Tissue specimens from patients with primary CRC treated at the Second Affiliated Hospital of Xuzhou Medical University were collected from January 2018 to December 2023. Among the 85 patients, there were 43 males and 42 females, with ages ranging from 35 to 69 years and a median age of 56 years. The Ethics Committee of Xuzhou Medical University approved the use of these clinical samples (approval no. 2023100505) and all patients provided written informed consent. The animal experiment of the present study was approved by the Animal Ethics Committee of Xuzhou Medical University (approval no. 2023110512; Xuzhou, China).

The CRC cell lines LOVO and HCT116, were provided from the Chinese Academy of Sciences' Cell Bank. LOVO and HCT116 cells were cultured in DMEM (cat. no. G4515-500ML; Wuhan Servicebio Technology Co., Ltd.). The media were supplemented with 10% fetal bovine serum (FBS; Wuhan Servicebio Technology Co., Ltd.), streptomycin and penicillin (×100). YWHAH-targeting siRNAs and a negative control (NC) were synthesized by Ruijie Bio (https://www.11467.com/shanghai/co/936727.htm). LOVO and HCT116 cells were transfected using Endo-Free Plasmid Midi Kit (Omega Corporation) with YWHAH siRNAs (15 pmol) or NC-siRNA at 37°C for 24-48 h, and the effects were assessed using reverse transcription-quantitative PCR (RT-qPCR). The siRNA sequences are provided in Table SI. The verification of knockdown and overexpression efficiency is shown in Fig. S1, and siR-2 was selected for subsequent studies.

The CRC cell and tissue RNAs were extracted using TRIzol^® (Thermo Fisher Scientific, Inc.). Reverse transcription reaction was used to convert the RNA to cDNA and YWHAH primers synthesized by Takara Bio USA, Inc according to the manufacturer's protocol. The cDNA was chilled for subsequent use. qPCR was conducted on a LongGene Q2000B, starting hybridization for 2 min at 50°C and subsequent amplification for 40 cycles at 95°C for 2 min, 95°C for 15 sec (denaturation), and 60°C for 60 sec (extension). Primer sequences for qPCR experiments are provided in Table SII. The 2^−∆∆Cq method was applied for mRNA analysis and GAPDH was used as the reference gene.

CCK-8 assays were conducted using a kit (Beyotime Institute of Biotechnology). CRC cells (5×10³ cells/well) were incubated in 96-well plates. Following reagent treatments, the medium in each well was substituted with 100 μl CCK-8 solution (comprising 90 μl fresh medium and 10 μl CCK-8 reagent). The color reaction starts at 37°C and was allowed to proceed for 2 h to maintain consistency. The measurement of optical density at 450 nm was performed using a spectrophotometer.

During Bisphenol A (BPA, 99%; MilliporeSigma) treatment, 5×10⁵ CRC cells per well were incubated in 6-well plates and cultured overnight; they were harvested 48 h later and resuspended in binding buffer. The cell suspension was incubated away from light for 20 min with Annexin V-fluorescein isothiocyanate and propidium iodide (Beyotime Institute of Biotechnology). Late apoptosis was subsequently assessed via flow cytometry (CellQuestPro software; BD FACSCalibur; BD Biosciences).

The proteins were extracted using RIPA buffer (Beyotime Institute of Biotechnology) with protease inhibitors, incubated at 4°C for 30 min and centrifuged (12,000 × g, 20 min, at 4°C). The protein concentration levels in the supernatants were assessed by BCA assays and 30 μg samples were electrophoresed using 8% SDS-PAGE gels prior to their transfer to nitrocellulose membrane. Following blocking (5% skimmed milk) at 37°C for 30 min, the membranes were incubated overnight at 4°C with primary antibodies (Table SIII) at a ratio of 1:5,000 according to the antibody instructions (GAPDH was diluted at a ratio of 1:5,000), followed by incubation for 30 min at room temperature with a Goat Anti-Rabbit IgG H&L secondary antibody (cat. no. 111-035-003; Jackson ImmunoResearch Laboratories, Inc.), which was diluted and prepared according to the antibody instructions. The protein bands were detected and visualized using the ChemiDocTM XRS⁺ system (Bio-Rad Laboratories, Inc.) and Ultra-sensitive ECL chemiluminescence kit (Beyotime Institute of Biotechnology). Image software (V.1.8.0; National Institutes of Health) was used for densitometric analysis.

A two-color fluorescence fusion protein, namely RFP-GFP-hLC3, was used to monitor autophagy. The fusion protein emits green and red fluorescence signals upon localization to autophagosomes, resulting in yellow signals in merged images during autophagy induction. The cells were plated onto glass cover slips (1×1.5 cm) in Corning 6-well plates overnight to adhere. A total of 6 h after transfection with siRNA and plasmids, cells were subsequently transfected with tandem RFP-GFP-hLC3 adenoviruses [pGMLV-CMV-RFP-GFP-hLC3-Puro Lentivirus, Genomeditech (Shanghai) Co., Ltd.] according to the manufacturer's instructions. Following the 24-h incubation, the cells were rinsed with PBS at room temperature, fixed in 4% paraformaldehyde at room temperature for 10-20 min, counterstained with 4',6-diamidino-2-phenylindole and subsequently covered with coverslips. Confocal images were obtained with the Olympus FV1000 laser scanning confocal fluorescence microscope. Quantification of autophagosomes with green and red fluorescence was performed using ImageJ software (V.1.8.0, National institutes of Health) by counting the LC3 puncta per cell.

To establish the model, the cells in the logarithmic growth phase were selected; the old culture medium was discarded from the dish, followed by washing once with PBS. The cells were subsequently digested with 0.25% trypsin for 1-2 min and FBS was added promptly to terminate digestion and prevent over-digestion. Following low-speed centrifugation (1,000 × g, 5 min) to collect the cells, they were washed twice with PBS, resuspended in serum-free medium, pipetted evenly, counted and adjusted to a density of 3×10⁷ cells/ml with sterile PBS. A total of 24 4-6-week-old healthy SPF-grade female BALB/c-nu nude mice with similar body weights SPF-grade female BALB/c-nu nude mice were randomly selected from the breeding cage, and 100-μl tumor cells (3×10⁶ cells) were subcutaneously incubated at the axilla of the right forelimb, with the injection site pressed with a sterile cotton swab to terminate bleeding prior to returning the mice to a sterilized cage for continuous feeding. The temperature was controlled at 20-26°C, and the humidity maintained between 40-70%. For the light/dark cycle, a 12/12-h light/dark cycle was adopted. The air exchange rate was 10-15-fold per h, and clean, special breeders provided food and sterile drinking water. The daily observations of the mental state of the mice, their activity, diet and defecation were conducted, while their body weights were measured weekly with an electronic balance and the longest and shortest diameters of the subcutaneous xenografts were obtained with a vernier caliper to calculate the volume using the following formula: V (mm³)=longest diameter (mm) × shortest diameter × shortest diameter × 0.5. This equation was used for plotting the growth curve with time on the abscissa and tumor volume on the ordinate (The maximum volume of a single tumor did not exceed 1,000 mm³). A total of 5 weeks later, the nude mice were weighed, euthanized by cervical dislocation and the xenografts were completely removed. The excised tumors were washed with sterile PBS, images were captured by experimental group on white paper, weighed and the data were recorded prior to storage at −80°C.

Cells were collected and gently blown into a fresh complete medium to obtain a uniform suspension. The cells were counted and spread onto 6-well plates in a 37°C 5% CO₂ incubator overnight before transfection. After 24 h, the cells were collected for analysis. Pancreatic enzyme digestion was performed at 1,000 rpm for 5 min, then the supernatant was removed. Cells were resuspended in 5-ml sterile PBS, a small sample was counted using a hemocytometer at 1,000 rpm, and cells were collected again after 5 min. In total, 3 ml of serum-free medium was added to adjust cell density to 2×10⁵/ml. Cells were added to sterile 24-well plates with 500-μl complete medium per well.; cells were transferred to 8-μm Transwell chambers using forceps and gently inserted into the plate. The cell suspension was mixed and 200 μl were added to the Transwell chamber. Incubation lasted for 48 h, then cells were rinsed twice with PBS. In total, 500 μl 4% PFA was added at room temperature for 20 min. PFA was removed, washing three times with PBS, and cells were stained with crystal violet for 20 min. Washing three times with PBS was performed to remove unbound dye. The Transwell chamber surface was gently wiped with a cotton swab to remove residual dye, then rinsed once with PBS and allowed to air-dry. Chambers were placed directly on a microscope slide and images of purple-stained cells were captured.

The MAPK/ERK signaling pathway inhibitor PD98059 was purchased from Beyotime Institute of Biotechnology, while its agonist U-46619 and the autophagy inducer RAPA were obtained from MedChemExpress. Additionally, 3-methoxyamphetamine (3-MA) was purchased from OriLeaf Biotechnology. Based on the concentration screening results, 2 μmol/l rapamycin, 5 mmol/l 3-MA, 20 μM PD98059, and 2 μM U46619 were selected for the experiment.

The tissues were treated with 4% paraformaldehyde at room temperature for 24 h, rinsed in running water and dehydrated using gradient alcohols. Dehydrated tissues were embedded in wax using an embedding machine, cooled at −20°C, trimmed and sectioned at 4-μm thickness slices. Following incubation at 40°C for 1-3 min (on a water surface to flatten), the sections were retrieved with glass slides, incubated at 60°C and stored. For immunohistochemical staining, the sections were incubated at 62°C for 1 h, the wax was removed and rehydrated prior to antigen retrieval in citrate buffer (pH 6.0) with microwave treatment (high-power for 1 min, medium-low for 9 min). Following cooling, the sections were washed with PBS and treated with 3% H₂O in methanol to block endogenous peroxidase activity, followed by PBS washing. Blocking was performed with diluted sheep serum (1:10) (ZSGM-BIO) for 1 h at 37°C. Primary antibody incubation with anti-rabbit YWHAH (1:1,000) was carried out overnight at 4°C, followed by 30 min at room temperature, and washing with PBST (1X PBS + 0.05% Tween-20). An HRP-labeled secondary antibody (1:1,000) was added for 30 min at 37°C, followed by PBST washing. DAB color development lasted for 1 min; it was terminated with water and examined under a microscope. Counterstaining with hematoxylin for 30 sec was followed by washing and examination. The sections underwent dehydration, clearing and mounting with neutral gum.

The RNA-sequencing expression (level 3) profiles and the corresponding clinical information for CRC were downloaded from The Cancer Genome Atlas (https://portal.gdc.cancer.gov/analysis_page?app=Downloads).

The data were subjected to analysis using SPSS version 21.0 (IBM Corp.) or GraphPad Prism version 10.1.2 (Dotmatics). The nominal and absolute values were assessed employing either paired t-tests or one-way ANOVA. All analyses were conducted utilizing R version 4.0.3 (https://www.r-project.org/), along with the appropriate R packages. P<0.05 was considered to indicate a statistically significant difference. Data are expressed as the mean ± standard deviation (SD). Error bars in all figures indicate SD from three biological replicates.

In a previous study using acRIP-seq, the involvement of YWHAH in CRC was established. The analysis with the R software markedly raised YWHAH levels in tumors relative to normal tissues (Fig. 1A). RT-qPCR confirmed this result in CRC tumor and paracancerous tissue (PCT) (Fig. 1B). Immunohistochemical analysis was performed on 85 CRC tissue pairs (tumor and PCT), revealing notably high YWHAH levels in tumors (P<0.001; Fig. 1D) and a strong association with poor overall survival (P=0.038; Fig. 1C). YWHAH expression was significantly linked to CRC tissue differentiation (P=0.024), tumor node metastasis (TNM) stage (P=0.011), N stage (P=0.028) and vascular invasion (P=0.04; Table I).

These studies are in consistency with previous research, suggesting that YWHAH is a major factor in CRC tumorigenesis and could function as a biomarker and a target for therapy.

In our previous study, colony formation assays were conducted, and the results showed that YWHAH overexpression markedly increased colony numbers in both cell lines (12). In the present study, the CCK-8 assay was performed to assess the influence of varying YWHAH expression levels on the proliferative capacity of CRC cells. As illustrated in Fig. 2A and B, YWHAH overexpression significantly enhanced the proliferative ability of LOVO cells, whereas YWHAH suppression markedly diminished their proliferative capacity (P<0.05). Similar results were observed in HCT116 cells.

To assess the oncogenic potential of YWHAH in vivo, subcutaneous xenograft models in nude mice were performed utilizing cells transfected with si-NC, si-YWHAH, overexpression (OE)-NC and OE-YWHAH constructs. Tumor growth was monitored on a weekly basis and growth curves were subsequently generated. Following a period of 5 weeks, the mice were euthanized, and the tumors were excised to measure their volumes. The findings indicated that tumors in the YWHAH overexpression group exhibited a significantly larger volume compared with those in the OE-NC group. Conversely, knockdown of YWHAH expression resulted in a significantly reduced tumor volume compared with the si-NC group (Fig. 2C-F). These in vitro and in vivo results collectively suggest that YWHAH facilitates the proliferation of CRC cells.

The authors' previous study found that knockdown of YWHAH expression levels significantly lowered the numbers of migratory and invasive cells in both cell lines (P<0.01). In contrast to these observations, YWHAH overexpression exhibited the opposite effect, increasing both these processes. These results suggested that YWHAH positively regulated the migratory and invasive capabilities of CRC cells (12).

To further assess the influence of YWHAH on the EMT of CRC cells, western blotting was utilized to assess the changes in EMT markers. These indicated a decrease in E-cadherin and an increase in N-cadherin levels in cells overexpressing YWHAH. Conversely, in knockdown cells, E-cadherin was elevated, while N-cadherin levels declined (Fig. 3A-D).

The induction of apoptosis is closely associated with tumor suppression. Flow cytometry was used to assess the function of YWHAH in modulating the apoptotic response in CRC cells. As illustrated in Fig. 4, knockdown of YWHAH expression via siRNA (si-YWHAH) significantly increased the apoptotic rate in both cell lines, while OE-YWHAH resulted in reduced apoptotic rates in these cell lines (Fig. 4A and B). Western blot analysis further revealed that knockdown of YWHAH expression led to an upregulation of caspase 3 and a decrease of Bcl-2/BAX protein levels in LOVO and HCT116 cell lines. Conversely, YWHAH overexpression significantly inhibited the expression of caspase 3 whilst it enhanced the Bcl-2/BAX protein in both cell lines (Fig. 4C-H). Collectively, these findings suggested that YWHAH overexpression suppresses apoptosis in CRC cells.

A dual-labeled LC3B construct enables the distinct visualization of autophagosomes and autolysosomes, capitalizing on the divergent pH stabilities of its two fluorescent components. Specifically, within the acidic environment of lysosomes, the fluorescent signals from the red (mRFP) and green (GFP) proteins undergo differential quenching. By utilizing the mRFP-GFP-LC3 dual fluorescence system, a more profound understanding of the role of YWHAH in autophagy was obtained. Fluorescence microscopy indicated significantly increased numbers of fluorescent puncta in YWHAH-knockdown CRC cells compared with those of the controls. These observations indicated that the disruption of YWHAH expression could enhance autophagy. Furthermore, the red fluorescent puncta were substantially more abundant than the green ones and their co-localization resulted in a predominantly red region, corroborating the efficient progression of autophagy. Conversely, the opposite effects were observed in YWHAH-OE CRC cells (Fig. 5A-D). These findings were subsequently validated by western blot analysis (Fig. 5E-F). Specifically, knockdown of YWHAH expression via siRNA led to an upregulation of autophagy related 7 (ATG7) and LC3II/I protein expression in both LOVO and HCT116 cells, while it resulted in a downregulation of P62 protein expression. In contrast to these observations, YWHAH overexpression significantly suppressed the levels of ATG7 and the LC3II/I ratio in both cell lines, while increasing P62 expression specifically in LOVO cells. In addition, YWHAH overexpression exhibited a tendency to increase P62 expression in the HCT116 cell line.

It has been found that YWHAH inhibits autophagy in CRC cells. To explore if this affects cell migration and invasion, experiments altering autophagic flux were conducted. Initially, autophagy was inhibited and the data indicated that si-YWHAH increased ATG7 and LC3II/I levels, while it decreased those of P62, confirming that it induced autophagy. In contrast to these observations, the addition of 3-methoxyamphetamine (3-MA) to both si-NC and si-YWHAH groups reduced ATG7 and LC3II/I levels, while it increased P62 levels (Fig. 6A and B), indicating that 3-MA suppressed autophagy in these cells.

The Transwell assay indicated that the si-NC + 3-MA group exhibited a significantly higher number of migratory and invasive cells than the si-NC group, suggesting increased migration and invasion following inhibition of autophagy. In the si-YWHAH + 3-MA group, cell migratory and invasive activities were lower than those noted in the si-NC + 3-MA group, yet higher than those of the si-YWHAH group, indicating that 3-MA reversed the inhibitory effects of si-YWHAH on CRC cell migration and invasion (Figs 6C-F and S2). This confirmed that inhibition of autophagy could counteract the suppressive impact of knockdown of YWHAH expression on these processes.

An in-depth analysis of migration and invasion in CRC cells was performed with stable knockdown and overexpression models of YWHAH by introducing the autophagy inducer Rapamycin (RAPA). In the OE-YWHAH group, the levels of ATG7 and LC3II/I were markedly reduced compared with those of OE-NC cells, while P62 levels were notably elevated, suggesting that OE-YWHAH inhibits autophagy in CRC cells, corroborating previous research findings. In contrast to these observations, OE-NC + RAPA cells exhibited a marked rise in ATG7 and LC3II/I levels together with reduced P62 levels compared with those noted in OE-NC cells. Similarly, OE-YWHAH + RAPA cells demonstrated markedly elevated ATG7 and LC3II/I levels and decreased P62 levels compared with OE-YWHAH cells, indicating that RAPA effectively activated autophagy in CRC cells (Fig. 7A and B). Furthermore, the results from the Transwell migration and invasion assays revealed that the number of migratory and invasive cells in the si-NC + RAPA cells was significantly decreased compared with that of OE-NC cells (Figs. 7C-F and S3), suggesting that autophagy activation led to a relative reduction in migratory and invasive activities.

In subsequent studies, the MAPK/ERK signaling pathway inhibitor PD98059 and the agonist U-46619 were used to evaluate the levels of the related pathway proteins ERK1/2, phosphorylated (p)-ERK1/2, and p-ETS Like-1 protein (ELK)-1 by western blotting. The aim of these experiments was to assess whether YWHAH exhibited regulatory effects on the ERK signaling pathway.

Previous studies have demonstrated that YWHAH overexpression inhibits autophagy, whereas its knockdown promotes autophagy. The present study explored the ERK pathway-related protein expression following alteration of the YWHAH levels. The results revealed that si-YWHAH increased p-ERK1/2 and p-ELK-1 expression levels, activating the MAPK/ERK pathway, while OE-YWHAH decreased their expression levels, suppressing the pathway. In addition, si-YWHAH reversed the inhibitory effect of PD98059 on the pathway, as p-ERK1/2 and p-ELK-1 levels were higher in si-YWHAH + PD98059 cells compared with those of the si-NC + PD98059 cells (Fig. 8). Subsequent investigations indicated that p-ERK1/2 and p-ELK-1 levels were significantly elevated in OE-NC + U46619 cells compared with those of the OE-NC cells. They were also higher in OE-YWHAH + U46619 cells compared with those of the OE-YWHAH cells; however, they were lower than those noted in the OE-NC + U46619 cells (Fig. 9). This suggested that YWHAH overexpression could counteract the activation of the signaling pathway by U46619. The protein expression levels were statistically significant, with the exception of t-ERK. Overall, these results indicated that YWHAH negatively regulated the ERK axis in CRC cells.

To further investigate the relationship between the regulation of autophagy by YWHAH in CRC and the involvement of the MAPK/ERK signaling pathway, the expression levels of autophagy-related proteins were investigated following inhibition and activation of the ERK signaling pathway.

Western blotting indicated that in si-YWHAH cells, ATG7 and LC3II/I levels were significantly elevated, while P62 levels were lower compared with those noted in si-NC cells (Fig. 8). The introduction of the inhibitor PD98059 to the si-YWHAH group decreased the levels of ATG7 and LC3II/I, while it increased those of P62, indicating reduced autophagy and reversal of the activation effect of si-YWHAH on autophagy in CRC cells (Fig. 8). Conversely, in the OE-YWHAH group, ATG7 and LC3II/I levels were lower, and P62 expression was higher. The addition of the activator U-46619 increased ATG7 and LC3II/I levels and reduced P62 expression (Fig. 9), suggesting enhanced autophagy and reversal of the inhibitory effect of OE-YWHAH on autophagy in these cells. In summary, these results indicated that in CRC cells, YWHAH regulated autophagy in a manner dependent on the MAPK/ERK axis.

To evaluate if YWHAH inhibits autophagy in CRC via the MAPK/ERK pathway, the expression levels of autophagic and signaling proteins were investigated in nude mouse tumors. In the si-YWHAH group, the expression levels of the autophagic markers ATG7 and LC3II/I were increased, while the levels of P62 were reduced, indicating enhanced autophagy. Conversely, in OE-YWHAH cells, the expression levels of ATG7 and LC3II/I were lowered, while those of P62 were increased, suggesting reduced autophagy. In addition, p-ERK1/2 and p-ELK-1 levels were increased in the si-YWHAH group and decreased in the OE-YWHAH group (Fig. 10A-F). These results confirmed that YWHAH inhibited autophagy in CRC via the MAPK/ERK pathway.