutes for Life Sciences, Chinese Academy of Sciences (Beijing, China). All cells were cultured in DMEM (Thermo Fisher, 12430054, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, SH30396.02, USA), except for the ASPC-1 cells, which were cultured in RPMI-1640 medium (Thermo Fisher, 11875119, USA) supplemented with 10% FBS in a humidified incubator supplemented with 5% carbon dioxide at 37\u0026deg;C, and the medium was changed every 3 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eTissue specimens were dewaxed in xylene and rehydrated in a graded series of ethanol. Afterwards, the tissue sections were placed in a pressure cooker at 100\u0026deg;C for 15 minutes to repair the antigens. Subsequently, the sections were incubated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 15 minutes at room temperature to block endogenous peroxidase activity. Next, the sections were blocked with goat serum (Thermo Fisher, 16210064, USA) for 30 minutes. Next, the sections were incubated with an anti-KIFC1 antibody (1:200, ORIGENE, TA38608) at 4\u0026deg;C overnight. Afterwards, the sections were incubated with the corresponding secondary antibodies. Both the staining intensity and area of positive staining for KIFC1 were evaluated by two pathologists in a mutually blinded manner. Staining was graded as 0 (negative), 1 (weakly positive), 2 (moderate) or 3 (strongly positive) based on the intensity of staining. The extent of staining was scored as 1 (\u0026lt;\u0026thinsp;10%), 2 (10\u0026ndash;40%), 3 (40%-75%) or 4 (\u0026gt;\u0026thinsp;75%). The intensity and extent of the staining were multiplied to obtain a total staining score. The KIFC1 score ranged from 0 to 12, which allowed the specimens to be categorized into a low-expression group (0\u0026ndash;3) and a high-expression group (3\u0026ndash;12).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData mining and bioinformatics analysis\u003c/h2\u003e \u003cp\u003eThe expression profiles of KIFC1 and BUB1 in PC and their respective survival curves were obtained from the Gene Expression Profiling Interactive Analysis (GEPIA) online database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). KIFC1 and BUB1 gene correlation analyses were performed with the Gene_Corr module of the TIMER2.0 database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://timer.cistrome.org/\u003c/span\u003e\u003cspan address=\"http://timer.cistrome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The PPIs were first identified from the STRING database and then mapped using Cytoscape v3.9.0 software. The GSE107160, GSE16515 and GSE15471 datasets were obtained from the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection\u003c/h2\u003e \u003cp\u003eSmall interfering RNA (siRNA) targeting KIFC1 and lentiviruses for the knockdown or overexpression of KIFC1 and BUB1 were purchased from General Biol (Anhui, China). Small interfering RNA (siRNA) was used to grow the cells in 6-well plates to 70\u0026ndash;80% confluence, and the cells were transfected with Lipofectamine 2000 (Invitrogen, 11668-019, USA) according to the manufacturer's protocol. After the cells reached 90% confluence, they were infected with lentivirus, followed by screening with puromycin to obtain stable overexpression and knockdown cell lines. The sequences of the siRNAs used were as follows: siRNA1: 5'-GGACUUAAAGGGUCAGUUATT-3' and siRNA2: 5'-CGGGAACGCCUUCGGGAAATT-3'.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR (qRT‒PCR)\u003c/h2\u003e \u003cp\u003eThe expression of KIFC1 was assessed by qRT‒PCR. Total RNA was isolated from PC cells using TRIzol reagent, and cDNA was obtained by reverse transcription according to the instructions of TRAN One-Step gDNA Removal and cDNA Synthesis SuperMix Kits (TransGen, AE311, China). qRT‒PCR was subsequently performed using SYBR\u0026reg; Premix Ex Taq II Kits (Takara, RR420B, USA). The 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method was used to calculate KIFC1 using GAPDH as an internal reference[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] for relative expression. The sequences of the primers used were as follows: KIFC1-F: 5'-GCAGGAACTCAAGGGCAA-3', \u003cem\u003eKIFC1\u003c/em\u003e-R 5'-GCTAAGGCGGGGTTGGAG-3'; and GAPDH-F: 5'-GGACCTGACCTGCCGTCTAG-3', GAPDH-R 5'-GTAGCCCAGGATGCCCTTGA-3'.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eCell or tissue samples were lysed using RIPA lysis buffer supplemented with protease inhibitors. The proteins were separated by SDS‒PAGE using either an 8% or 10% gel and then transferred to 0.22 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:m\\)\u003c/span\u003e\u003c/span\u003e PVDF membranes (Millipore, ISEQ00010, USA). The membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4\u0026deg;C. The strips were then washed with TBST and incubated with secondary antibodies for one hour at room temperature. Finally, the strips were visualized using a bioimaging system and analyzed with ImageJ software.\u003c/p\u003e \u003cp\u003ePrimary antibodies against KIFC1 (cat. no. TA384608, ORIGENE), BUB1 (cat. no. HA60053, HUABIO), β-catenin (cat. no. #8480, Celling Signaling), TCF-4 (cat. no. #2569, Celling Signaling), c-Myc (cat. no. #9402, Celling Signaling), cyclin D1 (cat. no. #55506, Celling Signaling), N-cadherin (cat. no. #13116, Celling Signaling), E-cadherin (cat. no. #3195, Celling Signaling), vimentin (cat. no. #5741, Celling Signaling), and GAPDH (cat. no. #5174, Celling Signaling) were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell Counting Kit-8\u003c/h2\u003e \u003cp\u003eThe transfected cells were inoculated into 96-well plates at a density of 4000 cells per well and cultured for 24, 48, or 72 hours. Then, 10 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:L\\:\\)\u003c/span\u003e\u003c/span\u003eof CCK-8 (Abcam, ab228554, USA) reagent was added to each well at the end of each incubation cycle. After a 2-hour incubation, the absorbance of each well at 450\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:nm\\:\\)\u003c/span\u003e\u003c/span\u003ewas measured using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eColony Formation Assay\u003c/h2\u003e \u003cp\u003eCells that had been transfected and resuspended were inoculated into six-well plates at a density of 800 cells per well. The cells were cultured for 14 days, and the cells in each well were fixed with paraformaldehyde for 30 minutes at room temperature and then stained with crystal violet for 10 minutes. A microscope was used to count the number of cell clusters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assay\u003c/h2\u003e \u003cp\u003eCell proliferation was assessed by a 5-ethynyl-2ʹdeoxyuridine (EdU) proliferation assay, and an EdU kit (UElandy, C6044S, China) was purchased from UElandy Biotechnology Co. in Suzhou, China. Transfected and resuspended cells were inoculated into 96-well plates at 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. Once the cells had attached to the wall, the EdU reagent was added to the medium, and the cells were incubated for two hours. After this, the cells were fixed with 4% paraformaldehyde and destained with glycine, and the cell membrane was permeabilized with 0.5% Triton X-100. YF594 or YF488 was added for 30 minutes at 25\u0026deg;C in the dark. After washing, Hoechst 33342 was added for another 30 minutes under the same conditions. Images were acquired using a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWound healing\u003c/h2\u003e \u003cp\u003eThe fused cells were treated with mitomycin (1\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\mu\\:g/mL\\)\u003c/span\u003e\u003c/span\u003e) for one hour after being switched to serum-free medium. Vertical scratches were made at the bottom using a 200 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:l\\:\\)\u003c/span\u003e\u003c/span\u003epipette gun tip. The shed cells were washed away with PBS, and the scratches were imaged at 0 h. Images were captured again 24 h after incubation, and the rate of cell migration was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTranswell assay\u003c/h2\u003e \u003cp\u003eFor the invasion assay, Matrigel was spread into Transwell chambers. Transfected cells were then resuspended in serum-free DMEM and added to the upper chambers, while DMEM containing 10% FBS was added to the lower chambers. The chambers were incubated for 48 hours in a 24-well plate. After incubation, the chambers were fixed with 4% paraformaldehyde for 25 minutes and stained with crystal violet for 5 minutes. Images were captured using a microscope, and the cells were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell Cycle Assay\u003c/h2\u003e \u003cp\u003eA total of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e posttransfection cells were collected and fixed with precooled 70% ethanol in a refrigerator at 4\u0026deg;C overnight. The following day, the cells were washed twice with PBS, stained with 0.5 ml of PI/RNase (Abcam, ab112116, USA) and resuspended. The cells were incubated for 30 minutes at room temperature in the dark. Afterward, the cell cycle distribution was analyzed using flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Experiments\u003c/h2\u003e \u003cp\u003eFemale BALB/c nude mice (6\u0026ndash;8 weeks old) weighing 15\u0026thinsp;\u0026plusmn;\u0026thinsp;1 g from Beijing SPF Biotechnology Co., Ltd., were used to construct a xenograft tumor model. All animal experiments were conducted at Nanchang Royo Biotech Co,. Ltd. The aim of this study was to investigate the effect of KIFC1 on tumor formation in vivo.\u003c/p\u003e \u003cp\u003eThe mice were injected subcutaneously with 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e PANC-1 cells (with stable KIFC1 knockdown) resuspended in 100\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\mu\\:L\\:\\)\u003c/span\u003e\u003c/span\u003eof serum-free DMEM. Tumor volumes were measured at five-day intervals, and images of the mice were captured after 30 days using a small animal live imager. After the mice were euthanized, the tumor tissue was removed for detection. All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanchang Royo Biotech Co,. Ltd (Nanchang, China, IACUC No. RYE2024033001)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll analyses were performed using SPSS 26.0 software. Survival curves were plotted using the Kaplan‒Meier method and compared using the log-rank test, and the association between KIFC1 and clinicopathology was tested using the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\chi\\:2\\)\u003c/span\u003e\u003c/span\u003e test. Bivariate correlations were calculated using Pearson's correlation coefficient. Comparisons between two groups were made using two independent samples t tests, and differences were considered statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eKIFC1 is highly expressed in PC tissue and is associated with advanced clinical stage and poor prognosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBoth the high-throughput sequencing and GSE107610 datasets indicated that KIFC1 is highly expressed in PC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To explore the expression and location of KIFC1 in PC patients, we performed immunohistochemistry (IHC) analysis on 62 PC patient samples. We observed almost no KIFC1 expression in normal tissues. On the other hand, KIFC1 expression was significantly high in neoplastic tissues, and it was detected mainly in the nuclei of tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This finding was supported by findings obtained from the online GEPIA database (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, by analyzing the clinicopathological features of 62 patients, we found that a high KIFC1 expression level was closely correlated with T stage (P\u0026thinsp;=\u0026thinsp;0.016), TNM stage (P\u0026thinsp;=\u0026thinsp;0.017) and histological grade (P\u0026thinsp;=\u0026thinsp;0.001) in patients with PC (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These findings indicate that KIFC1 expression is clearly related to poor clinical and pathological stage. Therefore, we analyzed the association between KIFC1 expression and patient survival by Kaplan‒Meier analysis, and the results demonstrated that KIFC1-high PC patients had a significantly worse overall survival (OS) than did KIFC1-low patients (log rank P\u0026thinsp;=\u0026thinsp;0.008; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). This result was also confirmed by the GEPIA database (log rank P\u0026thinsp;=\u0026thinsp;0.0059; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelationships between KIFC1 expression and the clinicopathological features of 62 PC patients.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eKIFC1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigh expression\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow expression\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.466\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.495\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17(68.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8(32.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22(59.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15(40.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.726\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.394\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23(67.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11(32.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16(57.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12(42.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT staging\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.016*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8(40.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12(60.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18(66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9(33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13(86.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2(13.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNerve infiltration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.777\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.378\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31(66.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16(34.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8(53.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7(46.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVenous invasion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.503\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.114\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25(71.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10(28.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14(51.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13(48.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistological grade\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11.519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6(31.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13(68.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow / Medium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33(76.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10(23.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph node status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.470\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15(57.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11(42.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24(66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12(33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNM staging\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.017*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7(36.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12(63.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21(72.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8(27.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11(78.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3(21.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eKIFC1 overexpression promotes PC cell growth in vitro\u003c/h2\u003e \u003cp\u003eThe KIFC1 protein and mRNA were examined by western blotting and qRT‒PCR, and the results revealed that KIFC1 was more highly expressed in PC cell lines than in normal pancreatic ductal epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and G). To confirm that KIFC1 promotes PC cell proliferation, two independent siRNAs and one KIFC1-overexpressing lentiviral vector were used to silence or overexpress KIFC1, respectively. SiRNA transfection and lentivirus infection were detected in PANC-1 and SW-1990 cells via western blotting, and the KIFC1 protein levels decreased and increased, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Compared with control cells, SW-1990 cells overexpressing KIFC1 exhibited greater cell viability, colony formation and proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, KIFC1 knockdown in PANC-1 cells had opposite effects on cell viability, colony formation and proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, a cell cycle assay demonstrated that KIFC1 promoted progression through the cell cycle from the G1 to the S and G2 phases. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKIFC1 promoted PC cell migration and invasion.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eScratch and Transwell assays were performed in PANC-1 and SW-1990 cells to examine the impact of KIFC1 on PC cell migration and invasion. A scratch assay indicated that KIFC1 overexpression markedly enhanced migration, while the opposite effect was observed in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, Transwell assays revealed that PC invasion capacity was closely related to KIFC1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). It is widely known that epithelial\u0026ndash;mesenchymal transition (EMT) is an indispensable factor that promotes cell migration and invasion[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, western blotting was used to verify the key role of EMT in PC cell migration and invasion. The results demonstrated that the epithelial marker E-cadherin was repressed; in contrast, the mesenchymal markers N-cadherin and vimentin were increased in KIFC1-overexpressing PC cells. However, silencing KIFC1 had the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Overall, the above results indicated that KIFC1 promoted PC cell migration and invasion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKIFC1 overexpression activated the Wnt/β/catenin pathway in PC cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn a previous study, the Wnt/β/catenin pathway was verified to contribute to PC malignancy[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The dephosphorylation of β-catenin prevents it from being degraded by ubiquitination, after which it accumulates in the nucleus and increases the expression of TCF/LEF. TCF/LEF subsequently interacts with downstream targets, such as c-Myc and cyclin D1[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Western blotting revealed that KIFC1 overexpression upregulated β-catenin, TCF4, c-My and cyclin D1, while KIFC1 depletion downregulated the expression of these genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Thus, the results indicated that KIFC1 might affect PC malignancy by activating the Wnt/β-catenin signaling pathway.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBUB1 interacts with KIFC1 at the protein level, is highly expressed in PC patients and is associated with poor prognosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUsing high-throughput sequencing, we screened 2922 upregulated genes. After genes that were not interrelated were eliminated, PPI analysis revealed that KIFC1 may interact with BUB1. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To investigate the relationship between KIFC1 and BUB1 in PC, we analyzed transcriptomic data from The Cancer Genome Atlas (TCGA) Spearman's correlation analysis confirmed a strong positive correlation between the expression of KIFC1 and that of BUB1 in PC (r\u0026thinsp;=\u0026thinsp;0.835; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover, interactions between KIFC1 and BUB1 were observed at the protein level via Co-IP assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). To confirm the relationship between BUB1 expression levels and clinical outcomes, we selected two datasets (GSE15471 and GSE16151) from the GEO database for paired-sample t tests. The results showed that BUB1 expression was significantly greater in tumor tissues than in adjacent normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and E). Additionally, the GEPIA database revealed high expression of BUB1 in patients with tumors, which adversely affected patient prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBUB1 mediates the effect of KIFC1 on the Wnt/β-catenin pathway and the malignancy of PC cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWestern blotting revealed that BUB1 was successfully knocked down or overexpressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). EdU experiments revealed that BUB1 knockdown decreased the proliferation of PC cells, whereas BUB1 overexpression had the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Similarly, a positive correlation between BUB1 expression and cell migration and invasion ability was observed in the scratch and Transwell assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and D). The knockdown and overexpression of KIFC1 in PANC-1 cells resulted in a similar trend for BUB1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). A lentivirus was used to knock down BUB1, which reversed the positive effects of KIFC1 overexpression on the proliferation, migration, and invasion ability of PANC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-D). Similarly, decreased protein levels of β-catenin, TCF-4, c-Myc, and cyclin D1 were observed in KIFC1-overexpressing cells upon BUB1 silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). These results demonstrated that reducing BUB1 expression inhibits the activation of the Wnt/β-catenin pathway induced by KIFC1 overexpression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eKIFC1 enhances PC growth and activation of the Wnt/β-catenin pathway in vivo\u003c/h2\u003e \u003cp\u003eWe established subcutaneous tumor models to explore the function of KIFC1 in regulating PC tumorigenesis. Tumors with silenced KIFC1 exhibited significantly lower growth rates, weights, and volumes than those in the control group. (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-C). Moreover, a small animal imaging system was used to evaluate tumor growth, and the results also verified that KIFC1 knockdown obviously suppressed cancer cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Furthermore, Western blotting revealed that KIFC1 regulates BUB1 at the protein level and affects the activation of the Wnt/β-catenin pathway in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). Thus, in vivo experiments confirmed that KIFC1 regulates BUB1 and the downstream Wnt/β-catenin pathway, promoting tumor growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePC is a highly malignant tumor of the gastrointestinal tract, with an incidence rate almost equal to the mortality rate[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Due to its insidious early onset, most cases are already in advanced stages when it is detected. PC can only be eliminated through surgery, but the chances of recurrence after the procedure are high, leading to a poor prognosis[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, finding potential molecular targets for PC may lead to new directions for its treatment. Through a series of experiments, we confirmed that KIFC1 is highly expressed in PC cell lines and patients. KIFC1 overexpression activates the Wnt/β-catenin pathway through BUB1, promoting malignant behavior and functions in PC cells. These findings indicate that the KIFC1-BUB1-Wnt pathway signaling axis could be a more effective target for PC therapy.\u003c/p\u003e \u003cp\u003eWith respect to KIFC1, growing evidence suggests that KIFC1 overexpression is strongly associated with cancer development, progression, and drug resistance[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Cancer cells typically exhibit centrosome amplification, and these cells survive and proliferate by clustering supernumerary centrosomes for bipolar division[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Coincidentally, KIFC1 can aggregate centrosomes to achieve bipolar division of cancer cells, promoting their survival and proliferation[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These findings demonstrate that KIFC1 may promote PC progression by facilitating centrosome aggregation in PC cells.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that the Wnt/β-catenin signaling pathway can promote PC tumorigenesis and drug resistance[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Targeting this pathway has become a crucial initiative for treating PC and other gastrointestinal tumors[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In head and neck carcinoma, KIFC1 has been identified as an 'activator' of the Wnt/β-catenin signaling pathway, promoting the development of head and neck squamous cell carcinoma[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Our experimental results suggest that KIFC1 may promote PC development by activating the Wnt/β-catenin signaling pathway. Further mechanistic exploration revealed that KIFC1 binds to BUB1 at the protein level. Knocking down BUB1 reversed the increase in cell function and Wnt/β-catenin pathway activity caused by KIFC1 overexpression, suggesting that KIFC1 may activate the Wnt/β-catenin pathway via BUB1. BUB1 functions as a protein kinase that phosphorylates APC (a component of the disruption complex)[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. We speculate that this phosphorylation may alter the activity of the disruption complex, preventing the degradation of most β-catenin. The retained β-catenin then enters the nucleus and binds to TCF/LEF proteins, transcribing downstream target genes. BUB1 is a crucial component in chromosome segregation, and BUB1 overexpression induces Aurora-B hyperactivation, resulting in chromosomal missegregation and aneuploidy. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In general, cancer cells with aneuploidy-containing and dispersed centromeres tend to undergo multipolarization, preventing them from dividing and leading to cell death[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. However, KIFC1 promotes the aggregation of dispersed centrosomes in cancer cells, facilitating polar division and ensuring cell survival[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The available evidence indicates that KIFC1 and BUB1 have molecular and cellular interactions that promote the proliferation, migration, and invasion of PC cells both in vitro and in vivo by activating the downstream Wnt/β-catenin pathway.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, in the clinic, KIFC1 is expressed at high levels in PC tissues and cells, which significantly reduces the overall survival and results in a poor prognosis for patients. Mechanistically, KIFC1 overexpression contributed to PC cell proliferation, migration, and invasion via the BUB1/Wnt/β-catenin pathway. (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eKIFC1\u003c/strong\u003e Kinesin family member C1\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003ePC\u003c/strong\u003e Pancreatic cancer\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eTCGA\u003c/strong\u003e The Cancer Genome Atlas Program\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eGEO\u003c/strong\u003e Gene Expression Omnibus\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAPC\u0026nbsp;\u003c/strong\u003eAdenomatosis polyposis coli\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eGEPIA\u003c/strong\u003e Gene Expression Profiling Interactive Analysis\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003ePPI\u003c/strong\u003e Protein‒Protein Interaction Networks\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEMT\u003c/strong\u003e Epithelial\u0026ndash;mesenchymal transition\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eIHC\u003c/strong\u003e Immunohistochemistry\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eTCF/LEF\u003c/strong\u003e T-cell factor/Lymphoid enhancing factor\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eOS\u003c/strong\u003e Overall Survival\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics Approval and Consent to Participate\u003c/h2\u003e \u003cp\u003e The use of human tissue specimens for this study was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University. Animal ethics were reviewed and approved by the Institutional Animal Care and Use Committee of Nanchang Royo Biotech Co., Ltd.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eAll authors agreed with submission of the manuscript for publication and agree to be accountable for all aspect of the manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by grants from the Natural Science Foundation of Jiangxi Province, China (No. 20232BAB216076, 20224BAB216050, 20232BAB206078 and 20224ACB206037); the Research Project of Education Department of Jiangxi Province, China (No. GJJ2200227 and GJJ2200237); the National Natural Science Foundation Incubation Project of The Second Affiliated Hospital of Nanchang University (2022YNF12020 and 2022YNFY12002); and the National Natural Science Foundation of China (No. 82360220 and 82360513).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.C: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, and Writing\u0026mdash;original draft. Y.X.Y: Resources, Validation, and Writing\u0026mdash;original draft. J.Y.W and L.J.R: Resources, Data curation and Validation. Y.Q.Z: Resources, Funding acquisition and Writing\u0026mdash;review \u0026amp; editing. Y.Z, R.X.L, M.Y.D and H.W: Resources, Validation, and Data curation. L.F: Resources, Supervision, Funding acquisition, Validation, and Writing\u0026mdash;review \u0026amp; editing. X.W.F: Resources, Supervision, Funding acquisition and Writing\u0026mdash;review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to express our gratitude to the editors and reviewers for their hard work and the authors for their efforts.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData will be made available on request by contacting the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Giaquinto AN, Jemal A (2024) Cancer statistics, 2024. CA Cancer J Clin 74:12\u0026ndash;49\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKindler HL (2018) A Glimmer of Hope for Pancreatic Cancer. N Engl J Med 379:2463\u0026ndash;2464\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalafa MP (2015) Defining borderline resectable pancreatic cancer: emerging consensus for an old challenge. 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Genes Dev 22:2189\u0026ndash;2203\u003c/span\u003e\u003c/li\u003e\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":"KIFC1, BUB1, Pancreatic cancer, Protein interactions, Tumorigenesis","lastPublishedDoi":"10.21203/rs.3.rs-4838885/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4838885/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Pancreatic cancer (PC) is a highly lethal tumor of the gastrointestinal tract. New molecular targets are urgently needed for its treatment. Kinesin family member C1 (KIFC1) is implicated in the development and progression of several types of cancer. Previously, our studies indicated that KIFC1 is overexpressed in hepatocellular carcinoma and activates the malignant behavior of hepatocellular carcinoma through the PI3K/AKT pathway. However, the molecular and functional mechanisms of KIFC1\u003cbr\u003e\nin PC have not been investigated.\u003c/p\u003e\n\u003cp\u003eMethods: In this study, high-throughput sequencing technology was utilized to characterize differential gene expression profiles in patients with PC. KIFC1 was revealed by screening up-regulated genes from our sequenced data and the Gene Expression Omnibus (GEO) database. Sixty-two PC tissues were analyzed to determine\u003cbr\u003e\nthe correlation of KIFC1 expression with the clinicopathological features and prognosis\u003cbr\u003e\nof patients. The role of KIFC1 in proliferation, migration and invasion in PC was verified both in vitro and in vivo. Bioinformatics analysis, coimmunoprecipitation (CoIP), and western blotting were performed to identify proteins that interact with KIFC1and further affect the downstream pathway.\u003c/p\u003e\n\u003cp\u003eResults: According to high-throughput sequencing and the GEO database, KIFC1 is highly expressed in PC. KIFC1 is highly expressed in PC tissues and cells and is positively correlated with poor patient prognosis and malignant cellular behavior. Silencing KIFC1 inhibited the proliferation, migration, and invasion of PC cells, and\u003cbr\u003e\noverexpression of KIFC1 had the opposite effect. Protein‒protein interaction (PPI) and\u003cbr\u003e\nCo-IP analyses indicated that KIFC1 interacts with and regulates BUB1. Overexpression of BUB1 can also promote the proliferation, migration, and invasion of PC cells. BUB1 acts as an intermediary in the activation of the Wnt/β-catenin pathway by KIFC1, leading to an increase in the malignant behaviors of PC cells. The reversal of Wnt/β-catenin activation and increase in cellular malignant behavior induced by KIFC1 overexpression are achieved by silencing BUB1. These biological functions of KIFC1 in PC were also confirmed in a nude mouse xenograft model.\u003c/p\u003e\n\u003cp\u003eConclusions: Our experiments demonstrated for the first time that KIFC1 can influence PC progression by regulating BUB1 to activate the Wnt/β-catenin pathway. Therefore, KIFC1 shows promise as an attractive therapeutic target for PC in the future.\u003c/p\u003e","manuscriptTitle":"KIFC1 overexpression promotes pancreatic carcinoma progression via the BUB1/WNT/β- catenin pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-08 19:33:53","doi":"10.21203/rs.3.rs-4838885/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":"c39fa39d-add7-4527-b9f6-54e6b56c6f06","owner":[],"postedDate":"September 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-17T18:08:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-08 19:33:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4838885","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4838885","identity":"rs-4838885","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}