This study accessed gene expression data from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/), specifically datasets GSE39582, encompassing CRC and normal colon tissue samples. Raw data files were retrieved and preprocessed to ensure accuracy. The Kaplan-Meier survival analysis investigated the relationship between CPNE1 expression and CRC prognosis, utilizing patient survival data and CPNE1 expression levels. Data processing and analysis were conducted using GraphPad Prism 7.0 software.

Four human CRC cell lines (SW480, SW620, HCT116, and HT29) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS) and antibiotics (100-units/mL penicillin and 100-µg/mL streptomycin). The cells were maintained at 37 ℃ in a humidified incubator with 5% CO₂.

The impact of CPNE1 knockdown on the survival of SW480 and HCT116 cells was evaluated using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories Co., Ltd.) in an opaque-walled assay plate (Thermo Fisher Scientific, Inc.), following the manufacturer’s protocol with some slight modifications. Specifically, SW480 and HCT116 cells were seeded into 6-well plates (1 × 10⁵ cells per well) and incubated for 24 hours at 37 ℃. The cells were then infected with Ad-EGFP or Ad-shCPNE1 adenovirus at a multiplicity of infection of 20. Twenty-four hours postinfection, the growth medium was replaced with a normal culture medium. The infected cells were then incubated for an additional 48 hours, detached using trypsin/EDTA, and replated in 96-well plates (5 × 10² cells per well). Cell viability was then assessed using the CCK-8 assay. To continuously monitor cell viability, the pro-substrate was added at the time of cell replating, and each sample was analyzed at least in triplicate.

Triplicate samples of SW480 and HCT116 cells infected with Ad-EGFP or Ad-shCPNE1 adenovirus were seeded into 6-well plates (5 × 10² cells per well). The culture plates were incubated at 37 ℃ for 9 days in a growth medium, which was replaced every 3 days. The resulting colonies were then stained with crystal violet (1%, w/v; supplied by Sigma-Aldrich and Merck KGaA) for 2 hours at 20 ℃ and counted under a microscope (Eclipse 80Ti, Nikon Instruments Inc.).

Cell migration assays were conducted using gap closure assay chambers from Ibidi GmbH. SW480 and HCT116 cells infected with Ad-EGFP or Ad-shCPNE1 adenovirus were trypsinized and resuspended in a growth medium containing 10% FBS. The cells were then seeded in the insert at a density of 1 × 10⁵ cells per well and allowed to adhere to the well walls overnight. The following day, the inserts were removed and images were acquired using light microscopy (3 images for each sample). The cells were then cultured in a serum-free culture medium for the indicated time, and images were acquired using an inverted microscope equipped with an image capture system from Nikon Corporation. The images were analyzed using automated image analysis software (Nikon NIS Elements version 5.1 software, NIS-Elements Advanced Research; Nikon Instruments Inc.).

Cells infected with Ad-EGFP or Ad-shCPNE1 adenovirus were seeded at a density of 1 × 10⁵ cells per well into the upper chamber of Transwell inserts with 8-µm pore polycarbonate filters (Costar; Corning, Inc.) coated with 10-µL Matrigel (BD Biosciences). The lower chamber was filled with DMEM containing 10% FBS, and the inserts were incubated at 37 ℃ for 24 hours. The invaded cells were fixed on the microscopic slide using 4% paraformaldehyde and stained with DAPI (4’,6-diamidino-2-phenylindole). Five random fields were observed and images were obtained using a fluorescence microscope (magnification, ×100) to count the cells.

Cell extracts were prepared by lysing cells in radioimmunoprecipitation assay lysis buffer (RIPA Lysis Buffer System; Santa Cruz Biotechnology, Inc.) containing protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific, Inc.). The cells were sonicated for 2 minutes and centrifuged at 12,000 ×g for 10 minutes at 4 ℃ to remove insoluble debris, and protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce, Thermo Fisher Scientific, Inc.). A total of 30 µg of protein per well was separated on a 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel and transferred onto a nitrocellulose membrane (EMD Millipore). After following a standard protocol for blocking and washing, the target proteins were detected by incubating the membrane with primary antibodies against CPNE1 (cat. no. ab155675; Abcam, Inc.), cyclin A (cat. no. sc-751; Santa Cruz Biotechnology, Inc.), cyclin B1 (cat. no. sc-245; Santa Cruz Biotechnology, Inc.), cyclin E (cat. no. sc-198; Santa Cruz Biotechnology, Inc.), vimentin (cat. no. sc-73259; Santa Cruz Biotechnology, Inc.), Slug (cat. no. ab51772; Abcam, Inc.), Snail (cat. no. ab53519; Abcam, Inc.), and β-actin (cat. no. A5441; Sigma-Aldrich; Merck KGaA). Protein bands were visualized using an enhanced chemiluminescence detection reagent (cat. no. #170-5061; Bio-Rad Laboratories, Inc.), and images were captured using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories).

Sections of 5 µm were cut from formalin-fixed, paraffin-embedded tissue blocks from 263 CRC patients. These sections were mounted on charged slides, deparaffinized, and rehydrated in a graded series of alcohol. To block endogenous peroxidase activity, the sections were incubated with 0.3% hydrogen peroxide for 30 minutes at 20 ℃. The antigens were retrieved by boiling the sections in 10-mM sodium citrate for 10 minutes at 90 ℃ and immunostaining was carried out with a rabbit polyclonal CPNE1 antibody (1:200) using the UltraVision LP Detection System (ref. no. TL-125-HL; Thermo Scientific, Inc.) and DAB Quanto (ref. no. TA-125-QHDX; Epredia, Inc.) according to the manufacturer’s instructions. The sections were lightly counterstained with Mayer’s hematoxylin and mounted onto the microscope.

Athymic nude mice (male, 5 weeks old) were purchased from KOATECH (Pyeongtaek, Korea) and were bred following the guidelines of the Gyeongsang National University Animal Laboratory. To establish the xenograft model, CRC cell line HCT116 with 200-µL saline was injected subcutaneously into the mice (10⁷ cells per mouse). After 8 days, the grafted nude mice were randomly divided into 2 groups (n = 9 each). Purified Ad-EGFP and Ad-shCPNE1 adenovirus, suspended in 20-µL saline, were injected intratumorally into the mice every 3 days (6 × 10⁹ PFU per tumor). Tumor volume was measured based on the length and width and calculated using the formula [V = (L × 2W) / 2].

Each experiment was conducted at least thrice independently, and the data are presented as the mean ± standard deviation. The difference between the 2 groups was assessed using the 2-tailed Student t-test. A P-value of <0.05 was considered to be significant.

To identify CPNE1 overexpression and overall survival (OS) rate in CRC, we investigated 2 GEO datasets (GSE41258, and GSE39582) from the National Library of Medicine. The relative expression of the CPNE1 mRNA in primary CRC tissues (373.4 ± 13.67, n = 186) was significantly higher than that in paracancerous colorectal tissues (176.8 ± 7.326, n = 54; P < 0.001). The relative expression of the CPNE1 mRNA was significantly higher in metastatic tissues (472.9 ± 23.7, n = 67) compared to that in paracancerous colorectal tissues (P < 0.001) and primary CRC tissues (P < 0.001) (Fig. 1A). Furthermore, the OS rate was used to predict the prognostic value of CPNE1 expression in CRC tissues. CPNE1 overexpression in CRC patients resulted in a shorter OS rate (Fig. 1B). Cancer progression and increased expression of CPNE1 mRNA were significantly correlated. Additionally, we used western blotting to determine whether CPNE1 protein is detected in human CRC cell lines (SW480, SW620, HCT116, and HT29) (Fig. 1C).

To evaluate CPNE1 expression in CRC, a total of 263 CRC samples were analyzed using immunohistochemistry, wherein the staining intensity score was divided into negative (0%–25%), weak (26%–50%), moderate (51%–75%), and strong (76%–100%) (Fig. 1D). The observed intensity scores were 31 negative, 74 weak, 127 moderate, and 31 strong. We found that CPNE1 expression was positively associated with the invasion depth (P = 0.012) and significantly associated with the lymph node metastasis status (P < 0.001), distant metastasis (P < 0.001), lymphatic invasion (P = 0.018), vascular invasion (P < 0.001), neural invasion (P = 0.001), KRAS (Kirsten rat sarcoma virus; P < 0.001), NRAS (neuroblastoma RAS viral oncogene homolog; P = 0.006), and microsatellite instability status (P < 0.001). Moreover, CPNE1 expression was higher in patients with advanced TNM stage than in those without (P < 0.001) (Table 1). However, we observed no significant correlation between CPNE1 expression and other clinical and pathological features, including the tumor site and tumor differentiation.

To investigate the role of CPNE1 in CRC cells, we constructed the adenoviral-mediated overexpression vector of CPNE1-specific shRNA. Western blotting showed the efficient knockdown of CPNE1 in CRC cell lines SW480 and HCT116 (Fig. 2A). To determine the effect of CPNE1 silencing on CRC cell proliferation, we performed the CCK-8 assay (Fig. 2B, C) and colony formation assay (Fig. 2D). CPNE1 knockdown in CRC cells showed decreased cell viability and colony formation ability (compared with the control cells). Furthermore, we found that the protein levels of cell cycle-associated proteins, including cyclin A, cyclin B1, and cyclin E, were markedly decreased in the CPNE1-silenced cells compared with the control cells (Fig. 2E). These results demonstrated that CPNE1 knockdown significantly inhibits CRC cell proliferation.

To compare the migrating and invasive abilities of CPNE1-silenced CRC cells and control cells, we performed the gap closure assay (Fig. 3A) and Transwell invasion assay (Fig. 3B). In the gap closure assay, CPNE1-silenced cells migrated and invaded much more slowly than the control cells. Additionally, western blotting was used to detect the expression of vimentin, Slug, and Snail, which are markers of invasion and metastasis, in the CPNE1-silenced CRC cells. These markers were expressed at lower levels in the CPNE1-silenced CRC cells (compared to the control cells) (Fig. 3C). Taken together, these results suggest that silencing the CPNE1 gene in CRC cells may affect their ability to migrate and invade, which the hallmarks of cancer malignancy. This is supported by western blotting results, which showed decreased expression of invasion and metastasis markers in the CPNE1-silenced CRC cells, indicating that silencing of CPNE1 may make the cells less malignant.

To further validate the biological role of CPNE1 in vivo, we used a mouse xenograft model; HCT116 cells were subcutaneously injected into the left flank of the male athymic nude mice. Knockdown of CPNE1 markedly suppressed tumor growth (Fig. 4A). The tumor growth curve was plotted according to the monitored volumes (Fig. 4B), and the tumor weight was measured (0.87 g vs. 0.52 g, P < 0.001) (Fig. 4C). Tissues excised from the xenograft tumors were analyzed to confirm CPNE1 and Ki-67 expression using hematoxylin and immunohistochemistry (Fig. 4D). These results indicated that CPNE1 silencing significantly suppresses CRC tumor growth in vivo.