The CRC cell lines SW480 and DLD1 were purchased from the American Type Culture Collection. Cells were cultured in Dulbecco's Modified Eagle's Medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; HyClone^™; Cytiva) and incubated at 37°C with 5% CO₂. Overexpression or knockdown lentiviral vectors encoding AL445238.2, USP4 or the corresponding empty vector were constructed using the pLVX-IRES-Puro plasmid backbone by Shanghai GeneChem Co., Ltd. Lentiviral particles were also produced and concentrated to a titer of 1×10⁸ TU/ml by Shanghai GeneChem Co., Ltd. For lentiviral transduction, cells were seeded at 30-40% confluency and transduced with lentiviral particles at a multiplicity of infection of 10-20 in the presence of 5 μg/ml polybrene (MilliporeSigma) at 37°C for 24 h. Stable transfected cells were selected and maintained with 2 μg/ml puromycin (MilliporeSigma) starting 72 h post-transfection. All of the cell lines were confirmed to be free from mycoplasma contamination and were authenticated by an expert before being used for experimentation. All shRNA sequences are listed in Table SI.

Total RNA was extracted from SW480 and DLD1 cells using TRIzol reagent (MilliporeSigma) and RNA purity and integrity were assessed by electrophoresis. RT was performed using random primers at 42°C for 1 h (RevertAid First Strand cDNA Synthesis Kit; Thermo Fisher Scientific, Inc.). The target gene was then amplified in a 20 μl reaction system using SYBR-Green qPCR Mix (Thermo Fisher Scientific, Inc.). The PCR conditions were as follows: 50°C for 2 min, 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 32 sec. Relative gene expression levels were normalized to the reference gene β-actin and quantified using the 2^−ΔΔCq method (24). The primer sequences for qPCR are provided in Table SII.

CRC cells were lysed in Cell Lysis Buffer for Western and IP without Inhibitors (NCM Biotech; Suzhou Xinsaimei Biotechnology Co., Ltd.) containing a protease and phosphatase inhibitor mixture (Beyotime Biotechnology). The protein concentration was determined using the BCA assay. Denatured proteins (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 4-20% gel and transferred to polyvinylidene fluoride membranes (MilliporeSigma). Membranes were blocked with 5% non-fat milk (BD Biosciences) in Tris-buffered saline containing 0.1% Tween-20 at room temperature for 1 h. Membranes were incubated overnight at 4°C with primary antibodies (dilution 1:1,000) and then washed and incubated at room temperature for 1 h with HRP-conjugated Goat Anti-Rabbit IgG (H+L) secondary antibody (dilution 1:5,000). Enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) was used to detect protein bands. GAPDH or β-actin were used as the internal control. Protein band intensities were quantified by densitometry using ImageJ software (version 1.53k; National Institutes of Health). Antibody information is provided in Table SIII.

Cells (1×10⁷) were harvested and lysed in IP lysis buffer (Pierce IP Lysis Buffer; Thermo Fisher Scientific, Inc.; cat. no. 87787) supplemented with protease inhibitor cocktail (1:100 dilution; Thermo Fisher Scientific, Inc.; cat. no. 78430) and phosphatase inhibitor cocktail (1:100 dilution; Thermo Fisher Scientific, Inc.; cat. no. 78428) on ice for 30 min with intermittent vortexing. Cell lysates were centrifuged at 14,000 × g for 15 min at 4°C, and the supernatant was collected. Protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.; cat. no. 23225). For each IP reaction, 500 μg of total protein lysate was pre-cleared with 20 μl of Protein A/G Magnetic Beads (Thermo Fisher Scientific, Inc.; cat. no. 88802) at 4°C for 1 h with rotation to reduce non-specific binding. After magnetic separation, the pre-cleared lysate was incubated with 2 μg of anti-USP4 antibody (cat. no. ab108931; Abcam) or normal rabbit IgG control antibody (cat. no. 2729; Cell Signaling Technology, Inc.) overnight at 4°C with gentle rotation. Subsequently, 30 μl of Protein A/G Magnetic Beads were added to each sample and incubated for 2 h at 4°C with rotation. The beads were then collected using a magnetic separator and washed five times with IP wash buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol). After the final wash, beads were resuspended in 30 μl of 2X SDS loading buffer and boiled at 95°C for 5 min to elute the immunocomplexes. The supernatant was collected by centrifugation at 1,000 × g for 1 min at room temperature and subjected to western blot analysis.

CRC cells (3×10³ cells/well) were seeded into a 96-well plate. After 24, 48 or 72 h of incubation, 10 μl CCK-8 reagent (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well and incubated at 37°C for 60 min. Cell proliferation was measured at 450 nm using a Varioskan LUX microplate reader.

Cytotoxicity was assessed by measuring LDH release into the culture medium using the CytoTox 96^® Non-Radioactive Cytotoxicity Assay (Promega Corporation; cat. no. G1780) according to the manufacturer's instructions. Briefly, cells were seeded in 96-well plates at a density of 5×10³ cells per well and cultured for 24, 48 or 72 h. At each time point, 50 μl of culture supernatant was transferred to a new 96-well plate and mixed with 50 μl of CytoTox 96^® Reagent. After incubating at room temperature for 30 min in the dark, the reaction was stopped by adding 50 μl of Stop Solution. Absorbance was measured at 490 nm using a microplate reader (BioTek Instruments, Inc.; Agilent Technologies, Inc.). LDH release was calculated as a percentage of maximum LDH release (determined by lysing cells with 1% Triton X-100). Background absorbance from culture medium alone was subtracted from all measurements. Each experiment was performed in triplicate and repeated three times independently.

Cells (1×10⁷) were collected and lysed in RIPA buffer (Beyotime Biotechnology) with a protease inhibitor mixture (NCM Biotech; Suzhou Xinsaimei Biotechnology Co., Ltd.). For each 1 ml of cell lysate, 10% was used as the input control. Biotinylated AL445238.2 sense probe and a scrambled negative control probe with no homology to any known transcripts were synthesized by GenePharma Co., Ltd. AL445238.2 sense and scrambled probes were incubated with the cell lysate at 4°C for 4 h. Next, streptavidin magnetic beads were added and the mixture was incubated at 4°C for 1 h. After boiling the magnetic beads in 0.1% sodium dodecyl sulfate solution for 3 min, protein interactions with AL445238.2 were detected by western blot. The control cells were treated with an equal volume of beads with scrambled probes to assess non-specific protein binding.

Apoptosis was detected using an Annexin V-FITC/PI dual-staining apoptosis detection kit (Absin Bioscience, Inc.) according to the manufacturer's instructions. In brief, cells were harvested, washed with pre-chilled phosphate buffered saline (PBS), resuspended in 100 μl binding buffer and sequentially stained with Annexin V-FITC (5 μl) for 10 min in the dark at room temperature, followed by PI (5 μl) for 5 min in the dark. After washing with PBS, the cells were resuspended and analyzed by flow cytometry.

Stemness was assessed by staining with a FITC-conjugated CD133 flow cytometric monoclonal antibody (cat. no. 372803; BioLegend, Inc.). After collection, the cells were resuspended in staining buffer (BioLegend, Inc.) at a concentration of 1.25×10⁶ cells/100 μl, incubated with 5 μl CD133 antibody in the dark for 20 min at room temperature, washed with PBS and resuspended in PBS for flow cytometry analysis.

Mitochondrial activity was assessed using the tetramethylrhodamine (TMRM) probe (cat. no. I34361 Invitrogen; Thermo Fisher Scientific, Inc.). After collecting the cells, the supernatant was discarded and the cells were incubated with DMEM containing a 1:1,000 dilution of the TMRM probe at 37°C with 5% CO₂ for 30 min. After washing with PBS, the cells were resuspended for flow cytometry analysis.

All flow cytometry data were collected using BD FACSCelesta and analyzed using FlowJo software (version 10; FlowJo LCC; BD Biosciences). Median fluorescence intensity was calculated using the median fluorescence value for each population.

Transwell migration assays were performed using Corning, Inc. Transwell chambers. In brief, cells (1×10⁶ cells/well) were seeded in the upper chambers of 24-well plates with 100 μl serum-free medium. The lower chambers were filled with 600 μl medium containing 30% FBS. After 24 h of incubation at 37°C, cells that migrated to the lower chambers were fixed with 4% paraformaldehyde at room temperature for 15 min, washed with PBS and stained with 0.1% crystal violet at room temperature for 20 min. The number of migrated cells was counted under a light microscope (Zeiss Axio Observer; Carl Zeiss AG) and quantified using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.). Images were acquired using ZEN imaging software (version 2.3 blue edition; Carl Zeiss AG).

Cells were seeded on glass slides in 24-well plates and cultured for 24 h. After washing with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min, followed by permeabilization with 0.5% Triton X-100 (PBS) for 15 min at room temperature. After blocking with PBS containing 5% normal goat serum (MilliporeSigma) at room temperature for 1 h, the cells were incubated with the primary antibody (Anti-USP4; 1:500; cat. no. ab181105; Abcam) overnight at 4°C, followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) secondary antibody (1:500; cat. no. A11008; Thermo Fisher Scientific, Inc.) for 1 h at room temperature in the dark. For FISH staining of AL445238.2, after permeabilization, cells were incubated with pre-hybridization buffer (Abnova Corporation; cat. no. U0028) at 37°C for 30 min, then hybridized with Alexa Fluor 555-labeled FISH probe provided by GenePharma Co., Ltd. at 37°C overnight in the dark. Following hybridization, cells were washed with SSC buffer (Thermo Fisher Scientific, Inc.; cat. no. J60839.K2; 2X SSC for 5 min, 1X SSC for 5 min and 0.5X SSC for 5 min) at 37°C. The slides were mounted using an anti-fade mounting medium containing 4',6-diamidino-2-phenylindole and analyzed under a fluorescence microscope.

Cells were dissociated into a single-cell suspension and seeded at a density of 5×10³ cells/well in a 6-well ultra-low attachment plate (Corning, Inc.). Cells were cultured in serum-free DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with epidermal growth factor (20 ng/ml; PeproTech, Inc.; Thermo Fisher Scientific, Inc.) and B27 (2%; Gibco; Thermo Fisher Scientific, Inc.). After 1-2 weeks of culture, the tumor spheroids were observed under a light microscope and the size and number of spheroids were measured using Image-Pro Plus software.

A nude mouse xenograft model was established using female BALB/c-nude immunodeficient mice aged 5-6 weeks (initial body weight: 18-20 g; GemPharmatech, Co., Ltd.). Mice were randomly assigned to 3 groups using a random number generator, with each mouse given a unique identification number before group allocation to ensure unbiased distribution. The groups were subcutaneously injected with SW480 cells transfected with empty vector, USP4 overexpression vector or AL445238.2 overexpression vector (n=4 per group). The mice were housed under a 12-h light/dark cycle with free access to food and water. Each mouse was subcutaneously injected with 5×10⁶ SW480 cells suspended in 100 μl PBS into the right abdominal flank. Animal health and behavior were monitored daily for signs of distress, weight loss or abnormal behavior. Tumor size was measured twice weekly using digital calipers and the tumor volume was calculated using the formula: Volume (mm³)=(length × width²)/2, where length is the longest diameter and width is the perpendicular diameter. After 12 weeks, the mice were euthanized using CO₂ following the AVMA Guidelines for the Euthanasia of Animals (25). Mice were placed in a clear euthanasia chamber and 100% CO₂ from a compressed gas cylinder was introduced using a gradual fill method with a displacement rate of 30% of the chamber volume per min. The CO₂ flow was maintained for at least 1 min after respiratory arrest and death was confirmed by physical examination (absence of heartbeat and respiration for at least 10 mi with graying of mucous membranes) before tumor harvest.

The sample size for the animal study was determined following the recommendations of the Animal Ethics Committee of Wenzhou Medical University (Wenzhou, China) to ensure the establishment of well-behaved animal models with adequate statistical power. During the experiment, the tumor diameter was not allowed to exceed 1.5 cm, nor was the tumor volume allowed to exceed 15% of the total body volume of the mouse. No animals reached these humane endpoints during the study.

Data from three independent experiments were analyzed using R (version 4.3.1; R Foundation for Statistical Computing). Results are expressed as the mean ± standard deviation. Comparisons between two groups were analyzed using unpaired Student's t-test. Multiple group comparisons were performed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

After confirming the overexpression or knockdown efficiency of AL445238.2 using qPCR (Fig. 1A), the impact of AL445238.2 on CRC cell proliferation was assessed using the CCK-8 assay and detection of LDH in the supernatant. AL445238.2 overexpression significantly enhanced the proliferation of DLD1 and SW480 CRC cells and reduced LDH levels in the supernatant, suggesting decreased cytotoxicity and cell membrane damage (Fig. 1B and C). Conversely, AL445238.2 knockdown weakened the proliferation of DLD1 and SW480 cells and increased LDH levels in the supernatant, suggesting enhanced cytotoxicity. Apoptosis analysis by flow cytometry revealed that overexpression of AL445238.2 reduced overall apoptosis in both DLD1 and SW480 cells, whereas knockdown of AL445238.2 increased overall apoptosis (Fig. 1D and E). Taken together, these results demonstrated that AL445238.2 promoted CRC cell proliferation while protecting cells from both cytotoxic damage and apoptotic cell death.

Flow cytometry analysis of CD133 (a stem cell marker) expression on DLD1 and SW480 cells showed that overexpression of AL445238.2 increased CD133 expression on the cell surface compared with the empty vector group, while knockdown of AL445238.2 decreased CD133 expression (Fig. 2A and B). These results suggest that AL445238.2 promotes stemness in CRC cells. Subsequently, TMRM (a probe for assessing mitochondrial membrane potential) flow cytometry analysis revealed that AL445238.2 overexpression enhanced TMRM fluorescence in DLD1 and SW480 cells, indicating increased mitochondrial activity, whereas AL445238.2 knockdown decreased mitochondrial activity (Fig. 2C and D).

To further explore the role of AL445238.2 in regulating CRC cell stemness, a spheroid formation assay was conducted. AL445238.2 overexpression in DLD1 and SW480 cells increased the diameter of tumor spheroids, whereas AL445238.2 knockdown reduced the spheroid size, suggesting a role in stemness enhancement (Fig. 2E).

RNA pull-down assays were performed to explore the molecular interactors of AL445238.2, which revealed that AL445238.2 bound to USP4 (Fig. 3A). Immunofluorescence confirmed the co-localization of AL445238.2 with USP4, indicating an interaction (Fig. 3B). The overexpression or knockdown efficiency of USP4 was confirmed using qPCR (Fig. 3C). Overexpression of AL445238.2 in SW480 cells upregulated Bcl2 and downregulated BAX and cleaved CASP3 (C-CASP3) expression, whereas knockdown of AL445238.2 reduced Bcl2 expression and increased BAX and C-CASP3 expression, suggesting that AL445238.2 contributed to cell survival (Fig. 3D).

The expression of the stemness markers aldehyde dehydrogenase 1 family member A1 (ALDH1A1), B lymphoma Mo-MLV insertion region 1 (Bmi1) and epithelial cell adhesion molecule (EpCAM) in stable SW480 and DLD1 cell lines was further examined. Overexpression of AL445238.2 increased the levels of ALDH1A1, Bmi1 and EpCAM, whereas knockdown of AL445238.2 reduced their expression, suggesting that AL445238.2 enhanced stemness in CRC cells (Fig. 3E and F). Co-IP experiments were performed to further how the AL445238.2-USP4 complex modulates the apoptotic pathway. It was found that USP4 can interact with Bcl2 (Fig. 3G). Knockdown of USP4 expression reduced Bcl2 expression in colon cancer cells, which could be reversed by MG132 (Fig. 3H). Further protein half-life experiments confirmed that USP4 overexpression increased the stability of Bcl2 protein and prolonged its half-life (Fig. 3I). Moreover, the co-expression of USP4 and AL445238.2 could further enhance the effect of USP4, extending the intracellular lifespan of Bcl2. Therefore, we conclude that AL445238.2-USP4 regulates the apoptotic pathway and exerts its subsequent functions by stabilizing Bcl2 protein.

The impact on CRC cell proliferation was evaluated using CCK-8 assays and LDH detection (Fig. 4A and B). USP4 overexpression significantly enhanced the proliferation of DLD1 and SW480 cells and reduced LDH levels in the supernatant, indicating reduced apoptosis. Conversely, USP4 knockdown weakened cell proliferation and increased LDH levels, suggesting an increase in apoptosis. Flow cytometry analysis confirmed that USP4 overexpression reduced apoptosis in DLD1 and SW480 cells, whereas USP4 knockdown increased apoptosis (Fig. 4C and D). Consistent with AL445238.2, USP4 overexpression in SW480 cells upregulated Bcl2 and downregulated BAX and C-CASP3 expression, whereas USP4 knockdown reduced Bcl2 expression and increased BAX and C-CASP3 expression, suggesting that USP4 suppresses cell apoptosis (Fig. 5A).

Flow cytometry analysis revealed that USP4 overexpression increased CD133 expression in DLD1 and SW480 cells, while USP4 knockdown decreased CD133 expression, suggesting that USP4 may promote stemness (Fig. 5B and C). TMRM flow cytometry showed that USP4 overexpression increased mitochondrial activity, whereas USP4 knockdown decreased mitochondrial activity (Fig. 5D and E). Spheroid formation assays confirmed that USP4 overexpression in DLD1 and SW480 cells increased the tumor spheroid diameter, whereas USP4 knockdown reduced the spheroid size (Fig. 5F).

Transwell migration assays demonstrated that both AL445238.2 and USP4 overexpression enhanced the migratory capacity of CRC cells (Fig. 6A-D). By contrast, AL445238.2 or USP4 knockdown impaired cell migration. Furthermore, when USP4 was knocked down while AL445238.2 was overexpressed, no significant increase in cell migration was observed compared with the control group (Fig. 6E and F). These findings suggest that AL445238.2 promotes CRC cell migration in a USP4-dependent manner.

In vivo tumorigenesis experiments using SW480 cells demonstrated that the overexpression of AL445238.2 or USP4 increased the growth rate, final tumor size and tumor weight of CRC cells in mice (Fig. 7A-D). These results suggest that both AL445238.2 and USP4 contribute to CRC cell proliferation in vivo.