Pan-cancer expression profiles and related clinical data were obtained from the UCSC Xena database (https://xenabrowser.net/datapages/), including data from the Genotype-Tissue Expression (GTEx) and the Cancer Genome Atlas (TCGA) project. At the same time, obtained three expression profiles of SNRPB2 (GSE20347, GSE53625, and GSE23400) from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/). The “ggplot2” package in R formed box plots illustrating the expression distribution. Using the R package ‘survival’, we conducted Kaplan-Meier analysis to investigate the impact of SNRPB2 on the prognosis of ESCA. According to the median of SNRPB2 expression, the patient was divided into a highly expressed group and a lowly expressed group.

A retrospective collection of 162 paraffin-embedded ESCA tissue samples was conducted between May 2004 and 2018 at the Pathology Department of Taizhou Hospital, Affiliated with Wenzhou Medical University(Linhai, China). All tissue samples underwent clinical pathological analysis and were diagnosed as ESCA. Table 1 contains detailed clinical data and specimen information. This study was conducted based on written informed consent from all patients, ensuring ethical standards and the right to informed participation, and received approval from the Ethics Committee of Taizhou Hospital in Zhejiang Province (No.K20210618).

IHC staining was performed following the established protocol (29). The 4 μm sections were obtained by cutting the paraffin-embedded tissue blocks and then attached them to glass slides. Following deparaffinization, rehydration, and microwave antigen retrieval, the slides were incubated overnight at 4°C with antibodies SNRPB2 (Thermo Fisher, PA5-84991) and Ki-67 (Roche, Cat. No. 790-4286). After a 30-minute incubation with a secondary antibody at room temperature, the slides underwent staining using a DAB substrate. Subsequently, counterstaining with hematoxylin was performed. The final staining score was determined based on staining intensity, which ranged from 0 for negative, 1+ for weak, 2+ for moderate, 3+ for strong. Scores of 0 to 1 were considered indicative of low expression, while scores between 2 and 3 were classified as high expression.

The Eca109 and Kyse150 cell lines were sourced from the Cell Bank of the Chinese Academy of Sciences in Shanghai, China. These cell lines were maintained in RPMI 1640 medium (Meilun Biology, Dalian, China) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C in a 5% CO2 atmosphere. To investigate whether SNRPB2 regulates the expression of c-Myc through β-catenin, SNRPB2-knockdown cells and Sh-NC cells were treated with the β-catenin agonist SKL2001(MCE HY-101085) at 20 µmol/L for 24 h. SNRPB2-overexpressed cells were treated with β-catenin inhibitor XAV-939 (MCE HY-15147) at 20 µmol/L for 24 h.

In order to obtain stable cell lines, three SNRPB2 gene shRNA interference sequences and overexpression sequences were respectively cloned into pLVshRNA-EGFP(2A) puro and pCDH-CMV-MCS-EF1 vectors. These constructs were co-transfected with corresponding helper plasmids into 293T cells. After 48 and 72 hours of transfection, collected the supernatant and filtered through a 0.45 μm filter for further purification. Then used the filtered supernatant was mixed with 4μg/ml Polybrene to infect ESCA cells. Infected cells were selected by treating them with puromycin at a concentration of 2μg/ml for 3 days, and subsequent experiments were conducted.

Total RNA was isolated from Eca109 and Kyse150 cells using TRIzol reagent, with the extraction process conducted in strict accordance with the manufacturer’s instructions. Total RNA content and concentration was quantified using a Nucleic Acid Quantification Analyzer (Nanodrop, USA). Subsequently, the RNA was converted into cDNA using a reverse transcription PCR kit under specific conditions: incubation at 37°C for 30 minutes, denaturation at 85°C for 15 seconds, and storage at 4°C indefinitely. The expression level of SNRPB2 was determined using qPCR with 18s rRNA serving as the internal reference. The relative expression levels of SNRPB2 were calculated using the formula 2^(-ΔΔCT). The primers for qPCR employed in this study were as follows:

ESCA cells were subjected to protein extraction using RIPA lysis buffer. The concentration of the extracted protein was then measured with a BCA protein assay kit (Beyotime, Jiangmen, China). Subsequently, twenty micrograms of protein were loaded onto a polyacrylamide gel for electrophoretic separation, and the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. For immunodetection purposes, primary antibodies including anti-SNRPB2 (1:1000; PA5-84991,thermo),anti-Vimentin(1:1000;5741,CST),anti-N-cadherin(1:1000;14215, CST), anti-E-cadherin(1:1000;GTX100443,Genetex),anti-ZO1(1:1000;GTX636491,G enetex),anti-β-catenin(1:1000,GTX633010, Genetex), anti-c-Myc(1:1000,9402, CST),anti-CCNB1(1:1000;CY5378,abways), anti-CCNA2(1:2000;CY5837,abways), anti-CDK1(1:1000;CY5061,abways), anti-CDK2(1:1000;CY5020,abways), anti-β-actin (1:5000; YT0099, Immunoway), and anti-GAPDH(1:10000;60004-1-Ig,proteintech) were used in this study. The membranes were incubated overnight at 4°C with the primary antibodies, after which they were washed thoroughly. Secondary antibodies (at a dilution of 1:10000) were then added to the membranes. Finally, ECL was employed for color development and Image Quant LAS 500 (GE, USA) was used to detect the protein bands. The resulting images were quantified using the specialized software ImageJ.

Cell Counting Kit-8 (GLPBIO, GK10001) was utilized to evaluate cellular proliferation capacity. A total of 3000 cells were added to each well of a 96-well plate. After 24, 48, and 72 hours, each well was supplemented with a mixture of 100μL medium without serum and 10μL CCK-8 reagents. The plates were then incubated at 37°C for 1.5 hours, after which the absorbance at 450 nm was measured with a microplate reader to assess cellular metabolic activity. Furthermore, the EDU cell proliferation assay kit (Beyotime, C0085S) was used to evaluate cell proliferation. Briefly, ESCA cells were seeded in 6-well plates and allowed to reach optimal growth. EDU solution (10 mM) was added and incubated for 2 hours at 37°C. Following fixation with 4% paraformaldehyde, the cells were washed, permeabilized, and subjected to fluorescent dye staining. Flow cytometry analysis was performed to determine the number of proliferating cells that were labeled with EDU.

Cellular proliferation was evaluated through a cell colony formation assay. A total of 1,000 cells were plated in 60 mm dishes and cultured for seven days in a 5% CO2 environment until visible colonies formed. Subsequently, following fixation with 4% paraformaldehyde for 20 minutes, the formed colonies underwent staining using a 1% crystal violet solution for 15 minutes. After thorough drying, the colonies were meticulously enumerated to quantify their proliferative capacity.

The cells were cultured in a 6-well plate until they reached full confluency of 100%. To create uniform scratches on the cell monolayer, a 200μL pipette tip was carefully used. Following the scratching, serum-free medium was added to the wells. Subsequently, the cells were imaged using an inverted microscope after 12 to 24 hours to observe and quantify their Migration into the wounded area.

For the cell invasion assay, the upper chamber was properly coated with extracellular matrix gel (Corning, 356234). After digesting the cells, a suspension in 100 µL of serum-free medium was prepared and then seeded into the upper chamber (Corning, Lowell, MA, USA). The lower chamber contained a 10% FBS culture medium, a chemoattractant to facilitate cell movement. Following a 24-hour incubation at 37°C, the cells in the lower chamber were fixed with 4% paraformaldehyde and subsequently stained with crystal violet. The number of invaded cells was quantified by counting in five randomly selected fields using an inverted microscope.

For cell cycle assay, Eca109 and Kyse150 cells were collected and subsequently fixed in ice-cold ethanol (70%) at a temperature of 4°C for 24 hours. Then, the cells were incubated with propidium iodide (10 mg/ml, Beyotime) for 30 min at room temperature in a dark place, and the distribution of the cell cycle distribution was analyzed using flow cytometry. For the apoptosis assay, cells were harvested and subjected to staining with Annexin V APC/7-AAD (Elabscience, China) according to the manufacturer’s instructions. The percentage of cell apoptosis was measured, and the results were analyzed by FlowJo V10 software.

BALB/c nude mice were obtained from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). All procedures involving the mice were conducted in accordance with the guidelines and regulations set forth by the Animal Care Committee of Taizhou Hospital (No. tyz-2022130). To establish subcutaneous tumor models, male BALB/c nude mice aged 4 weeks were randomly assigned to four groups, each containing six mice. The groups included a blank group, a negative control (NC) group, and two experimental groups: SNRPB2-shRNA2 and SNRPB2-shRNA3. The digested cells were mixed with extracellular matrix gel in a 1:1 ratio and resuspended. Subsequently, each nude mouse was injected subcutaneously with 100 μL of the cell suspension-extracellular matrix gel mixture. A total of 1 × 10⁶ cells were inoculated into each mouse to ensure consistent cell dosage. Tumor images were captured after a 4-week incubation period, and its diameter was measured. Subsequently, the tumor masses were fixed, embedded, and subjected to immunohistochemical staining for further analysis.

The presentation of all experimental data followed the mean ± standard deviation (SD) format. Statistical analyses were conducted using either GraphPad Prism (Version 8.0, CA, USA) or the R programming language. Each cell experiment was independently repeated three times. Comparisons between the two groups were assessed using either an unpaired two-tailed t-test or a chi-square test. A p-value of less than 0.05 was deemed statistically significant.

The expression of SNRPB2 was analyzed across 33 tumor types through integration of samples from the GTEx and TCGA databases. Results revealed that SNRPB2 is upregulated in 27 tumor types, including ACC, BLCA, BRCA, CESC, CHOL, COAD, DLBC, ESCA, GBM, HNSC, KIRC, KIRP, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, READ, SKCM, STAD, TGCT, THCA, THYM, UCEC, and UCS ( Figure 1A ).

To further validate these findings, we analyzed data from the TCGA databases specifically for ESCA, which consistently demonstrated a significant increase in SNRPB2 expression levels in ESCA tissues compared to normal tissues ( Figure 1B ). This observation was corroborated when analyzing paired samples ( Figure 1C ). Additionally, our analysis revealed a positive correlation between SNRPB2 expression levels and the staging of ESCA ( Figure 1D ). Further validation was conducted using multiple ESCA tissues datasets within the GEO database, all of which showed elevated expression levels of SNRPB2 ( Figure 1E ). To evaluate the prognostic relevance of SNRPB2 in ESCA, Kaplan-Meier survival analysis revealed that high SNRPB2 expression in the TCGA and GSE53625 cohorts was associated with significantly poorer prognosis compared to low-expression groups ( Figure 1F ). These results indicated that SNRPB2 has the potential to be an important prognostic biomarker for ESCA.

IHC staining was conducted on ESCA tissues, and adjacent non-tumorous samples were matched to validate bioinformatic findings, systematically comparing SNRPB2 protein expression levels. The results showed a marked increase in SNRPB2 protein expression levels in ESCA tissues compared to adjacent non-cancerous tissues, confirming consistency with the bioinformatics data. Additionally, a significant correlation was observed between SNRPB2 protein expression and the staging of ESCA patients ( Figure 1G ).

To interrogate SNRPB2 function in ESCA cells, stable knockdown cell lines were generated, with efficient depletion confirmed by qPCR ( Figure 2A ) and Western blot analysis ( Figure 2B ). Subsequently, two shRNA sequences exhibiting the highest interference efficiency were chosen for follow-up experiments. The results of the CCK-8 assay exhibited a remarkable decline in the proliferative capacity of Eca109 and Kyse150 cell lines following SNRPB2 knockdown, in comparison to the Sh-NC cells ( Figure 2C ). Further assessment using the EDU assay confirmed that SNRPB2 knockdown significantly suppressed the proliferative ability of Eca109 and Kyse150 cells ( Figure 2D ). Furthermore, quantitative analysis of the colony formation assay indicated a noticeable decrease in the number of colonies formed by SNRPB2-deficient cells compared to the Sh-NC cells ( Figure 2E ). Flow cytometry analysis revealed that SNRPB2 knockdown caused cell cycle arrest in the G2/M phase and promoted apoptosis ( Figures 2F, G ). In addition, we utilized scratch assays and Transwell invasion assays to assess the effects of SNRPB2 knockdown on tumor migration and invasion. The quantification of migrated and invaded cells revealed a substantial reduction in both in vitro Migration ( Figure 3A ) and invasion capability ( Figure 3B ) of ESCA cells following SNRPB2 knockdown.

To further substantiate the involvement of SNRPB2 in ESCA progression, SNRPB2 overexpression experiments were conducted in ESCA cells. The efficacy of overexpression was validated through qPCR ( Figure 4A ) and Western blot ( Figure 4B ). Subsequently, comprehensive CCK-8 and EDU assays unequivocally demonstrated that SNRPB2 overexpression exerted a profound stimulatory effect on the proliferative capacity of ESCA cells ( Figures 4C, D ). Furthermore, the colony formation experiments illustrated the augmenting impact of SNRPB2 on the cellular proliferation process ( Figure 4E ). Flow cytometry analysis demonstrated that overexpression of SNRPB2 induced G2/M phase transition and attenuated cell apoptosis ( Figures 4F, G ). Strikingly, scratch assays convincingly revealed that SNRPB2 overexpression significantly expedited the migratory potential of ESCA cells ( Figure 5A ), while Transwell assays effectively substantiated its capability to enhance the invasive prowess of these cells ( Figure 5B ).

The EMT signaling pathway exerts a pivotal role during the progression of tumor growth and the subsequent development of metastasis (30). Based on preliminary phenotypic analyses, we hypothesized that SNRPB2 may promote ESCA cells’ malignant proliferation and metastasis through the EMT process. As expected, Western blot analysis was performed to investigate the expression of key markers associated with epithelial and mesenchymal cells during the EMT process. The findings indicated that in the SNRPB2 knockdown group, the levels of mesenchymal markers, including Vimentin and N-cadherin, were significantly reduced, whereas the expression of epithelial markers, such as ZO-1 and E-cadherin, showed a notable increase compared to the Sh-NC group ( Figure 3C ). Conversely, in the SNRPB2 overexpression group, there was an upregulation of Vimentin and N-cadherin expression, accompanied by a suppression of ZO-1, E-cadherin expression ( Figure 5C ). These results highlight the crucial function of SNRPB2 in the in vitro proliferation and migration of ESCA cells, which is facilitated by its regulation of the EMT process.

In the subsequent study, we explored in vivo role of SNRPB2 in the progression of ESCA. Both SNRPB2 knockdown and control Eca109 cells were subcutaneously inoculated into nude mouse models. The results indicated that, in comparison to the Sh-NC group, the tumor volume in the shRNA-treated group was significantly smaller (p < 0.01), accompanied by a lighter tumor weight (p < 0.01), and a slower growth rate ( Figures 6A-D ). Histological examination using HE staining demonstrated a remarkable decrease in tumor cell density in the SNRPB2 knockdown group. Furthermore, immunohistochemical analysis showed consistent downregulation of SNRPB2 expression levels and Ki-67 expression in the shRNA-treated group. These findings further confirm that SNRPB2 knockdown can effectively inhibit the malignant proliferative capacity of ESCA cells in vivo ( Figures 6E, F ).

In order to delve deeper into the potential signaling pathways through which SNRPB2 promotes the progression of ESCA, SNRPB2-related gene analysis was conducted. A heatmap was generated to display the top 50 genes that exhibited the strongest correlation with SNRPB2 ( Figure 7A ). KEGG analyses were performed on 687 genes with a correlation coefficient greater than 0.3. The results indicated their involvement in pathways such as the Ribosome, Spliceosome, Oxidative phosphorylation, RNA degradation, and protein export ( Figure 7B ). Furthermore, GSEA enrichment analysis was conducted, revealing that SNRPB2 can activate pathways such as MYC targets pathway V1, E2F target, DNA repair, and Oxidative phosphorylation, while inhibiting pathways like p53 pathway and Hypoxia ( Figures 7C, D ). Abnormal expression of c-Myc is generally considered to be closely associated with cell proliferation, cycle, migration and invasion, while β-catenin is an important factor activating the expression of c-Myc in cancer cells. Additionally, β-catenin is also an upstream regulator for EMT (31). Therefore, we studied the correlation between SNRPB2 expression and β-catenin, c-Myc and the related cell cycle regulatory proteins. The experimental results showed that compared to the Sh-NC group, the knockdown of SNRPB2 significantly decreased the expression of β-catenin, c-Myc, CCNA2, CCNB1, CDK1, CDK2, while overexpression of SNRPB2 upregulated the expression of these genes ( Figures 7E, F ). These findings indicate that SNRPB2 can modulate the expression of proteins associated with the β-catenin/c-Myc signaling cascade.

Rescue experiments were conducted using the β-catenin agonist SKL2001 to investigate whether SNRPB2 influences ESCA cell proliferation, migration, and invasion via the β-catenin/c-Myc signaling pathway. Colony formation assays revealed that treatment with SKL2001 partially restored the reduced colony-forming capability associated with SNRPB2 knockdown ( Figure 8A ). Furthermore, after a 24-hour treatment with SKL2001, we harvested ESCA cells for migration and invasion assays. The results indicated that the SNRPB2 knockdown + SKL2001 group demonstrated significantly enhanced migration and invasion compared to the SNRPB2 knockdown group alone (P < 0.05) ( Figures 8B, C ). Following the 24-hour SKL2001 treatment, Proteins were collected from Eca109 and Kyse150 cells to assess the expression levels of β-catenin and c-Myc. As depicted in Figure 8D , SKL2001 effectively rescued the downregulation of both β-catenin and c-Myc that resulted from SNRPB2 knockdown.

The β-catenin inhibitor XAV-939 was used to verify that SNRPB2 overexpression impacts ESCA cell proliferation, migration, and invasion via the β-catenin/c-Myc signaling pathway. Colony formation assays demonstrated that treatment with XAV-939 partially mitigated the increased colony-forming ability linked to SNRPB2 overexpression ( Figure 9A ). After exposing Eca109 and Kyse150 cells to XAV-939 for 24 hours, we conducted migration and invasion assays. The results indicated that in the SNRPB2 overexpression group treated with XAV-939, both migration and invasion abilities were significantly diminished compared to the group with SNRPB2 overexpression alone (P < 0.05) ( Figures 9B, C ). Proteins were collected from the Eca109 and Kyse150 cell lines following a 24-hour treatment with XAV-939 to assess the expression levels of β-catenin and c-Myc. As depicted in Figure 9D , the inhibitor effectively diminished the elevated expression of both β-catenin and c-Myc caused by SNRPB2 overexpression.