Yin Yang 1-associated protein 1: Clinical significance and underlying functional mechanisms in gastric cancer

Abstract

BACKGROUND

Gastric adenocarcinoma remains a lethal malignancy with limited prognostic biomarkers and therapeutic targets. Yin Yang 1-associated protein 1 (YY1AP1) is a regulatory partner of Yin Yang 1, which has emerged as a protein of interest in the context of various biological processes and diseases. This study aimed to investigate the role of YY1AP1 in gastric adenocarcinoma.

AIM

To investigate the clinical significance and functional role of YY1AP1 in gastric adenocarcinoma and evaluate its potential as an independent prognostic biomarker.

METHODS

A total of 118 paraffin-embedded and 29 fresh gastric adenocarcinoma samples were analyzed. Immunohistochemistry was used to assess YY1AP1 expression and categorize samples into YY1AP1 high-expression group and YY1AP1 low-expression group. Reverse transcription-quantitative polymerase chain reaction was performed on fresh samples to measure mRNA levels. Online datasets were used to independently validate prognostic value. MKN45 cells were subjected to YY1AP1 overexpression and knockdown; analyzed for p21, c-myc, and snail; and assessed for proliferation and migration.

RESULTS

YY1AP1 was upregulated in tumor tissues. High YY1AP1 expression was correlated with decreased p21 levels and increased c-myc and snail levels. In vitro, YY1AP1 overexpression in MKN45 cells resulted in reduced p21 expression and enhanced c-myc and snail expression, concomitant with increased cell proliferation and migration. Conversely, YY1AP1 knockdown exhibited the opposite effects. Clinically, elevated YY1AP1 levels signify poor prognosis in our cohort and online dataset.

CONCLUSION

YY1AP1 significantly influences the pathogenesis of gastric adenocarcinoma by modulating key cancer-related pathways. This study provides the first comprehensive analysis of YY1AP1 as an independent prognostic factor in gastric cancer. The expression of YY1AP1 is correlated with aggressive tumor behavior, highlighting its potential as a prognostic biomarker and a target for therapeutic intervention.

Key Words: Yin Yang 1-associated protein 1; Gastric adenocarcinoma; Tumor proliferation; Oncogene; Prognostic biomarker

Core Tip: This study identifies Yin Yang 1-associated protein 1 (YY1AP1) as an independent prognostic biomarker in gastric cancer. We demonstrate that high YY1AP1 expression, linked to tumor progression and poor survival, promotes cancer cell proliferation and migration by modulating p21, c-myc, and snail pathways. These findings highlight YY1AP1’s potential as both a therapeutic target and a tool for risk stratification.

INTRODUCTION

Gastric cancer, a prevalent malignancy worldwide, remains a significant public health challenge because of its high morbidity and mortality rates[1]. Gastric cancer is characterized by its heterogeneous nature, resulting from various genetic and environmental factors. The disease often progresses asymptomatically, and late diagnosis is a common issue that contributes to poor prognosis and survival rates[2,3]. Although surgical resection and systemic chemotherapy are the primary treatments, the 5-year survival rate for advanced disease remains below 30%, highlighting an urgent need for novel biomarkers and therapeutic targets[4]. The complexity of gastric cancer pathogenesis necessitates ongoing research to identify novel biomarkers and therapeutic targets that may lead to early detection and enhance treatment efficacy[5].

Yin Yang 1-associated protein 1 (YY1AP1) has emerged as a protein of interest in the research of various biological processes and diseases. Initially identified as a regulatory partner of Yin Yang 1 (YY1)[6], a transcription factor involved in numerous cellular processes including proliferation, differentiation, and apoptosis, YY1AP1 has been implicated in several cellular mechanisms beyond its initial scope. For example, YY1AP1 has been implicated in cellular senescence and organ development[7,8]. Its role in the development of the cardiovascular system has been documented, which indicates its critical function in embryonic development and tissue homeostasis[9]. Recent studies have begun to elucidate its role in cancer biology, although its function in gastric cancer remains underexplored.

In cancer research, YY1AP1 is involved in the modulation of gene expression and affects tumor progression and metastasis. YY1AP1 is a chromatin-associated protein that regulates gene expression by interacting with YY1, influencing processes such as cell cycle progression, differentiation, and senescence. Its dysregulation has been associated with developmental disorders. Studies have shown its varying roles across different types of cancers. For instance, in colorectal cancer and hepatocellular carcinoma, YY1AP1 overexpression is associated with poor prognosis, likely via the regulation of cell cycle and apoptosis-related genes[10,11]. Conversely, in breast cancer, YY1 inhibits cancer cell growth and invasion[12]. The molecular functions of YY1AP1 appear to be context-dependent, as evidenced by its tumor-suppressive role in breast cancer models[13].

Despite its emerging significance in other malignancies, the clinical relevance and biological function of YY1AP1 in gastric adenocarcinoma remain largely unexplored. Given the established yet conflicting roles of YY1AP1 in cellular regulation and tumorigenesis, a systematic investigation in gastric cancer is warranted. This study was designed to comprehensively determine the expression pattern, clinical relevance, and biological functions of YY1AP1 in gastric adenocarcinoma. We aimed to assess YY1AP1 expression in patient tissues and correlate it with clinicopathological features; evaluate its prognostic value using our cohort and independent public datasets; investigate its functional role in modulating cell proliferation, migration, and key oncogenic pathways in vitro; and for the first time, determine its potential as an independent prognostic factor for gastric cancer.

MATERIALS AND METHODS

Patients and samples

This study was conducted on a cohort of 147 patients with gastric adenocarcinoma. The samples comprised 118 formalin-fixed, paraffin-embedded (FFPE) tissue samples and 29 fresh tissue samples. FFPE and fresh tissue samples included matched tumor tissues and adjacent nontumor gastric mucosa. The samples were obtained from patients who underwent surgical resection for gastric adenocarcinoma at Dazhou Dachuan District People’s Hospital (Dazhou Third People’s Hospital).

Immunohistochemical analysis

The FFPE tissue samples were sliced to 4 μm-thick sections for immunohistochemical analysis. Sections were deparaffinized in xylene and rehydrated through a graded series of ethanol. Antigen retrieval was performed using citrate buffer (pH 6.0) under high pressure. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. The FFPE tissue sections were incubated with a primary antibody against YY1AP1 (rabbit polyclonal, 21563-1-AP, dilution 1:200, Proteintech, IL, United States) overnight at 4 °C. After washing, the sections were incubated with a horseradish peroxidase-conjugated secondary antibody. Diaminobenzidine was used as the chromogen, with the reaction monitored and stopped after 3-5 minutes. The sections were then counterstained with hematoxylin. For negative controls, the primary antibody was omitted. YY1AP1 expression levels were evaluated by two independent pathologists blinded to the clinical data. A semi-quantitative scoring system was applied, considering staining intensity (graded as negative, weak, moderate, or strong) and the percentage of positive tumor cells. Samples were categorized into high- and low-expression groups based on the combined assessment.

Reverse transcription-quantitative polymerase chain reaction

Fresh gastric adenocarcinoma and adjacent nontumor tissues were snap-frozen in liquid nitrogen immediately after resection and stored at -80 °C until RNA extraction. Total RNA was extracted using TRIZOL reagent, and complementary DNA was synthesized using a reverse transcription kit. Reverse transcription-quantitative polymerase (RT-qPCR) chain reaction was performed using the qPCR System with SYBR Green to quantify YY1AP1 mRNA expression[14]. The relative expression levels of YY1AP1 were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase and calculated using the 2^-ΔΔCt method.

Cell culture

The MKN45 gastric cancer cell line was used for in vitro experiments. MKN45 is a well-characterized human gastric adenocarcinoma cell line known for its aggressive growth and metastatic behavior, which make it suitable for studying the molecular mechanisms of gastric cancer. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂[15].

Overexpression and knockdown experiments

To investigate the functional role of YY1AP1 in gastric cancer cells, we conducted overexpression and knockdown experiments in MKN45 cells. For overexpression, a full-length human YY1AP1 complementary DNA was cloned into the pcDNA3 expression vector (Invitrogen, CA, United States) using standard molecular cloning techniques, as previously described[7]. The construct was verified by sequencing before transfection. MKN45 cells were transfected with either the YY1AP1 expression vector or transfection reagent (Lipofectamine 3000) according to the manufacturer’s instructions. Following transfection, cells were selected with G418 for 2 days. Successful overexpression of YY1AP1 was confirmed by RT-qPCR and western blot analyses. For the knockdown assay, small interfering RNA targeting YY1AP1 was obtained from Santa Cruz Biotechnology (Cat. #sc-78797, TX, United States). MKN45 cells were transfected with YY1AP1 small interfering RNA or transfection reagent (Lipofectamine 3000) according to the manufacturer’s protocol. The knockdown efficiency was assessed 48 hours post-transfection by RT-qPCR and western blot analyses to confirm the reduction in YY1AP1 expression.

MTT assay for cell proliferation

MTT assay was used to assess cell proliferation following YY1AP1 overexpression and knockdown. Transfected MKN45 cells were seeded in 96-well plates at a density of 5000 cells per well and allowed to adhere overnight. After 24, 48, 72, and 96 hours of incubation, 20 μL of MTT solution (5 mg/mL) was added to each well. The plates were then incubated for 4 hours at 37 °C to allow viable cells to reduce MTT to formazan. The medium was carefully removed, and 150 μL of dimethyl sulfoxide was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader. The absorbance values, indicative of cell proliferation, were plotted to assess the proliferation rate of the cells under different experimental conditions.

Matrigel-Transwell assay for cell invasion

Matrigel-Transwell assay was conducted to evaluate the migratory and invasive capabilities of MKN45 cells after YY1AP1 manipulation. Transwell inserts (8 μm pore size) were coated with Matrigel and placed in 24-well plates. MKN45 cells, post-transfection, were resuspended in serum-free medium and seeded into the upper chamber of the Transwell insert (2.5 × 10⁴ cells per insert). The lower chamber was filled with medium containing 10% fetal bovine serum as a chemoattractant. Cells were incubated for 48 hours and allowed to invade through the pores. Invasive cells on the lower surface of the insert were fixed with methanol, stained with crystal violet, and counted under a microscope.

Western blot assay for protein analysis

Western blot assay was performed to analyze the protein expression levels of YY1AP1, p21, c-myc, and snail. Total protein was first extracted from the MKN45 cells using radio immunoprecipitation assay buffer, and the concentration was determined using a bicinchoninic acid protein assay kit. Equal amounts of protein were separated by sodium-dodecyl sulfate gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat milk and incubated with primary antibodies against YY1AP1 (rabbit polyclonal, 21563-1-AP, dilution 1:1000, Proteintech, IL, United States), p21 (mouse monoclonal, sc-817, dilution 1:500, Santa Cruz Biotechnology, TX, United States), c-myc (rabbit monoclonal, 5605S, dilution 1:1000, Cell Signaling Technology, MA, United States), snail (rabbit monoclonal, 3879S, dilution 1:1000, Cell Signaling Technology, MA, United States), and glyceraldehyde-3-phosphate dehydrogenase (rabbit monoclonal, 5174S, dilution 1:2000, Cell Signaling Technology, MA, United States) overnight at 4 °C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized by using an enhanced chemiluminescence detection system.

Statistical analysis

Statistical analyses were performed to evaluate the significance of the findings in this study. The normality of data distribution was assessed using the Shapiro-Wilk test. For cellular assays, data were normalized to control values and expressed as mean ± SD. Comparisons between two groups were performed using Student’s t-test. One-way ANOVA, followed by a post hoc Tukey’s test, was used for comparisons involving more than two groups. Kaplan-Meier survival curves were generated to assess cancer-specific survival, and differences between curves were analyzed using the log-rank test. Pearson’s correlation analysis was conducted to determine the relationship between YY1AP1 expression and clinicopathological features. A P value of less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS Statistics software.

RESULTS

YY1AP1 expression in gastric adenocarcinoma tissues

The expression of YY1AP1 was analyzed in 118 FFPE and 29 fresh gastric adenocarcinoma samples, including tumor and adjacent nontumor tissues. In the immunohistochemistry analysis of FFPE samples, YY1AP1 expression was significantly higher in tumor tissues than in adjacent nontumor tissues (Figure 1A and B). YY1AP1 was highly expressed in 68% (80/118) of the tumor samples, whereas only 32% (38/118) exhibited low YY1AP1 expression. Consistent with the immunohistochemistry results, RT-qPCR chain reaction analysis revealed a marked upregulation of YY1AP1 mRNA in tumor tissues compared with adjacent nontumor tissues (P < 0.001, Figure 1C). The expression level of YY1AP1 mRNA in tumor tissues was higher than that in adjacent nontumor tissues. This increase was observed in the majority of the cancer samples analyzed. These findings indicate a significant upregulation of YY1AP1 in gastric adenocarcinoma tissues and suggest its potential role in tumor pathogenesis and progression.

Figure 1 Immunohistochemical analysis and reverse transcription-quantitative polymerase chain reaction of Yin Yang 1-associated protein 1 in gastric adenocarcinoma. A and B: High Yin Yang 1-associated protein 1 protein expression is evident in tumor tissues compared to adjacent non-tumor tissues, as shown by the intensity of immunohistochemical staining; C: Reverse transcription-quantitative polymerase chain reaction revealed the different mRNA level of Yin Yang 1-associated protein 1 in tumor tissues compared to adjacent non-tumor tissues. YY1AP1: Yin Yang 1-associated protein 1.

Correlation of YY1AP1 expression with clinicopathological features

The analysis of YY1AP1 expression in 118 patients with gastric adenocarcinoma revealed significant correlations with several clinicopathological features (Table 1). Notably, a significant association was observed between YY1AP1 expression and tumor size. YY1AP1 exhibited high expression more frequently in tumors exceeding 5.0 cm (P = 0.017) than in other sizes. Similarly, elevated YY1AP1 expression was significantly associated with advanced T stages (T3-T4) than with early stages (T1-T2), with P = 0.012. The expression of YY1AP1 was also significantly correlated with the tumor-node-metastasis stage of cancer. High YY1AP1 expression was more common in advanced stages (III-IV) than in early stages (I-II) of gastric adenocarcinoma (P = 0.018). However, no significant correlations were found between YY1AP1 expression and other clinicopathological features such as age, gender, tumor location, and differentiation level. These findings suggest that YY1AP1 expression could be linked to tumor progression and might serve as an indicator of advanced disease in gastric adenocarcinoma.

Table 1 Correlations between Yin Yang 1-associated protein 1 expression with patients’ features.

Clinicopathologic features	Cases (n = 118)	YY1AP1 level		P value
		Low (n = 56)	High (n = 62)
Age				0.670
≤ 55 years	44	22	22
> 55 years	74	34	40
Gender				0.443
Female	36	19	17
Male	82	37	45
Localization				0.643
Upper	16	6	10
Middle	55	26	29
Lower	47	24	23
Tumor size				0.017
≤ 5.0 cm	58	34	24
> 5.0 cm	60	22	38
T stage				0.012
T1-T2	49	30	19
T3-T4	69	26	43
Differentiation				0.982
Well	12	6	6
Moderate	57	27	30
Poor	49	23	26
TNM stage				0.018
I-II	56	33	23
III-IV	62	23	39

TNM: Tumor-node-metastasis; YY1AP1: Yin Yang 1-associated protein 1.

Kaplan-Meier cancer-specific survival analysis and multivariate analysis

Kaplan-Meier analysis explored the effect of various clinicopathological features on the cancer-specific survival of patients with gastric adenocarcinoma (Table 2 and Figure 2). A significant difference in survival based on T stage was noted. Patients with early-stage tumors (T1-T2) exhibited notably better survival outcomes compared with those with advanced-stage tumors (T3-T4) (P = 0.018, Figure 2E), which highlighted the critical effect of tumor invasion depth on prognosis. Well-differentiated tumors exhibited markedly superior survival outcomes compared with poorly differentiated tumors (P = 0.002, Figure 2F). Similarly, patients in early tumor-node-metastasis stages (I-II) demonstrated higher survival rates than those in later stages (III-IV) (P = 0.013, Figure 2G), highlighting the importance of tumor staging in survival prognosis. Notably, patients with low YY1AP1 expression levels showed significantly improved survival compared with those with high expression levels (P = 0.014, Figure 2H), which suggested that YY1AP1 could be a potential prognostic marker for gastric adenocarcinoma. No significant survival differences were observed with respect to age (P = 0.457, Figure 2A), gender (P = 0.621, Figure 2B), tumor size (P = 0.172, Figure 2D), and localization (P = 0.229, Figure 2C). Thus, YY1AP1 expression and tumor pathology play critical roles in determining survival outcomes in gastric adenocarcinoma.

Figure 2 Kaplan-Meier survival curves regarding cancer-specific survival in gastric adenocarcinoma patients were plotted and compared using log-rank test. A: Age; B: Gender; C: Tumor localization; D: Tumor size; E: T stage; F: Tumor differentiation; G: Tumor-node-metastasis stage; H: Yin Yang 1-associated protein 1 level. YY1AP1: Yin Yang 1-associated protein 1; TNM: Tumor-node-metastasis.

Table 2 Kaplan-Meier cancer-specific survival analysis, mean ± SD.

Clinicopathologic features	OS months	5-year OS, %	P value
Age, years
≤ 55	63.9 ± 3.4	55.6	0.457
> 55	60.3 ± 2.4	64.6
Gender
Female	58.7 ± 3.5	53.9	0.621
Male	62.7 ± 2.5	64.4
Localization
Upper	48.2 ± 4.0	33.1	0.229
Middle	64.3 ± 2.8	66.8
Lower	60.1 ± 3.0	58.9
Tumor size, cm
≤ 5.0	65.0 ± 2.5	64.1	0.172
> 5.0	57.6 ± 3.0	57.5
T stage
T1-T2	68.0 ± 2.7	74.8	0.018
T3-T4	56.6 ± 2.8	49.0
Differentiation
Well	68.0 ± 5.3	81.8	0.002
Moderate	66.3 ± 2.5	72.1
Poor	51.9 ± 2.8	42.3
TNM stage
I-II	67.6 ± 2.5	65.4	0.013
III-IV	56.0 ± 2.9	57.1
YY1AP1 level
Low	67.4 ± 2.4	72.7	0.014
High	56.0 ± 3.0	48.4

OS: Overall survival; YY1AP1: Yin Yang 1-associated protein 1; TNM: Tumor-node-metastasis.

Multivariate analysis aimed to evaluate the independent prognostic value of various clinicopathological features, including YY1AP1 expression (Table 3). Accordingly, the differentiation status of tumors (poor vs well/moderate) emerged as a significant independent predictor of survival, and poorly differentiated tumors were associated with a substantially elevated risk of adverse outcomes (hazard ratio = 3.175, 95% confidence interval: 1.633-6.175, P = 0.001). High YY1AP1 expression was also identified as an independent prognostic factor and significantly associated with an increased risk of poor survival in patients with gastric adenocarcinoma (hazard ratio = 2.155, 95% confidence interval: 1.116-4.159, P = 0.022). These findings suggest that the differentiation status of the tumor and YY1AP1 expression levels are key independent factors affecting the survival of patients with gastric adenocarcinoma.

Table 3 Multivariate analysis.

Clinicopathologic features	HR	95%CI	P value
T stage (T3-T4 vs T1-T2)	1.385	0.681-2.818	0.368
Differentiation (poor vs well/moderate)	3.175	1.633-6.175	0.001
TNM stage (III-IV vs I-II)	1.637	0.839-3.194	0.149
YY1AP1 (high vs low)	2.155	1.116-4.159	0.022

HR: Hazard ratio; CI: Confidence interval; YY1AP1: Yin Yang 1-associated protein 1; TNM: Tumor-node-metastasis.

Effect of YY1AP1 based on online gastric cancer datasets

To further validate and extend the clinical significance of YY1AP1 observed in our patient cohort, we conducted survival analyses. The results highlight the clinical significance of YY1AP1 expression in gastric cancer across multiple datasets, including GSE14210, GSE15459, GSE22377, GSE29272, GSE51105, and GSE62254. Elevated YY1AP1 expression was correlated with poor outcomes in first-progression survival (Figure 3A), post-progression survival (Figure 3B), and overall survival (Figure 3C). Thus, YY1AP1 plays a critical role in tumor progression and resistance to treatment. Patients with high YY1AP1 expression exhibited accelerated disease progression and reduced survival times, highlighting its potential as a biomarker for predicting prognosis in gastric cancer.

Figure 3 Kaplan-Meier survival curves based on Yin Yang 1-associated protein 1 expression in patients. A: First-progression survival: Patients with high Yin Yang 1-associated protein 1 (YY1AP1) expression exhibited significantly shorter first-progression survival compared to those with low YY1AP1 expression (P = 0.028); B: Post-progression survival: High YY1AP1 expression was associated with reduced post-progression survival compared to low expression (P = 0.0067); C: Overall survival: High YY1AP1 expression correlated with worse overall survival (P = 0.013). The X-axis represents survival time in months, while the Y-axis shows the survival probability. Below each plot, the number of patients at risk is indicated for each group at specific time points. Data were extracted from GSE14210, GSE15459, GSE22377, GSE29272, GSE51105, and GSE62254. YY1AP1: Yin Yang 1-associated protein 1.

Subgroup analyses further refined the effect of YY1AP1 expression by analyzing its prognostic relevance across different clinical and molecular subgroups (Figure 4). The association between high YY1AP1 expression and poor survival was particularly pronounced in diffuse-type gastric cancer, male patients, and human epidermal growth factor receptor 2-negative tumors, which indicated that YY1AP1 may contribute to highly aggressive disease phenotypes. The significant association with poor outcomes in patients undergoing surgery alone suggested that high YY1AP1 expression may identify patients at high risk of recurrence, who could benefit from intensive postoperative therapies.

Figure 4 Kaplan-Meier survival analyses stratified by clinicopathological features based on Yin Yang 1-associated protein 1 expression. A: High Yin Yang 1-associated protein 1 (YY1AP1) expression showed a trend toward shorter survival, though not statistically significant (P = 0.052) in gastric cancers with intestinal type; B: High YY1AP1 expression was associated with significantly worse survival (P = 0.026) in gastric cancers with diffuse type; C: No significant survival difference between high and low YY1AP1 expression groups (P = 0.17) in female patients; D: High YY1AP1 expression was linked to shorter survival (P = 0.005) in male patients; E: High YY1AP1 expression correlated with poorer outcomes (P = 0.035) in the gastric cancer patients who only accepted surgery treatment; F: No significant survival difference based on YY1AP1 expression (P = 0.093) for those patients who underwent 5-fluorouracil based adjuvant therapy after surgical treatment; G: A marginal survival trend was noted for high expression (P = 0.056), however without significance those patients who underwent other adjuvant therapy after surgical treatment; H: In human epidermal growth factor receptor 2-negative patients, high YY1AP1 expression was significantly associated with worse survival (P = 0.0079); I: In human epidermal growth factor receptor 2-positive patients, no significant difference in survival outcomes (P = 0.34). Data were extracted from GSE14210, GSE15459, GSE22377, GSE29272, GSE51105, and GSE62254. YY1AP1: Yin Yang 1-associated protein 1; 5-Fu: 5-fluorouracil; HER2: Human epidermal growth factor receptor 2.

YY1AP1 expression did not significantly influence survival in human epidermal growth factor receptor 2-positive patients or those treated with 5-fluorouracil-based adjuvant therapy, which implied that its oncogenic role may be context-dependent or overshadowed by the effects of targeted and chemotherapy-based treatments in these subgroups. These findings suggest that YY1AP1 could serve as a prognostic biomarker to identify high-risk patients and guide personalized treatment strategies. Future studies focusing on the biological mechanisms by which YY1AP1 promotes tumor progression and resistance to therapy may reveal new opportunities for therapeutic intervention.

YY1AP1 promotes gastric adenocarcinoma cell proliferation and invasion

In the cell experiments, the effects of YY1AP1 on p21, c-myc, and snail expression, as well as cell proliferation and invasion, were investigated using the MKN45 gastric cancer cell line. Following YY1AP1 overexpression, a significant decrease in p21 expression and an increase in c-myc and snail levels were observed, which was associated with enhanced proliferation and invasion capabilities compared with control cells, as demonstrated by EdU, MTT, and Transwell experiments. Conversely, YY1AP1 knockdown resulted in an increase in p21 levels and a decrease in c-myc and snail expression (Figure 5A and B). MKN45 cells with overexpressed YY1AP1 demonstrated enhanced proliferation and invasion capabilities compared with control cells, as evidenced by EdU, MTT, and Transwell experiments. By contrast, cells with YY1AP1 knockdown exhibited reduced proliferation (Figure 5C and D) and invasion (Figure 5E). These results indicate that YY1AP1 plays a critical role in modulating the expression of key genes involved in cell cycle regulation and metastasis, and its expression is closely linked to the proliferative and migratory behaviors of gastric adenocarcinoma cells.

Figure 5 Effects of Yin Yang 1-associated protein 1 overexpression and knockdown in MKN45 cells on key cancer-related markers and cellular behaviors were tested. A and B: The data on mRNA and protein expression levels of Yin Yang 1-associated protein 1 (YY1AP1), p21, c-myc, and snail; C and D: EdU experiments showed that overexpressing YY1AP1 can significantly promote gastric cancer cell proliferation while silencing YY1AP1 exerts opposite effects, which had also been confirmed by MTT assays; E: Transwell assays showed the migration and invasion impacts of YY1AP1 in gastric cancer cells. YY1AP1: Yin Yang 1-associated protein 1; siRNA: Small interfering RNA; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

DISCUSSION

Our findings elucidate the role of YY1AP1 in gastric adenocarcinoma, providing valuable insights into its underlying molecular mechanisms. By integrating evidence from our own clinical cohort, in vitro functional experiments, and validation through independent public databases, this study provides a comprehensive assessment of YY1AP1 in gastric cancer. The significant upregulation of YY1AP1 in tumor tissues compared with adjacent nontumor tissues suggests its potential role in tumor progression and aggressiveness. The correlation between high YY1AP1 expression and advanced tumor stages, large tumor size, and poor survival outcomes aligns with previous studies in other cancers, where YY1AP1 has been associated with tumor progression and metastasis. In colorectal cancer and renal cancer, YY1AP1 expression has been linked to varying effects on cell proliferation and invasion[10,16]. This variability highlights the complex, context-dependent nature of YY1AP1’s function in cancer biology. Our multivariate analysis identified high YY1AP1 expression as an independent prognostic factor, a novel finding in gastric adenocarcinoma.

Notably, the findings highlight the heterogeneity of cancer pathogenesis and the importance of tissue-specific investigations. Our integrated approach, combining clinical, in vitro, and bioinformatic analyses, strengthens the evidence for YY1AP1 as a pivotal factor in gastric cancer progression. The inverse relationship between YY1AP1 expression and p21 levels and the direct relationship with oncogenes c-myc and snail in this study further reinforce the complex role of YY1AP1 in modulating key pathways in gastric adenocarcinoma[17-19]. The enhanced proliferative and migratory capabilities observed in YY1AP1-overexpressing cells support the hypothesis that YY1AP1 may play a role in the aggressiveness of malignancies[20]. This finding is particularly significant in the context of metastasis, a major challenge in the treatment of gastric cancer.

The findings have several implications for gastric adenocarcinoma research and treatment. First, YY1AP1 could serve as a novel biomarker for early detection and prognosis and aid in the stratification of patients based on risk and guiding treatment decisions. Second, targeting YY1AP1, possibly through gene therapy or small-molecule inhibitors, may develop into a therapeutic strategy, especially in cases where conventional treatments are ineffective[21].

However, this study has limitations. The retrospective design and the specific patient cohort limit the generalizability of the results. Future studies involving large, diverse populations and longitudinal data are necessary to validate the findings. Although we employed MTT and EdU assays, which are established methods for assessing cell proliferation, the inclusion of additional proliferation markers, such as Ki-67 staining by flow cytometry, would strengthen our conclusions in future work. The exact molecular mechanisms through which YY1AP1 affects tumor biology in gastric adenocarcinoma require further elucidation. Although our data demonstrated a clear association between YY1AP1 and the regulation of p21, c-myc, and snail, further studies, such as rescue experiments, are needed to establish a direct causal relationship between these molecular changes and the observed phenotypes of proliferation and migration. Despite these limitations, our work established YY1AP1 as a clinically relevant biomarker and a potential therapeutic target in gastric adenocarcinoma, warranting further investigation. The upstream regulators of YY1AP1 deserve further investigation, such as microRNA-1193, which has been reported in gliomas[22].

CONCLUSION

This study highlights the significant role of YY1AP1 in gastric adenocarcinoma. The upregulation of YY1AP1 in tumor tissues and its correlation with advanced disease stages and poor survival outcomes suggest its potential as a biomarker and therapeutic target. YY1AP1 modulates key genes and pathways that affect cell proliferation and invasion, emphasizing its critical role in the pathogenesis of gastric adenocarcinoma.