SNRPB2 facilitates esophageal squamous cell carcinoma oncogenesis and progression via E2F4 stabilization

SNRPB2 facilitates esophageal squamous cell carcinoma oncogenesis and progression via E2F4 stabilization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article SNRPB2 facilitates esophageal squamous cell carcinoma oncogenesis and progression via E2F4 stabilization Feng Xu, Chen-cheng Zhu, Chen Lu, Guang-yao Ning, Ren-quan Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5382410/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Esophageal cancer (ESCA) has poor prognosis. Small nuclear ribonucleoprotein polypeptide B2 (SNRPB2) involves in pre-mRNA splicing a component of the spliceosome. However, the potential role of SNRPB2 in tumors remain s poorly understood. This study aimed to determine the clinical relevance and prognostic value of SNRPB2 in patients with ESCA. Methods SNRPB2 mRNA expression levels and genetic alterations were analyzed using GEPIA2 and cBioPortal. The SNRPB2 protein were detected by immunohistochemical staining using paraffin-embedded tissue specimens of esophageal squamous cell carcinoma (ESCC). Cell experiments were performed to verify the role of SNRPB2 in ESCC cells. TIMER, GO , and GSEA analyses were performed to investigate potential biological functions of SNRPB2. Cycloheximide (CHX) chase was used to test protein stability. Results SNRPB2 mRNA was highly expressed in ESCA and associated with tumor progression and clinical prognosis. SNRPB2 protein was highly expressed in ESCC and significantly correlated with vessel carcinoma embolus, lymph node metastasis, clinical stage, and tumor grade. in vitro and in vivo experiments showed that the knockdown of SNRPB2 significantly suppressed proliferation, migration, and invasion. GSEA showed that SNRPB2 inhibits the Rb/E2F pathway . SNRPB2 positively correlates with E2F4 by increasing E2F4 protein stability . E2F4 overexpression dramatically eliminated the effects of SNRPB2 knockdown on ESCC tumor progression. Conclusion SNRPB2 promotes the proliferation, migration, and invasion of ESCC cells by increasing E2F4 protein stability. SNRPB2 has a prognostic role and immunoregulatory potential in ESCA, and is an important factor for prediction, evaluation, and individualized treatment. SNRPB2 esophageal cancer cancer biomarker clinical relevance prognostic factor tumor progression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Esophageal cancer (ESCA) is one of the most lethal malignancies. 1 Surgical resection of the tumor from the primary site has been the standard treatment, especially for localized esophageal squamous cell carcinoma (ESCC), which is the main histological type of ESCA in China. 2 Nowadays, proteogenomics and single-cell transcriptomic analyses have elucidated cancer-driving waves in ESCC progression and revealed the molecular characterization of dietary habit-associated signatures. 3 , 4 However, the intrinsic characteristics of the molecular and clinical perspectives associated with the risk factors of ESCC progression are still unknown. Therefore, further study of the comprehensive understanding of ESCC molecular targets will help overcome the current challenges in precision therapeutic s and improve outcomes for ESCC patients. Small nuclear ribonucleoprotein polypeptide B2 (SNRPB2), known as U2 snRNP B'' or U2 small nuclear ribonucleoprotein B'', is one of the unique proteins that comprise the U2 snRNP. 5 SNRPB2 has unique RNA binding property, with the high degree of sequence and structural conservation, 6 involved in precursor messenger RNA (pre-mRNA) splicing as component of the spliceosome in physiological state. 7 , 8 Pre-mRNA splicing are key for eukaryotic gene expression and cellular function while splicing alterations can lead to various diseases including blindness, 9 autoimmunity disease and cancer. 10 , 11 In tumors, alternative splicing frequently plays critical roles in tumour genesis, development and metastasis, whose regulatory factors may be considered as prognostic biomarkers and therapeutic targets for cancer intervention. 12 SNRPB2 as an early growth-inducible gene essential for the regulation of pre-mRNA splicing and gene expression, is widely expressed in primary cortical neurons, epithelial cells and fibroblasts. 13 , 14 However, the potential role of SNRPB2 in tumors remain ill-defined, necessitating further evaluation. In this study, we conducted a comprehensive analysis of the expression and prognosis for SNRPB2 in ESCC using online databases, and experiment in vitro and in vivo . SNRPB2 mRNA expression levels and their prognostic values were analyzed using Gene Expression Profiling Interactive Analysis 2 (GEPIA2), cBioPortal, and Tumor Immune Evaluation Resources (TIMER). SNRPB2 protein expression levels, clinicopathological characteristics, and clinical relevance were determined by western blot ting and immunohistochemistry using paraffin-embedded tissue specimens and ESCC cell lines , respectively. In addition, we explored the molecular mechanisms by which SNRPB2 promotes the occurrence and progression of ESCC using gene set enrichment analysis (GSEA) and validated the results using a co-immunoprecipitation (Co-IP) assay. 2. Materials and Methods 2.1 Cell lines and lentivirus-mediated gene knockdown The ESCC cell lines (KYSE-150, KYSE-140, KYSE-450, and TE-1) and the human immortalized esophageal epithelial cell line (NE-1) were purchased from the Cell Bank of the Chinese Academy (Shanghai, China). All ESCC cells were cultured in RPMI-1640 supplemented with 10% FBS and 100IU/ml Penicillin-Streptomycin solution with 5% CO 2 at 37℃. For lentiviral gene knockdown, a pLKO.1 shRNA was generated against human SNRPB2 target sequences. pLKO lentiviral vectors containing shRNA were transfected into 293T cells together with psPAX2 and pMD2.G by calcium phosphate transfection (Sigma -Aldrich). The shRNA sequences were transfected into the TE-1 and KYSE-150 cells. shSNRPB2#1: Forward:5’-CCGGCCCAAGGAAATTCAACACCAACTCGAGTTGGTGTTGAATTTCCTTGGGTTTTT-3’ Reverse:5’-AATTCAAAAACCCAAGGAAATTCAACACCAACTCGAGTTGGTGTTGAATTTCCTTGGG-3’ shSNRPB2#2: Forward:5’-CCGGCCATGCTATGAAGATCACCTACTCGAGTAGGTGATCTTCATAGCATGGTTTTT-3’ Reverse:5’-AATTCAAAAACCATGCTATGAAGATCACCTACTCGAGTAGGTGATCTTCATAGCATGG-3’ shE2F4#1: Forward:5’-CCGGCCCTCTCTTCATTTCGGCTTTCTCGAGAAAGCCGAAATGAAGAGAGGGTTTTT-3’ Reverse:5’-AATTCAAAAACCCTCTCTTCATTTCGGCTTTCTCGAGAAAGCCGAAATGAAGAGAGGG-3’ shE2F4#2: Forward:5’-CCGGCGGATTTACGACATTACCAATCTCGAGATTGGTAATGTCGTAAATCCGTTTTT-3’ Reverse:5’-AATTCAAAAACGGATTTACGACATTACCAATCTCGAGATTGGTAATGTCGTAAATCCG-3’ 2.2 Patient tissue sample collection One hundred and twenty-five paraffin-embedded ESCC tissues and paired normal esophageal epithelial tissues adjacent to the cancer tissue were collected from patients who underwent surgery at the First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China) between 2015 and 2018. None of the patients had a history of other malignant tumors or had received preoperative interventions such as radiotherapy or chemotherapy. Each patient provided written informed consent, and the study was approved by the institutional review board (2022AH051185). 2.3 GEPIA2, cBioPortal and TIMER analysis A correlative prognostic analysis of SNRPB2 from the GEPIA2 dataset, including overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI), was conducted using the log-rank test at a median cut-off of 50% for both the SNRPB2 low and SNRPB2 high cohorts. The cBioPortal for Cancer Genomics ( http://cbioportal.org ) served as an open online resource for visualizing multidimensional cancer genomics datasets. 15 The TCGA-ESCA dataset was selected for further analysis of SNRPB2 mRNA. Tumor immune evaluation resources (TIMER) ( https://cistrome.shinyapps.io/timer ), including B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells, were used for immune cell infiltration analysis. 16 2.4 GO and GSEA enrichment analysis Enrichment analysis was conducted using R package "clusterProfiler" to investigate the potential functions of SNRPB2. Gene Ontology (GO) and gene set enrichment analyses (GSEA) were used for pathway enrichment analysis in ESCA. For both GO and GSEA analyses, the cut-off criteria were P < 0.05, and P . adjust < 0.5, respectively. The Benjamini-Hochberg method was u sed to calculate adjusted P -values. 2.5 Xenograft tumor model assay Four-week-old male athymic BALB/c nude mice (Weitonglihua Co., Ltd., China) were randomly divided into four groups. An equivalent number of TE-1 cells (3×10 6 cells/200 µL) were resuspended in PBS and injected into the armpit area of each mouse. Tumor size was measured every three days. After 21 days, the mice were euthanized and the tumors were carefully removed, weighed, and photographed. All animal procedures were done in accordance with protocols approved by the experimental animal ethics committee, Anhui Medical University (LLSC20231142) . 2.6 Immunohistochemical analysis The in situ protein expression levels of SNRPB2 and E2F4 in paraffin-embedded ESCC tissue sections were assessed by immunohistochemistry using rabbit polyclonal antibodies against SNRPB2 (1:100, 21244-1-AP, Proteintech) and E2F4 (1:100, 10923-1-AP, Proteintech). Five fields were randomly observed at a high magnification under a microscope. The staining intensity of SNRPB2 and E2F4 in tumor cells was classified as follows: 0 (no staining); 1 (light yellow); 2 (medium yellow); and 3 (dark yellow). The percentage of stained cells was categorized as follows: 0 (no positive tumor cells), 1 ( 60% positive cells). The total score, which rang ed from 0 to 6, was calculated by summing the two scores. Samples with staining scores of 0–3 were classified as having low SNRPB2 or E2F4 expression , whereas those with scores > 3 were classified as having high SNRPB2 or E2F4 17 . 2.7 Western blot assay Whole cell lysates were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) containing a complete protease inhibitor cocktail. The total protein was loaded and separated by SDS-PAGE, followed by transfer onto polyvinylidene fluoride membranes (Millipore, USA). Immunodetection was performed using antibodies against β-actin (1:5000, 81115-1-RR; Proteintech) and SNRPB2 (1:2000). Horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) were used for digital chemiluminescence detection (GE Healthcare). Blot s are representative of three independent experiments. 2.8 Cell proliferation assay Cell proliferation was assessed using cell counting kit-8 (CCK‐8) description (Beyotime). Cells (5×10 3 cells/well) in logarithmic growth phase were seeded in 96-well plates and incubated at 37°C. After 24, 48, 72, 96 h and 120h, 10µL of CCK8 was added to each well and incubated for 2h. Cell proliferation was determined by measuring the absorption value of whole wells, which was directly detected at 450 nm. 2.9 Clone formation assay The cells were inoculated into six-well plates (1,000 cells/well) in triplicate and cultured for 14 days. Cells number more than 50 or cell size between 0.3 and 1.0 mm were considered as a single clone. All colonies were fixed with 4% paraformaldehyde for 10 min , stained with Giemsa, and photographed using a digital camera. 2.10 Wound healing assay The cells were collected and inoculated into six-well plates (5×10 5 cells/well) in 2 ml medium and cultured at 37°C with 5% CO 2 . When the cell s reached more than 90% confluence, a pipetting tip of 100µL was used to scratch the six-well plate vertically. Cells were washed twice with PBS and cultured in an incubator with 5% CO 2 at 37°C. Photographs were taken at 0 and 48 h under a microscope, and the experiment was repeated thrice. 2.11 Transwell assay The migration and invasion capacities of ESCC cells were determined using a Transwell chamber (Corning) precoated with Matrigel. After transfection, ESCC cells were collected, counted, and incubated in the upper chamber with 100 µL RPMI-1640 medium without FBS in a 24-well plate (5×10 4 cells/well). 600µL medium supplemented with 20% FBS was added to the lower chamber. After incubation at 37°C and 5% CO 2 for 24 h, non -metastatic cells were removed using a cotton swab. 500µL Giemsa was added for staining and the migratory ability of the cells was analyzed. Representative images were selected from three independent experiments, of which five fields per chamber were randomly selected to count cell numbers. The magnification used was 200× . 2.12 Co-immunoprecipitation (Co-IP) The cell extracts were resuspended and lysed on ice using an IP buffer. Protein A/G magnetic beads (Bimake, China) were added to cell lysate s and incubated for 2 h at 4°C. IgG was added to the negative control group, whereas IP antibody was added to the experimental group for 12 h at 4°C. Immunoprecipitated proteins were eluted by boiling in SDS loading buffer (2×) for western blot analysis as described above. 2.13 Cycloheximide (CHX) chase assay Cells were seeded in six-well plates (1×10 5 cells/well) and pretreated with CHX (100 µg/mL) for 0–4 h. Cells were harvested for western blot analysis , as described above. 2.14 Statistical analysis SPSS (version 22.0 ) was used for data analysis. Chi-square test and paired-samples t-test were used for variable comparison, with P < 0.05 regarded as statistically significant. 3. Results 3.1 High expression of SNRPB2 mRNA was correlated with worse outcome in patients with ESCA Differentially expressed genes were identified by comparing ESCA and para-carcinoma esophageal tissue samples from the GEPIA2 database. We found that SNRPB2 mRNA was significantly upregulated in esophageal cancer significantly and correlated with the clinical stage and prognosis (Supplementary material Table S1-2 ). To further determine the prognostic relevance of SNRPB2 in ESCA, patients from multiple ESCA datasets were classified into SNRPB2 high and SNRPB2 low groups and their survival (including OS, DSS, and PFI) was analyzed using Kaplan-Meier curves. As shown in Fig. 1 a, the SNRPB2 low group had significantly longer OS and DSS than the SNRPB2 high group (both P 0.05). These findings suggest that SNRPB2 mRNA expression might correlate with ESCA prognosis, whereas SNRPB2 overexpression might indicate worse outcome s in patients with ESCA. 3.2 Expression level of SNRPB2 mRNA in ESCA and its association with tumor stage To explore the expression of SNRPB2 mRNA in ESCA and normal esophageal tissue samples from TCGA, we analyzed para-carcinoma tissue from patients with ESCA and normal esophageal tissue from tumor-free subjects separately. We found that SNRPB2 mRNA was highly expressed in ESCA samples compared to normal samples, and similar results were observed in paired samples (both P < 0.05) ( Fig. 1 b ) . To further examine the clinical relevance of SNRPB2 in ESCA from TCGA database, we analyzed the correlation of its expression with tumor stage. As shown in Fig. 1 b, no significant correlation between SNRPB2 mRNA expression and tumor stage was observed in this dataset ( P > 0.05). 3.3 Expression level of SNRPB2 protein in ESCC To fu rther validate the above results , we performed immunohistochemical staining of paraffin-embedded tissue specimens to detect the expression of SNRPB2 protein in ESCC. SNRPB2 protein is l ocated in the nucleus and is extensively expressed in normal squamous epithelial cell s and ESCC ( Fig. 1 c ) . The expression rate of SNRPB2 in ESCC was 64.0%, which was significantly higher than that in adjacent tissues (24.0%, P < 0.05) ( Table 1 ) . The results showed that SNRPB2 protein was significantly highly expressed in ESCC samples compared to paired normal samples ( Fig. 2 b ) , whose expression trends were in consistent with the findings obtained from TCGA. Table 1 The expression levels of SNRPB2 protein in tumor and para-carcinoma tissues of patients with esophageal squamous cell carcinoma. Parameter n SNRPB2 P Value Low expression High expression Para-carcinoma tissue 125 95 (76.0%) 30 (24.0%) < 0.001 Esophageal squamous cancer 125 45 (36.0%) 80 (64.0%) 3.4 Association between the expression of SNRPB2 protein and the clinicopathological characteristics of patients with ESCC To explore the clinical significance of SNRPB2 expression in ESCC, we correlated its expression with the clinicopathological characteristics of ESCC patients . Interestingly, the expression levels of SNRPB2 protein were positively correlated with high tumor grade ( P < 0.05) ( Table 2 , Fig. 1 c ) . Further more , the expression levels of SNRPB2 protein in ESCC were significantly correlated with vessel carcinoma embolus, lymph node metastasis, and clinical stage (all P 0.05) ( Table 2 , Fig. 1 d ) . Kaplan-Meier analysis showed that ESCC patients with SNRPB2 high expression had significantly poorer overall survival compared with SNRPB2 low group ( P < 0.05) ( Fig. 1 e ) . Collectively, the expression of SNRPB2 may be associated with ESCC progression, which is associated with poor prognosis in ESCC. Table 2 The relationship between SNRPB2 expression and the clinicopathological features of esophageal squamous cell carcinoma. Parameter n SNRPB2 P Value Low expression High expression Age (years) 0.601 ≤ 60 30 12 (40.0%) 18 (60.0%) > 60 95 33 (34.7%) 62 (65.3%) Sex 0.824 male 93 34 (36.6%) 59 (63.4%) female 32 11 (34.4%) 21 (65.6%) Smoking 0.920 Yes 34 12 (35.3%) 22 (64.7%) No 91 33 (36.3%) 58 (63.7%) Drinking 0.781 Yes 37 14 (37.8%) 23 (62.2%) No 88 31 (35.2%) 57 (64.8%) BMI 0.840 ≤ 28 113 41 (36.3%) 72 (63.7%) > 28 12 4 (33.3%) 8 (66.7%) Grade 0.001 1 + 2 73 35 (47.9%) 38 (52.1%) 3 52 10 (19.2%) 42 (80.8%) Vessel carcinoma embolus 0.012 + 66 17 (25.8%) 49 (74.2%) - 59 28 (47.5%) 31 (52.5%) Nerve invasion 0.068 + 58 16 (27.6%) 42 (72.4%) - 67 29 (43.3%) 38 (56.7%) Lymph node metastasis < 0.001 + 80 19 (23.7%) 61 (76.3%) - 45 26 (57.8%) 19 (42.2%) Stage 0.018 I + II 52 25 (48.1%) 27 (51.9%) III + IV 73 20 (27.4%) 53 (72.6%) Tumor location 0.507 Upper + Lower 35 11 (31.4%) 24 (68.6%) Middle 90 34 (37.8%) 56 (62.2%) 3.5 SNRPB2 facilitated ESCC progression To investigate the potential role of SNRPB2 in ESCC, we first examined the expression levels of the SNRPB2 protein in various ESCC cells and human immortalized esophageal epithelial cells. As shown in Figs. 2 a, the expression levels of the SNRPB2 protein were variable in different ESCC cell lines and were significantly higher than those in human immortalized esophageal epithelial cells. Based on the expression level s , SNRPB2 was knocked down in the TE-1 and KYSE-150 cells. Western blot analysis suggested that shSNRPB2#2 significantly inhibit ed the protein expression of SNRPB2, which was used for subsequent experiments ( Figs. 2 b ) . Colony formation and cell proliferation assay s indicated that knockdown SNRPB2 remarkably suppressed cell proliferation ( Figs. 2 c-d ) . Transwell assays indicated that the expression of SNRPB2 was related with the migration and invasion of ESCC cells ( Figs. 2 e ) . Wound healing assays indicated that the migration rates of TE-1 and KYSE-150 cells with SNRPB2#2 knockdown were 21% and 16%, respectively , lower than those of the control group ( 80% and 70%, respectively), suggesting that SNRPB2 promoted the migration of ESCC cells ( Figs. 2 f ) . These results imply that SNRPB2 acts as an oncogene and facilitates ESCC progression. 3.6 Genetic alterations and immunological correlation of SNRPB2 in ESCA We conducted an online database analysis to investigate genetic alterations in SNRPB2 that may be associated with tumorigenesis via cBioPortal. Genetic alterations were detected in 1.1% of SNRPB2 gene s in ESCA, including 5 amplifications, 2 deep deletions, 1 truncating mutation, and 1 missense mutation (Figure S1a) . No alterations in SNRPB2 were detected in ESCC based on an analysis of 227 samples from two studies (Figure S1b) . These results suggested that SNRPB2 is structurally conserved in ESCC cells. TIMER 2.0 was used to analyze the relationship between SNRPB2 and multiple tumor-infiltrating immune cells in ESCC. SNRPB2 expression was significant ly positive ly correlated with CD8 + T cells (cor = 0.173, P < 0.05) and macrophages (cor = 0.236, P < 0.05) (Figures S2) . These results suggest that the potential role of SNRPB2 in ESCA may be related to tumor immunity. 3.7 SNRPB2 inhibited Rb/E2F pathway in ESCA via increasing stability of E2F4 protein GO and GSEA analyses were performed to reveal the promoting effect of SNRPB2 on ESCC cells. The pathway enrichment results suggested that SNRPB2 significantly inhibited the Rb/E2F pathway in ESCA and was involved in the process of digestion in the apical part of the cell ( Fig. 3 a-b ) . Furthermore, bioinformatic analysis indicated that the mRNA expression of SNRPB2 was positively correlated with E2F4, which is know n as the transcription inhibitory factor of the Rb/E2F pathway ( Fig. 3 c ) . As shown in Figs. 3 d, consistent with SNRPB2, the mRNA expression level of E2F4 was significantly upregulated in ESCA. To verify the interactions between SNRPB2 and E2F4, a co-immunoprecipitation assay was performed to confirm their intera ction in TE-1 and KYSE-150 cells. The results of the co-IP assay demonstrated that SNRPB2 interacts with E2F4 ( Fig. 4 e-f ) . We first knocked down the expression of SNRPB 2 with shRNA in TE-1 and KYSE-150 cells and observed that E2F4 protein expression was downregulated after SNRPB2 knockdown ( Fig. 4 g ) . However, SNRPB2 expression level s showed no significant change after E2F4 knockdown (Figure S3c) . As it was observed to SNRPB2 affected the protein level of E2F4, a CHX chase experiment was conducted to investigate the impact of SNRPB2 on the stability of E2F4. The results demonstrated that SNRPB2 extended the half-life of E2F4 ( Fig. 4 h ) . 3.8 E2F4 counteracted the inhibition effects of shSNRPB2 on the biology in ESCC To investigate the role of E2F4 in SNRPB2-promoted proliferation, migration, and invasion of ESCC cells, we constructed overexpression plasmid of E2F4 to cotransfect with shSNRPB2 in TE-1 and KYSE-150 cells (Figures S3a-b) . The results showed that the overexpress ion of E2F4 ( shCtrl + E2F4) group increased cell proliferation and clone formation compared to the shCtrl + Vector group. The shSNRPB2 + E2F4 group also showed increased proliferation and clone formation of TE-1 and KYSE-150 cells compared to the shSNRPB2 + Vector group ( Fig. 4 a-b ) . In wound healing and transwell assays, overexpression of shCtrl + E2F4 promoted the migration and invasion of TE-1 cells compared to the shCtrl + Vector group, whereas the sh SNRPB2 + E2F4 group rescued the migration and invasion of TE-1 cells compared to the shSNRPB2 + Vector group ( Figs. 4 c-d ) . As shown in Figs. 5 , E2F4 overexpression also blocked shSNRPB2-decreased ESCC cell progression in vivo . These results suggest that E2F4, which is positively regulated by SNRPB2, could be a target gene that plays a role in the progression of ESCC. Finally, we assessed IHC staining in specimens from 125 patients with ESCC to evaluate whether the SNRPB2-E2F4 axis exists in human ESCC specimens. The results suggested that SNRPB2 expression was significantly positively correlated with E2F4 expression (r = 0.517, P < 0.05) ( Fig. 6 , Table 3 ) . Table 3 The correlation between SNRPB2 and E2F4 protein expression in esophageal squamous cell carcinoma. SNRPB2 E2F4 n r Value P Value Low expression High expression Low expression 34 11 45 0.517 < 0.001 High expression 18 62 80 n 52 73 125 4. Discussion Increasing evidence indicates that pre-mRNA splicing provides the potential to produce diversity at RNA and protein levels. 12 Pre-mRNA splicing is also the key to the pathology of numerous diseases, especially cancers, which may be considered as new biomarkers and therapeutic targets for cancer intervention. 12 , 18 ESCA developing through multistage epithelial cancer formation, is a unique and substantial heterogeneous malignancy. 19 , 20 The improvement of ESCA poorly survival outcome is needed to multi-disciplinary evaluation and multi-modal therapy based on the exploration of ideal therapeutic targets and essential prognostic biomarkers. 21 SNRPB2 is essential for the formation of the functional 17S U2 snRNP and prespliceosome assembly in vivo , resulting in cell death as a consequence of blocked its pre-mRNA splicing steps in gene expression. 22 Pre-mRNA processing factor SNRPB2 locates in the nucleus, associated with vimentin-containing intermediate filaments, particularly those surrounding the nucleus. 23 SNRPB2 is involved in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms. 24 However, the roles of SNRPB2 in cancer have rarely been studied. In this study, we conducted an in-depth analysis of SNRPB2 expression and evaluated its feasibility of using SNRPB2 as a prognostic biomarker for ESCC. SNRPB2 is critical for the regulation of gene expression and early growth induction, inducing the process of intron removal from pre-mRNA. 13 , 25 In our study, SNRPB2 protein detected by immunohistochemistry was significantly higher in ESCC samples than in paired normal samples, which was consistent with the online database analysis. The expression levels of the SNRPB2 protein in ESCC were significantly correlated with lymph node metastasis and clinical stage , suggesting that SNRPB2 may be associated with ESCC tumor progression. Deregulated gene expression is a hallmark of cancer, and the progression of heterogeneous tumor s is influenced by multiple factors. 26 , 27 Therefore, we analyzed the genetic alterations in SNRPB2 and performed pathway enrichment analysis. SNRPB2 inhibits the Rb/E2F pathway in ESCA by increasing E2F4 protein stability Our study further revealed that SNRPB2 interacted with E2F4 to promote ESCC cell proliferation. Rb/E2F pathway involves a complex interaction across E2F family members in the regulation of cell cycle progression. 28 In the context of tumorigenesis, the function of E2F4, a transcriptional repressor, was consistently altered, contributing to uncontrolled cell proliferation and cancer progression. 29 In our study, E2F4 was positively regulated by SNRPB2, which acted as an oncogene in the progression of ESCC. Additionally, E2F4 counteracted the inhibitory effects of shSNRPB2 on ESCC biology. However, further investigation s are necessary to elucidate the mechanisms by which SNRPB2 enhances E2F4 stability. Alternative splicing of pre-mRNA plays an important role in immune cell development and regulation of immune activity. 30 Cancer cells have general alterations in the splicing process, which contribute to tumor immune responses and progression. 31 In this study, we investigated the correlation between SNRPB2 and immune cells using bioinformatics analysis. In ESCA, SNRPB2 expression levels positive ly correlat ed with TILs, MHC molecules, and multiple chemokines. This preliminary analysis underscores the need for future studies to elucidate the potential role of SNRPB2 as a therapeutic target for immunotherapy in ESCA. In summary, SNRPB2 protein expression is significant ly upregulat ed in ESCC and is associated with tumor progression and clinical prognosis. Our study provides comprehensive evidence that SNRPB2 is a feasible prognostic marker and a therapeutic target for ESCC. It may also play a role in the disease prediction, evaluation , and individualized treatment of ESCC. Declarations Acknowledgement We would like to express our gratitude to all members of the Department of Thoracic Surgery at the First Affiliated Hospital of Anhui Medical University for their valuable support and contributions to this study. We also appreciate the technical assistance and insightful discussions provided by our colleagues. Special thanks to all the patients and their families who participated in this research. Funding Statement This research was supported by the Anhui Province Higher Education Science Research Project (2022AH051185). The funding body had no role in the design of the study, data collection, analysis, interpretation, or writing of the manuscript. Conflict of interest The authors have no conflicts of interest to report. Ethics Statement The studies involving human participants were reviewed and approved by the First Affiliated Hospital of Anhui Medical University, Hefei 230032, Anhui, China (2022AH051185). All patients provided written informed consent to participate in the study. All animal procedures were done in accordance with protocols approved by the experimental animal ethics committee, Anhui Medical University (LLSC20231142). Author Contributions F.X., C.Z. contributed to the design and execution of the experiments. C.L. was responsible for data collection and analysis. G.N. provided critical insights for the interpretation of results. R.Z. supervised the entire project, contributed to study design, and provided guidance throughout the manuscript preparation. All authors participated in drafting the manuscript, reviewed the final version, and approved it for submission. References Siegel RL, Miller KD, Wagle NS, et al. Cancer statistics, 2023. CA Cancer J Clin. 2023;73(1):17-48. Zhu H, Ma X, Ye T, et al. Esophageal cancer in China: Practice and research in the new era. Int J Cancer. 2023;152(9):1741-1751. Li L, Jiang D, Zhang Q, et al. 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TIMER: a web server for comprehensive analysis of tumor‐infiltrating immune cells. Cancer Res. 2017;77:e108-110. Shen YT, Huang X, Zhang G, et al. Pan-Cancer Prognostic Role and Targeting Potential of the Estrogen-Progesterone Axis. Front Oncol. 2021;11:636365. Di C, Syafrizayanti, Zhang Q, et al. Function, clinical application, and strategies of Pre-mRNA splicing in cancer. Cell Death Differ. 2019;26(7):1181-1194. Li J, Li L, You P, et al. Towards artificial intelligence to multi-omics characterization of tumor heterogeneity in esophageal cancer. Semin Cancer Biol. 2023;91:35-49. Chen Y, Zhu S, Liu T, et al. Epithelial cells activate fibroblasts to promote esophageal cancer development. Cancer Cell. 2023;41(5):903-918. Waters JK, Reznik SI. Update on Management of Squamous Cell Esophageal Cancer. Curr Oncol Rep. 2022;24(3):375-385. Tanackovic G, Krämer A. Human splicing factor SF3a, but not SF1, is essential for pre-mRNA splicing in vivo. Mol Biol Cell. 2005;16(3):1366-1377. Craggs G, Finan PM, Lawson D, et al. A nuclear SH3 domain-binding protein that colocalizes with mRNA splicing factors and intermediate filament-containing perinuclear networks. J Biol Chem. 2001;276(32):30552-30560. Lee Y, Rio DC. Mechanisms and Regulation of Alternative Pre-mRNA Splicing. Annu Rev Biochem. 2015;84:291-323. Hluchý M, Gajdušková P, Ruiz de Los Mozos I, et al. CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1. Nature. 2022 Sep;609(7928):829-834. Huang KK, Huang J, Wu JKL, et al. Long-read transcriptome sequencing reveals abundant promoter diversity in distinct molecular subtypes of gastric cancer. Genome Biol. 2021;22(1):44. Camacho-Vanegas O, Narla G, Teixeira MS, et al. Functional inactivation of the KLF6 tumor suppressor gene by loss of heterozygosity and increased alternative splicing in glioblastoma. Int J Cancer. 2007;121(6):1390-5. Chen X, Chen J, Shao B, et al. Relationship between cancer mutations and parameter sensitivity in Rb pathway. J Theor Biol. 2016;404:120-125. Xiao W, Wang J, Wang X, et al. Therapeutic targeting of the USP2-E2F4 axis inhibits autophagic machinery essential for zinc homeostasis in cancer progression. Autophagy. 2022;18(11):2615-2635. Su Z, Huang D. Alternative Splicing of Pre-mRNA in the Control of Immune Activity. Genes (Basel). 2021;12(4):574. Bonnal SC, López-Oreja I, Valcárcel J. Roles and mechanisms of alternative splicing in cancer - implications for care. Nat Rev Clin Oncol. 2020;17(8):457-474. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterialfigures.docx SupplementarymaterialTables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5382410","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":376297172,"identity":"4b92c925-3cd0-4d29-9ca6-e08f079d8e64","order_by":0,"name":"Feng Xu","email":"","orcid":"","institution":"the First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Xu","suffix":""},{"id":376297173,"identity":"606da23f-9827-4ce1-9c87-9adb01b252a8","order_by":1,"name":"Chen-cheng Zhu","email":"","orcid":"","institution":"the First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chen-cheng","middleName":"","lastName":"Zhu","suffix":""},{"id":376297174,"identity":"fe43f809-06cc-44ec-a430-4b8aa6e88213","order_by":2,"name":"Chen Lu","email":"","orcid":"","institution":"the First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Lu","suffix":""},{"id":376297175,"identity":"e3c5dad3-d1e4-48c4-baa9-a82eebcb26d7","order_by":3,"name":"Guang-yao Ning","email":"","orcid":"","institution":"the First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guang-yao","middleName":"","lastName":"Ning","suffix":""},{"id":376297176,"identity":"2daf3bed-f0c2-4426-ac7a-9983ac87c458","order_by":4,"name":"Ren-quan Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIiWNgGAWjYDACCVSuHAM/M/PhB6RoMWaQbGdLMyBNi8F5HgUJ7GohQH5287OHX/fY5PHdSH724GebQeLmwzwMBgw1NtG4tDDOOWZuLPMsrVjyRpq5YS9Qy7bDvAceMBxLy23AoYVZIsFMWuLA4cQNNxLMJHjb/gC18CUYMDYcxqmFTSL9G1DLf6CW9G+Sf0EOa+YxkMCnhUcix0zyw4EDQC05ZtK8QC0bmAlokZDIKZNmOJCcOPPMmzJpmXMGxjMOAwM5AY9f5Gekb5P8ccAuse84kPGmzEC2v//w4QcfamxwagEHAQ+IPADEjGwMjmCVCXiUgwDjD5gWhj8M9gQUj4JRMApGwQgEAIJTX4WuxRHOAAAAAElFTkSuQmCC","orcid":"","institution":"the First Affiliated Hospital of Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ren-quan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-11-03 14:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5382410/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5382410/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70385316,"identity":"02d99853-653f-47b5-b64c-ae192210965d","added_by":"auto","created_at":"2024-12-02 17:06:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":876679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSNRPB2 was highly expressed in ESCA which was associated with clinical characteristics and prognosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Kaplan‒Meier curves were used to analyze the influence of SNRPB2 mRNA on OS, DSS and PFI in ESCA patients. \u003cstrong\u003e(b)\u003c/strong\u003eExpression level of SNRPB2 mRNAin ESCA and its association with tumor stage. From left to right: The expression of SNRPB2 mRNA in ESCA and normal esophageal tissue samples, the expression of SNRPB2 mRNA in ESCA and para-carcinoma tissue samples, the correlation between SNRPB2 mRNA expression and ESCA clinical stage. \u003cstrong\u003e(c)\u003c/strong\u003e Representative images of SNRPB2 protein expression in tumor and para-carcinoma tissues of ESCC. Upper 3 images show a case of low grade ESCC. From left to right: para-carcinoma tissue (100×), low grade ESCC (100×) and low grade ESCC (400×). Lower 3 images show a case of high grade ESCC. From left to right: para-carcinoma tissue (100×), high grade ESCC (100×) and high grade ESCC (400×). \u003cstrong\u003e(d)\u003c/strong\u003e Left: The expression levels of SNRPB2 protein in tumor and normal tissues of patients with ESCC. Right: The relationship between SNRPB2 expression and the clinicopathological features of ESCC. \u003cstrong\u003e(e)\u003c/strong\u003e The correlation between SNRPB2 protein expression and OS of ESCC (n=125). (*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5382410/v1/f91938cdbfee0f59fdfd1302.png"},{"id":70385327,"identity":"ccfe7f38-aacc-4109-8125-d87061793bfc","added_by":"auto","created_at":"2024-12-02 17:07:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":970368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSNRPB2 promoted proliferation,\u003c/strong\u003e \u003cstrong\u003emigration and invasion of ESCC cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eSNRPB2 protein expressions in four ESCC cell lines and human immortalized esophageal epithelial cells examined by western blot.\u003cstrong\u003e(b) \u003c/strong\u003eThe effect of SNRPB2 knockdown with shRNAswas verified by western blot.\u003cstrong\u003e (c) \u003c/strong\u003eThe number of colony cells decreased significantly when cells were treated with SNRPB2 shRNAs.\u003cstrong\u003e(d) \u003c/strong\u003eThe cell viability significantly decreased when cells were treated with SNRPB2 shRNAs. \u003cstrong\u003e(e-f)\u003c/strong\u003e TE-1 and KYSE-150 cells transfected with SNRPB2 shRNAs were evaluated by transwell migration assay, matrigel invasion assay, and wound healing experiment. All data were presented as the mean ± SDs (n = 3). (**\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5382410/v1/16c38c311e6da0a7f652b73c.png"},{"id":70385336,"identity":"baf3fc43-6028-487a-8a4b-142be42d4941","added_by":"auto","created_at":"2024-12-02 17:07:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":694394,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSNRPB2 inhibited Rb/E2F pathway in ESCA and interacted with E2F4 in ESCC cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003ePathway enrichment: GSEA enrichment analyses of co‐expressed genes indicating an association of SNRPB2with the top six signaling pathways in the TCGA-ESCAcohort. \u003cstrong\u003e(b)\u003c/strong\u003e GO functional enrichment analysis of SNRPB2 and its interactors were performed. \u003cstrong\u003e(c)\u003c/strong\u003e Correlation of the mRNA levels of SNRPB2 and E2F4 in TCGA-ESCA tumor samples. \u003cstrong\u003e(d)\u003c/strong\u003e The expression level of E2F4 mRNA in ESCA and normal esophageal tissue samples. \u003cstrong\u003e(e-f)\u003c/strong\u003e The Co‐IP assay demonstrated the specific interactions between SNRPB2 and E2F4.\u003cstrong\u003e (g)\u003c/strong\u003e Correlation of the protein expression levels of SNRPB2 and E2F4 in TE-1 and KYSE-150 cells. \u003cstrong\u003e(h)\u003c/strong\u003e The protein level of E2F4 after treated with CHX was measured by western blot.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5382410/v1/f7af5ba451aab51da54b8531.png"},{"id":70385335,"identity":"57740839-e7e5-433f-9291-0f4af5548be3","added_by":"auto","created_at":"2024-12-02 17:07:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":797514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eE2F4 overexpression rescued the effects of shSNRPB2 on proliferation, migration and invasion of ESCC cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a-b) \u003c/strong\u003eshCtrl+E2F4 group increased the proliferation and clone formation of ESCC compared with shCtrl+Vector group, and shSNRPB2+E2F4 group also increased the proliferation and clone formation of ESCC compared with shSNRPB2+Vectorgroup.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c-d) \u003c/strong\u003eshCtrl+E2F4 group promoted the migration and invasion of ESCC compared with shCtrl+Vector group, and shSNRPB2+E2F4group accelerated the migration and invasion of ESCC compared with shSNRPB2+Vector group. (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5382410/v1/9d059a9eac74d573332b98e2.png"},{"id":70385395,"identity":"2dde95ce-18bb-42be-a22c-2ee339b2c7c0","added_by":"auto","created_at":"2024-12-02 17:08:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":208524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eE2F4 overexpression blocked shSNRPB2‐decreased ESCC cell progression \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFive nude mice in each group were subcutaneously injected with TE-1 cells with shCtrl+Vector, shSNRPB2+Vector, shCtrl+E2F4, and shSNRPB2+E2F4. The mean tumor volume (cm\u003csup\u003e3\u003c/sup\u003e) and weight (g) were measured three weeks later. (***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5382410/v1/1415acf46f702aec0b90cc20.png"},{"id":70385308,"identity":"350e8b69-0fb9-4824-b3ab-f5fad6d21c45","added_by":"auto","created_at":"2024-12-02 17:06:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":550236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative images of SNRPB2 and E2F4 protein expression in ESCC specimens.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5382410/v1/2c2281385ec6c6f7245ebe68.png"},{"id":75443052,"identity":"46535028-4675-4d6b-a20e-02c3d0a6b7e7","added_by":"auto","created_at":"2025-02-04 16:01:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5612719,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5382410/v1/e22aa347-e8ee-4b34-a800-7ecde6965f28.pdf"},{"id":70385342,"identity":"0d717b2e-4344-47ce-92cc-4ef35e8a2459","added_by":"auto","created_at":"2024-12-02 17:07:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25662867,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterialfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-5382410/v1/0afa01e39b08296866fee254.docx"},{"id":70385392,"identity":"07f05247-f751-49a4-8c6f-4dab26dd578d","added_by":"auto","created_at":"2024-12-02 17:08:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24000,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5382410/v1/698a415e672498a2753c113e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"SNRPB2 facilitates esophageal squamous cell carcinoma oncogenesis and progression via E2F4 stabilization","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEsophageal cancer (ESCA) is one of the most lethal malignancies.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Surgical resection of the tumor from the primary site has been the standard treatment, especially for localized esophageal squamous cell carcinoma (ESCC), which is the main histological type of ESCA in China.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Nowadays, proteogenomics and single-cell transcriptomic analyses \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ehave elucidated cancer-driving waves in ESCC progression\u003c/span\u003e and revealed the molecular characterization of dietary habit-associated signatures.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e However, the intrinsic characteristics of the molecular and clinical perspectives associated with the risk factors of ESCC progression are still unknown. Therefore, further study of the comprehensive understanding of ESCC molecular targets will help overcome \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe current challenges in precision\u003c/span\u003e therapeutic\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es\u003c/span\u003e and improve outcomes for ESCC patients.\u003c/p\u003e \u003cp\u003eSmall nuclear ribonucleoprotein polypeptide B2 (SNRPB2), known as U2 snRNP B'' or U2 small nuclear ribonucleoprotein B'', is one of the unique proteins that comprise the U2 snRNP.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e SNRPB2 has unique RNA binding property, with the high degree of sequence and structural conservation,\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e involved in precursor messenger RNA (pre-mRNA) splicing as component of the spliceosome in physiological state.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Pre-mRNA splicing are key for eukaryotic gene expression and cellular function while splicing alterations can lead to various diseases including blindness,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e autoimmunity disease and cancer.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e In tumors, alternative splicing frequently plays critical roles in tumour genesis, development and metastasis, whose regulatory factors may be considered as prognostic biomarkers and therapeutic targets for cancer intervention.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e SNRPB2 as an early growth-inducible gene essential for the regulation of pre-mRNA splicing and gene expression, is widely expressed in primary cortical neurons, epithelial cells and fibroblasts.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e However, the potential role of SNRPB2 in tumors remain ill-defined, necessitating further evaluation.\u003c/p\u003e \u003cp\u003eIn this study, we conducted a comprehensive analysis of the expression and prognosis for SNRPB2 in ESCC using online databases, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand experiment\u003c/span\u003e \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. SNRPB2 mRNA expression levels and their prognostic values were analyzed using Gene Expression Profiling Interactive Analysis 2 (GEPIA2), cBioPortal, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand\u003c/span\u003e Tumor Immune Evaluation Resources (TIMER). SNRPB2 protein expression levels, clinicopathological characteristics, and clinical relevance \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewere\u003c/span\u003e determined by western blot\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eting\u003c/span\u003e and immunohistochemistry using paraffin-embedded tissue specimens and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eESCC cell lines\u003c/span\u003e, respectively. In addition, we explored the molecular mechanisms by which SNRPB2 promotes the occurrence and progression of ESCC using gene set enrichment analysis (GSEA) and validated \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe results using\u003c/span\u003e a co-immunoprecipitation (Co-IP) assay.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell lines and lentivirus-mediated gene knockdown\u003c/h2\u003e \u003cp\u003eThe ESCC cell lines (KYSE-150, KYSE-140, KYSE-450, and TE-1) and the human immortalized esophageal epithelial cell line (NE-1) were purchased from the Cell Bank of the Chinese Academy (Shanghai, China). All ESCC cells were cultured in RPMI-1640 supplemented with 10% FBS and 100IU/ml Penicillin-Streptomycin solution with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃.\u003c/p\u003e \u003cp\u003eFor lentiviral gene knockdown, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ea pLKO.1 shRNA was generated against human\u003c/span\u003e SNRPB2 target sequences. pLKO lentiviral vectors containing shRNA were transfected into 293T cells together with psPAX2 and pMD2.G by calcium phosphate transfection (Sigma\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e-Aldrich).\u003c/span\u003e The shRNA sequences were transfected into \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe\u003c/span\u003e TE-1 and KYSE-150 cells.\u003c/p\u003e \u003cp\u003eshSNRPB2#1: Forward:5\u0026rsquo;-CCGGCCCAAGGAAATTCAACACCAACTCGAGTTGGTGTTGAATTTCCTTGGGTTTTT-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse:5\u0026rsquo;-AATTCAAAAACCCAAGGAAATTCAACACCAACTCGAGTTGGTGTTGAATTTCCTTGGG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eshSNRPB2#2: Forward:5\u0026rsquo;-CCGGCCATGCTATGAAGATCACCTACTCGAGTAGGTGATCTTCATAGCATGGTTTTT-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse:5\u0026rsquo;-AATTCAAAAACCATGCTATGAAGATCACCTACTCGAGTAGGTGATCTTCATAGCATGG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eshE2F4#1:\u003c/p\u003e \u003cp\u003eForward:5\u0026rsquo;-CCGGCCCTCTCTTCATTTCGGCTTTCTCGAGAAAGCCGAAATGAAGAGAGGGTTTTT-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse:5\u0026rsquo;-AATTCAAAAACCCTCTCTTCATTTCGGCTTTCTCGAGAAAGCCGAAATGAAGAGAGGG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eshE2F4#2:\u003c/p\u003e \u003cp\u003eForward:5\u0026rsquo;-CCGGCGGATTTACGACATTACCAATCTCGAGATTGGTAATGTCGTAAATCCGTTTTT-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse:5\u0026rsquo;-AATTCAAAAACGGATTTACGACATTACCAATCTCGAGATTGGTAATGTCGTAAATCCG-3\u0026rsquo;\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Patient tissue sample collection\u003c/h2\u003e \u003cp\u003eOne hundred and twenty-five paraffin-embedded ESCC tissues and paired normal esophageal epithelial tissues adjacent to the cancer tissue were collected from patients who underwent surgery at the First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China) between 2015 and 2018. None of the patients had a history of other malignant tumors or had received preoperative interventions such as radiotherapy or chemotherapy. Each patient provided written informed consent, and the study was approved by the institutional review board (2022AH051185).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 GEPIA2, cBioPortal and TIMER analysis\u003c/h2\u003e \u003cp\u003eA correlative prognostic analysis of SNRPB2 from the GEPIA2 dataset, including overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI), was conducted using the log-rank test at a median cut-off of 50% for both \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe SNRPB2\u003c/span\u003e\u003csup\u003elow\u003c/sup\u003e and SNRPB2\u003csup\u003ehigh\u003c/sup\u003e cohorts. The cBioPortal for Cancer Genomics (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cbioportal.org\u003c/span\u003e\u003cspan address=\"http://cbioportal.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) served as an open online resource for visualizing multidimensional cancer genomics datasets.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e The TCGA-ESCA dataset was selected for further analysis of SNRPB2 mRNA. Tumor immune evaluation resources (TIMER) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cistrome.shinyapps.io/timer\u003c/span\u003e\u003cspan address=\"https://cistrome.shinyapps.io/timer\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), including B cells, CD8\u0026thinsp;+\u0026thinsp;T cells, CD4\u0026thinsp;+\u0026thinsp;T cells, macrophages, neutrophils, and dendritic cells, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewere used for immune cell infiltration analysis.\u003c/span\u003e\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eGO\u003c/span\u003e and GSEA enrichment analysis\u003c/h2\u003e \u003cp\u003eEnrichment analysis \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewas conducted using\u003c/span\u003e R package \"clusterProfiler\" to investigate the potential functions of SNRPB2. Gene Ontology (GO) and gene set enrichment analyses (GSEA) were used for pathway enrichment analysis in ESCA. For both GO and GSEA analyses, the cut-off criteria were \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand\u003c/span\u003e \u003cem\u003eP\u003c/em\u003e. adjust\u0026thinsp;\u0026lt;\u0026thinsp;0.5, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003erespectively. The Benjamini-Hochberg method was u\u003c/span\u003esed to calculate adjusted \u003cem\u003eP\u003c/em\u003e-values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Xenograft tumor model assay\u003c/h2\u003e \u003cp\u003eFour-week-old male athymic BALB/c nude mice (Weitonglihua Co., Ltd., \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eChina) were randomly divided into four groups. An equivalent number of TE-1 cells\u003c/span\u003e (3\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/200 \u0026micro;L) were resuspended in PBS and injected into the armpit area of each mouse. Tumor size was measured every three days. After 21 days, the mice were euthanized and the tumors were carefully removed, weighed, and photographed. All animal procedures were done in accordance with protocols approved by the experimental animal ethics committee, Anhui Medical University \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e(LLSC20231142)\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunohistochemical analysis\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein situ\u003c/em\u003e protein expression levels of SNRPB2 and E2F4 in paraffin-embedded ESCC tissue sections were assessed by immunohistochemistry using rabbit polyclonal antibodies against SNRPB2 (1:100, 21244-1-AP, Proteintech) and E2F4 (1:100, 10923-1-AP, Proteintech). Five fields were randomly observed at \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ea high magnification under\u003c/span\u003e a microscope. The staining intensity of SNRPB2 and E2F4 in tumor cells was classified as follows: 0 (no staining); 1 (light yellow); 2 (medium yellow); and 3 (dark yellow). The percentage of stained cells was categorized \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eas follows: 0 (no positive tumor cells), 1 (\u0026lt;\u0026thinsp;20% positive cells), 2 (20%\u003c/span\u003e-60% positive cells), and 3 (\u0026gt;\u0026thinsp;60% positive cells). The total score, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewhich rang\u003c/span\u003eed from 0 to 6, was calculated by summing the two scores. Samples with staining scores of 0\u0026ndash;3 were classified as having low SNRPB2 or E2F4 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eexpression\u003c/span\u003e, whereas those with scores\u0026thinsp;\u0026gt;\u0026thinsp;3 were classified as having high SNRPB2 or E2F4\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Western blot assay\u003c/h2\u003e \u003cp\u003eWhole cell lysates were prepared using \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eradioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) containing a\u003c/span\u003e complete protease inhibitor cocktail. The total protein was loaded and separated by SDS-PAGE, followed by transfer onto polyvinylidene fluoride membranes (Millipore, USA). Immunodetection was performed using antibodies against β-actin (1:5000, 81115-1-RR; Proteintech) and SNRPB2 (1:2000). Horseradish peroxidase-conjugated secondary antibodies (Cell Signaling \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eTechnology) were used for digital chemiluminescence detection (GE\u003c/span\u003e Healthcare). Blot\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es\u003c/span\u003e are representative of three independent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Cell proliferation assay\u003c/h2\u003e \u003cp\u003eCell proliferation was assessed using cell counting kit-8 (CCK‐8) description (Beyotime). Cells (5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well) in logarithmic growth phase were seeded in 96-well plates and incubated at 37\u0026deg;C. After 24, 48, 72, 96 h and 120h, 10\u0026micro;L \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eof CCK8\u003c/span\u003e was added to each well and incubated for 2h. Cell proliferation was determined by measuring the absorption value of whole wells, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewhich was directly detected at 450 nm.\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Clone formation assay\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe\u003c/span\u003e cells were inoculated into six-well plates (1,000 cells/well) in triplicate and cultured for 14 days. Cells number more than 50 or cell size between 0.3 and 1.0 mm were considered as a single clone. All colonies were fixed with 4% paraformaldehyde for 10 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003emin\u003c/span\u003e, stained with Giemsa, and photographed using a digital camera.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Wound healing assay\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe\u003c/span\u003e cells were collected and inoculated into six-well plates (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) in 2 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eml\u003c/span\u003e medium and cultured at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eWhen\u003c/span\u003e the cell\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es\u003c/span\u003e reached more than 90% confluence, a pipetting tip of 100\u0026micro;L was used to scratch the six-well plate \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003evertically. Cells were washed\u003c/span\u003e twice with PBS and cultured in \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ean\u003c/span\u003e incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Photographs were taken at 0 and 48 h under a microscope, and the experiment was repeated thrice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Transwell assay\u003c/h2\u003e \u003cp\u003eThe migration and invasion \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ecapacities of ESCC cells\u003c/span\u003e were determined using \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ea\u003c/span\u003e Transwell chamber (Corning) precoated with Matrigel. After transfection, ESCC cells were collected, counted, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand incubated in the upper chamber with 100\u003c/span\u003e\u0026micro;L RPMI-1640 medium without FBS in a 24-well plate (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well). 600\u0026micro;L medium supplemented with 20% FBS was added to the lower chamber. After incubation at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 h, non\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e-metastatic cells were removed\u003c/span\u003e using a cotton swab. 500\u0026micro;L Giemsa was added for staining and the migratory ability of \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe cells was analyzed.\u003c/span\u003e Representative images were selected from three independent experiments, of which five fields per chamber were randomly selected to count cell numbers. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe\u003c/span\u003e magnification \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eused was 200\u0026times; .\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Co-immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe\u003c/span\u003e cell extracts were resuspended and lysed on ice using \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ean IP\u003c/span\u003e buffer. Protein A/G magnetic beads (Bimake, China) were added to cell lysate\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es and incubated for 2 h\u003c/span\u003e at 4\u0026deg;C. IgG was added to the negative control group, whereas IP antibody was added to the experimental group for 12 h at 4\u0026deg;C. Immunoprecipitated proteins were eluted by boiling in SDS loading buffer (2\u0026times;) for western blot analysis as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Cycloheximide (CHX) chase assay\u003c/h2\u003e \u003cp\u003eCells were seeded in six-well plates (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) and pretreated with CHX (100 \u0026micro;g/mL) for 0\u0026ndash;4 h. Cells \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewere harvested for western blot analysis\u003c/span\u003e, as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Statistical analysis\u003c/h2\u003e \u003cp\u003eSPSS \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e(version\u003c/span\u003e 22.0\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e) was used for data analysis. Chi-square test\u003c/span\u003e and paired-samples t-test were used for variable comparison, with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 regarded as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e3.1 High expression of SNRPB2 mRNA was correlated with worse outcome in patients with ESCA\u003c/h2\u003e\n \u003cp\u003eDifferentially expressed genes were identified by comparing ESCA and para-carcinoma esophageal tissue samples from \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe\u003c/span\u003e GEPIA2 database. We found that SNRPB2 mRNA \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ewas significantly\u003c/span\u003e upregulated in esophageal cancer significantly and correlated with the clinical stage and prognosis \u003cstrong\u003e(Supplementary material Table S1-2\u003c/strong\u003e). To further determine the prognostic relevance of SNRPB2 in ESCA, patients from multiple ESCA datasets were classified into SNRPB2\u003csup\u003ehigh\u003c/sup\u003e and SNRPB2\u003csup\u003elow\u003c/sup\u003e groups and their survival (including OS, DSS, and PFI) was analyzed using Kaplan-Meier curves. As shown in Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003ea, the SNRPB2\u003csup\u003elow\u003c/sup\u003e group had significantly longer OS and DSS than the SNRPB2\u003csup\u003ehigh\u003c/sup\u003e group (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the PFI showed no difference in this dataset (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These findings suggest that SNRPB2 mRNA \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eexpression might\u003c/span\u003e correlate with ESCA prognosis, whereas SNRPB2 overexpression might indicate worse outcome\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003es in patients with ESCA.\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e3.2 Expression level of SNRPB2 mRNA in ESCA and its association with tumor stage\u003c/h2\u003e\n \u003cp\u003eTo explore the expression of SNRPB2 mRNA in ESCA and normal esophageal tissue samples from TCGA, we analyzed para-carcinoma tissue from \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003epatients with\u003c/span\u003e ESCA and normal esophageal tissue from tumor-free subjects separately. We found that SNRPB2 mRNA was highly expressed in ESCA \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003esamples compared\u003c/span\u003e to normal samples, and similar results were observed in paired samples (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eb\u003cstrong\u003e)\u003c/strong\u003e. To further examine the clinical relevance of SNRPB2 in ESCA from TCGA database, we analyzed the correlation of its expression with tumor stage. As shown in Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eb, no significant correlation between SNRPB2 mRNA \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eexpression and tumor stage\u003c/span\u003e was observed in this dataset (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003e3.3 Expression level of SNRPB2 protein in ESCC\u003c/h2\u003e\n \u003cp\u003eTo fu\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003erther validate the above results\u003c/span\u003e, we performed immunohistochemical staining of paraffin-embedded tissue specimens to detect the expression of SNRPB2 protein in ESCC. SNRPB2 protein \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eis l\u003c/span\u003eocated in the nucleus and is extensively expressed in normal squamous epithelial cell\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003es and ESCC\u003c/span\u003e \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003ec\u003cstrong\u003e)\u003c/strong\u003e. The expression rate of SNRPB2 in ESCC was 64.0%, which was significantly higher than that in adjacent tissues (24.0%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cstrong\u003e(\u003c/strong\u003eTable\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e. The results showed that SNRPB2 protein was significantly highly expressed in ESCC samples compared to paired normal samples \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eb\u003cstrong\u003e)\u003c/strong\u003e, whose expression trends were in consistent with the findings obtained from TCGA.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe expression levels of SNRPB2 protein in tumor and para-carcinoma tissues of patients with esophageal squamous cell carcinoma.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSNRPB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eValue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003cp\u003eexpression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003cp\u003eexpression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePara-carcinoma tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95 (76.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 (24.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEsophageal squamous cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45 (36.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80 (64.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003e3.4 Association between the expression of SNRPB2 protein and the clinicopathological characteristics of patients with ESCC\u003c/h2\u003e\n \u003cp\u003eTo explore the clinical significance of SNRPB2 expression in ESCC, we correlated its expression with \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe clinicopathological characteristics of\u003c/span\u003e ESCC \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003epatients\u003c/span\u003e. Interestingly, the expression levels of SNRPB2 protein were positively correlated with high tumor grade (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cstrong\u003e(\u003c/strong\u003eTable\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003ec\u003cstrong\u003e)\u003c/strong\u003e. Further\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003emore\u003c/span\u003e, the expression levels of SNRPB2 protein in ESCC were significantly correlated with vessel carcinoma embolus, lymph node metastasis, and clinical stage (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but not with patient age, sex, BMI, smoking or drinking history, tumor location, or nerve invasion (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cstrong\u003e(\u003c/strong\u003eTable\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003ed\u003cstrong\u003e)\u003c/strong\u003e. Kaplan-Meier analysis showed that ESCC patients with SNRPB2 high expression had significantly poorer overall survival compared with SNRPB2\u003csup\u003elow\u003c/sup\u003e group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003ee\u003cstrong\u003e)\u003c/strong\u003e. Collectively, the expression of SNRPB2 may be associated with ESCC progression, which is associated with poor prognosis in ESCC.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe relationship between SNRPB2 expression and the clinicopathological features of esophageal squamous cell carcinoma.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSNRPB2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eValue\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003cp\u003eexpression\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003cp\u003eexpression\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.601\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026le;\u0026thinsp;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12 (40.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18 (60.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33 (34.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e62 (65.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.824\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34 (36.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59 (63.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11 (34.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21 (65.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmoking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.920\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12 (35.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22 (64.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33 (36.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e58 (63.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDrinking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.781\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14 (37.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23 (62.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31 (35.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e57 (64.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBMI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.840\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026le;\u0026thinsp;28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41 (36.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e72 (63.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4 (33.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8 (66.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35 (47.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38 (52.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10 (19.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e42 (80.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVessel carcinoma embolus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.012\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17 (25.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e49 (74.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28 (47.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31 (52.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNerve invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.068\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16 (27.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e42 (72.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29 (43.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38 (56.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLymph node metastasis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19 (23.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e61 (76.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26 (57.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19 (42.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.018\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI\u0026thinsp;+\u0026thinsp;II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25 (48.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27 (51.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIII\u0026thinsp;+\u0026thinsp;IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20 (27.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53 (72.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTumor location\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.507\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUpper\u0026thinsp;+\u0026thinsp;Lower\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11 (31.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24 (68.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMiddle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34 (37.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e56 (62.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003e3.5 SNRPB2 facilitated ESCC progression\u003c/h2\u003e\n \u003cp\u003eTo investigate the potential role of SNRPB2 in ESCC, we first examined the expression levels of \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe\u003c/span\u003e SNRPB2 protein in various ESCC cells and human immortalized esophageal epithelial cells. As shown in Figs.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003ea, the expression levels of \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe SNRPB2\u003c/span\u003e protein were variable in different ESCC cell lines and were significantly higher than those in human immortalized esophageal epithelial cells.\u003c/p\u003e\n \u003cp\u003eBased on the expression level\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003es\u003c/span\u003e, SNRPB2 was knocked down in \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe\u003c/span\u003e TE-1 and KYSE-150 cells. Western blot analysis suggested that shSNRPB2#2 significantly inhibit\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eed\u003c/span\u003e the \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eprotein expression of\u003c/span\u003e SNRPB2, which was used for subsequent experiments \u003cstrong\u003e(\u003c/strong\u003eFigs.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eb\u003cstrong\u003e)\u003c/strong\u003e. Colony formation and cell proliferation assay\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003es\u003c/span\u003e indicated that knockdown SNRPB2 remarkably suppressed cell proliferation \u003cstrong\u003e(\u003c/strong\u003eFigs.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003ec-d\u003cstrong\u003e)\u003c/strong\u003e. Transwell assays indicated that the expression of SNRPB2 was related with the migration and invasion of ESCC cells \u003cstrong\u003e(\u003c/strong\u003eFigs.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003ee\u003cstrong\u003e)\u003c/strong\u003e. Wound healing assays indicated that the migration \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003erates of\u003c/span\u003e TE-1 and KYSE-150 cells with SNRPB2#2 knockdown were 21% and 16%, \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003erespectively\u003c/span\u003e, lower than \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethose of the control group (\u003c/span\u003e80% and 70%, respectively), suggesting that SNRPB2 promoted the migration of ESCC cells \u003cstrong\u003e(\u003c/strong\u003eFigs.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003ef\u003cstrong\u003e)\u003c/strong\u003e. These results imply that SNRPB2 acts as an oncogene and facilitates ESCC progression.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\"\u003e\n \u003ch2\u003e3.6 Genetic alterations and immunological correlation of SNRPB2 in ESCA\u003c/h2\u003e\n \u003cp\u003eWe conducted an online database analysis to investigate genetic alterations in SNRPB2 that may be associated with tumorigenesis via cBioPortal. Genetic alterations were detected in 1.1% of SNRPB2 gene\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003es\u003c/span\u003e in ESCA, including 5 amplifications, 2 deep deletions, 1 truncating mutation, and 1 missense mutation \u003cstrong\u003e(Figure S1a)\u003c/strong\u003e. No alterations in SNRPB2 were detected in ESCC based on an analysis of 227 samples from two studies \u003cstrong\u003e(Figure S1b)\u003c/strong\u003e. These results suggested that SNRPB2 is structurally conserved in ESCC \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ecells.\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eTIMER 2.0 was used to analyze the relationship between SNRPB2 and multiple tumor-infiltrating immune cells in ESCC. SNRPB2 expression was significant\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ely\u003c/span\u003e positive\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ely\u003c/span\u003e correlated with CD8\u0026thinsp;+\u0026thinsp;T cells (cor\u0026thinsp;=\u0026thinsp;0.173, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and macrophages (cor\u0026thinsp;=\u0026thinsp;0.236, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cstrong\u003e(Figures S2)\u003c/strong\u003e. These results suggest that the potential role of SNRPB2 in ESCA may be related to tumor immunity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\"\u003e\n \u003ch2\u003e3.7 SNRPB2 inhibited Rb/E2F pathway in ESCA via increasing stability of E2F4 protein\u003c/h2\u003e\n \u003cp\u003e\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eGO and GSEA analyses were performed\u003c/span\u003e to reveal the promoting effect of SNRPB2 on ESCC cells. The pathway enrichment \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eresults suggested that SNRPB2 significantly\u003c/span\u003e inhibited \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe Rb/E2F pathway in ESCA\u003c/span\u003e and was involved in the process of digestion in the apical part of \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe cell\u003c/span\u003e \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003ea-b\u003cstrong\u003e)\u003c/strong\u003e. Furthermore, bioinformatic analysis indicated that the mRNA expression of SNRPB2 was positively correlated with E2F4, which \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eis know\u003c/span\u003en as the transcription inhibitory factor of \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe\u003c/span\u003e Rb/E2F pathway \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003ec\u003cstrong\u003e)\u003c/strong\u003e. As shown in Figs.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003ed, consistent with SNRPB2, the mRNA expression level of E2F4 was significantly upregulated in ESCA. To verify the interactions between SNRPB2 and E2F4, a co-immunoprecipitation assay was performed to confirm their intera\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ection in\u003c/span\u003e TE-1 and KYSE-150 cells. The results of the co-IP assay demonstrated that SNRPB2 interacts with E2F4 \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003ee-f\u003cstrong\u003e)\u003c/strong\u003e. We first knocked down \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe expression of SNRPB\u003c/span\u003e2 with shRNA in TE-1 and KYSE-150 cells and observed that E2F4 protein expression was downregulated after SNRPB2 knockdown \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eg\u003cstrong\u003e)\u003c/strong\u003e. However, SNRPB2 expression level\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003es showed no significant change after E2F4 knockdown\u003c/span\u003e \u003cstrong\u003e(Figure S3c)\u003c/strong\u003e. As it was observed to SNRPB2 affected the protein level of E2F4, a CHX chase experiment was conducted to investigate the impact of SNRPB2 on the stability of E2F4. \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eThe\u003c/span\u003e results demonstrated that SNRPB2 extended the half-life of E2F4 \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eh\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\"\u003e\n \u003ch2\u003e3.8 E2F4 counteracted the inhibition effects of shSNRPB2 on the biology in ESCC\u003c/h2\u003e\n \u003cp\u003eTo investigate the role of E2F4 in SNRPB2-promoted proliferation, migration, \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eand invasion of ESCC cells, we constructed overexpression\u003c/span\u003e plasmid of E2F4 to cotransfect with shSNRPB2 in TE-1 and KYSE-150 cells \u003cstrong\u003e(Figures S3a-b)\u003c/strong\u003e. \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eThe\u003c/span\u003e results showed that \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe overexpress\u003c/span\u003eion \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eof E2F4 (\u003c/span\u003eshCtrl\u0026thinsp;+\u0026thinsp;E2F4) group increased cell proliferation and clone formation compared to \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe\u003c/span\u003e shCtrl\u0026thinsp;+\u0026thinsp;Vector group. The shSNRPB2\u0026thinsp;+\u0026thinsp;E2F4 group also \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eshowed increased\u003c/span\u003e proliferation and clone formation of TE-1 and KYSE-150 cells compared to \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe\u003c/span\u003e shSNRPB2\u0026thinsp;+\u0026thinsp;Vector group \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003ea-b\u003cstrong\u003e)\u003c/strong\u003e. In wound healing and transwell assays, overexpression \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eof\u003c/span\u003e shCtrl\u0026thinsp;+\u0026thinsp;E2F4 promoted the migration and invasion of TE-1 cells compared to \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe\u003c/span\u003e shCtrl\u0026thinsp;+\u0026thinsp;Vector group, whereas \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe sh\u003c/span\u003eSNRPB2\u0026thinsp;+\u0026thinsp;E2F4 group rescued the migration and invasion of TE-1 cells compared to \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ethe\u003c/span\u003e shSNRPB2\u0026thinsp;+\u0026thinsp;Vector group \u003cstrong\u003e(\u003c/strong\u003eFigs.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003ec-d\u003cstrong\u003e)\u003c/strong\u003e. As shown in Figs.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003e, E2F4 overexpression also blocked shSNRPB2-decreased ESCC cell progression \u003cem\u003ein vivo\u003c/em\u003e. These results suggest that E2F4, \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ewhich is positively regulated by\u003c/span\u003e SNRPB2, could be a target gene that plays a role in the progression of ESCC.\u003c/p\u003e\n \u003cp\u003eFinally, we assessed IHC staining in specimens from 125 patients \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003ewith ESCC to evaluate whether the\u003c/span\u003e SNRPB2-E2F4 axis exists in human ESCC specimens. The results suggested that SNRPB2 \u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eexpression was significantly positively correlated with\u003c/span\u003e E2F4 expression (r\u0026thinsp;=\u0026thinsp;0.517, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan\u003e6\u003c/span\u003e, Table\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n \u003cdiv\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe correlation between SNRPB2 and E2F4 protein expression in esophageal squamous cell carcinoma.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSNRPB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eE2F4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003er Value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eP Value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLow expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHigh expression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLow expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e0.517\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHigh expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIncreasing evidence indicates that pre-mRNA splicing provides the potential to produce diversity at RNA and protein levels.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Pre-mRNA splicing is also the key to the pathology of numerous diseases, especially cancers, which may be considered as new biomarkers and therapeutic targets for cancer intervention.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e ESCA developing through multistage epithelial cancer formation, is a unique and substantial heterogeneous malignancy.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e The improvement of ESCA poorly survival outcome is needed to multi-disciplinary evaluation and multi-modal therapy based on the exploration of ideal therapeutic targets and essential prognostic biomarkers.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSNRPB2 is essential for the formation of the functional 17S U2 snRNP and prespliceosome assembly \u003cem\u003ein vivo\u003c/em\u003e, resulting in cell death as a consequence of blocked its pre-mRNA splicing steps in gene expression.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Pre-mRNA processing factor SNRPB2 locates in the nucleus, associated with vimentin-containing intermediate filaments, particularly those surrounding the nucleus.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e SNRPB2 is involved in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e However, the roles of SNRPB2 in cancer have rarely been studied. In this study, we conducted an in-depth analysis of SNRPB2 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eexpression and\u003c/span\u003e evaluated its feasibility of using SNRPB2 as a prognostic biomarker for ESCC.\u003c/p\u003e \u003cp\u003eSNRPB2 is critical for the regulation of gene expression and early growth induction, inducing the process of intron removal from pre-mRNA.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e In our study, SNRPB2 protein detected by immunohistochemistry was significantly higher in ESCC samples than in paired normal samples, which was consistent with the online database analysis. The expression levels of \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe SNRPB2 protein in ESCC were significantly correlated with lymph node metastasis and clinical stage\u003c/span\u003e, suggesting that SNRPB2 may be associated with ESCC tumor progression.\u003c/p\u003e \u003cp\u003eDeregulated gene expression is a hallmark of cancer, and the progression of heterogeneous tumor\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es\u003c/span\u003e is influenced by multiple factors.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Therefore, we analyzed the genetic alterations in SNRPB2 and performed pathway enrichment analysis. SNRPB2 inhibits \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe\u003c/span\u003e Rb/E2F pathway in ESCA by increasing E2F4 protein stability Our study further revealed that SNRPB2 interacted with E2F4 to promote ESCC cell proliferation. Rb/E2F pathway involves a complex interaction across E2F family members in the regulation of cell cycle progression.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e In the context of tumorigenesis, the function of E2F4, a transcriptional repressor, was consistently altered, contributing to uncontrolled cell proliferation and cancer progression.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e In our study, E2F4 was positively regulated by SNRPB2, which acted as an oncogene in the progression of ESCC. Additionally, E2F4 counteracted the inhibitory effects of shSNRPB2 on ESCC biology. However, further investigation\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es\u003c/span\u003e are necessary to elucidate the mechanisms by which SNRPB2 enhances E2F4 stability.\u003c/p\u003e \u003cp\u003eAlternative splicing of pre-mRNA plays an important role in immune cell development and regulation of immune activity.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Cancer cells have general alterations in the splicing process, which contribute to tumor immune responses and progression.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e In this study, we investigated the correlation between SNRPB2 and immune cells using bioinformatics analysis. In ESCA, SNRPB2 expression levels positive\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ely correlat\u003c/span\u003eed with TILs, MHC molecules, and multiple chemokines. This preliminary analysis underscores the need for future studies to elucidate the potential role of SNRPB2 as a therapeutic target for immunotherapy in ESCA.\u003c/p\u003e \u003cp\u003eIn summary, SNRPB2 protein expression is significant\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ely upregulat\u003c/span\u003eed in ESCC and is associated with tumor progression and clinical prognosis. Our study provides comprehensive evidence that SNRPB2 is a feasible prognostic marker and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ea therapeutic target for ESCC. It may also play a role in the disease prediction, evaluation\u003c/span\u003e, and individualized treatment of ESCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to all members of the Department of Thoracic Surgery at the First Affiliated Hospital of Anhui Medical University for their valuable support and contributions to this study. We also appreciate the technical assistance and insightful discussions provided by our colleagues. Special thanks to all the patients and their families who participated in this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Anhui Province Higher Education Science Research Project (2022AH051185). The funding body had no role in the design of the study, data collection, analysis, interpretation, or writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to report.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe studies involving human participants were reviewed and approved by the First Affiliated Hospital of Anhui Medical University, Hefei 230032, Anhui, China (2022AH051185). All patients provided written informed consent to participate in the study. All animal procedures were done in accordance with protocols approved by the experimental animal ethics committee, Anhui Medical University (LLSC20231142).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF.X., C.Z. contributed to the design and execution of the experiments. C.L. was responsible for data collection and analysis. G.N. provided critical insights for the interpretation of results. R.Z. supervised the entire project, contributed to study design, and provided guidance throughout the manuscript preparation. All authors participated in drafting the manuscript, reviewed the final version, and approved it for submission.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Miller KD, Wagle NS, et al. Cancer statistics, 2023. CA Cancer J Clin. 2023;73(1):17-48.\u003c/li\u003e\n\u003cli\u003eZhu H, Ma X, Ye T, et al. Esophageal cancer in China: Practice and research in the new era. Int J Cancer. 2023;152(9):1741-1751.\u003c/li\u003e\n\u003cli\u003eLi L, Jiang D, Zhang Q, et al. Integrative proteogenomic characterization of early esophageal cancer. Nat Commun. 2023;14(1):1666.\u003c/li\u003e\n\u003cli\u003eZhang X, Peng L, Luo Y, et al. 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Coordinated alterations in RNA splicing and epigenetic regulation drive leukaemogenesis. Nature. 2019;574(7777):273-277.\u003c/li\u003e\n\u003cli\u003eRogalska ME, Vivori C, Valc\u0026aacute;rcel J. Regulation of pre-mRNA splicing: roles in physiology and disease, and therapeutic prospects. Nat Rev Genet. 2023;24(4):251-269.\u003c/li\u003e\n\u003cli\u003ePeng Q, Zhou Y, Oyang L, Wu N, Tang Y, Su M, Luo X, Wang Y, Sheng X, Ma J, Liao Q. Impacts and mechanisms of alternative mRNA splicing in cancer metabolism, immune response, and therapeutics. Mol Ther. 2022 Mar 2;30(3):1018-1035.\u003c/li\u003e\n\u003cli\u003eLaitinen J, Saris P, H\u0026ouml;ltt\u0026auml; E, Pettersson I. U2-snRNP B\u0026quot; protein gene is an early growth-inducible gene. J Cell Biochem. 1995;58(4):490-498.\u003c/li\u003e\n\u003cli\u003eMuresan Z, Muresan V. A phosphorylated, carboxy-terminal fragment of beta-amyloid precursor protein localizes to the splicing factor compartment. Hum Mol Genet. 2004 Mar 1;13(5):475-488.\u003c/li\u003e\n\u003cli\u003eGao J, Aksoy BA, Dogrusoz U, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 2013;6(269):pl1.\u003c/li\u003e\n\u003cli\u003eLi T, Fan J, Wang B, et al. TIMER: a web server for comprehensive analysis of tumor‐infiltrating immune cells. Cancer Res. 2017;77:e108-110. \u003c/li\u003e\n\u003cli\u003eShen YT, Huang X, Zhang G, et al. Pan-Cancer Prognostic Role and Targeting Potential of the Estrogen-Progesterone Axis. Front Oncol. 2021;11:636365.\u003c/li\u003e\n\u003cli\u003eDi C, Syafrizayanti, Zhang Q, et al. Function, clinical application, and strategies of Pre-mRNA splicing in cancer. Cell Death Differ. 2019;26(7):1181-1194.\u003c/li\u003e\n\u003cli\u003eLi J, Li L, You P, et al. Towards artificial intelligence to multi-omics characterization of tumor heterogeneity in esophageal cancer. Semin Cancer Biol. 2023;91:35-49.\u003c/li\u003e\n\u003cli\u003eChen Y, Zhu S, Liu T, et al. Epithelial cells activate fibroblasts to promote esophageal cancer development. Cancer Cell. 2023;41(5):903-918.\u003c/li\u003e\n\u003cli\u003eWaters JK, Reznik SI. Update on Management of Squamous Cell Esophageal Cancer. Curr Oncol Rep. 2022;24(3):375-385.\u003c/li\u003e\n\u003cli\u003eTanackovic G, Kr\u0026auml;mer A. Human splicing factor SF3a, but not SF1, is essential for pre-mRNA splicing in vivo. Mol Biol Cell. 2005;16(3):1366-1377.\u003c/li\u003e\n\u003cli\u003eCraggs G, Finan PM, Lawson D, et al. A nuclear SH3 domain-binding protein that colocalizes with mRNA splicing factors and intermediate filament-containing perinuclear networks. J Biol Chem. 2001;276(32):30552-30560. \u003c/li\u003e\n\u003cli\u003eLee Y, Rio DC. Mechanisms and Regulation of Alternative Pre-mRNA Splicing. Annu Rev Biochem. 2015;84:291-323.\u003c/li\u003e\n\u003cli\u003eHluch\u0026yacute; M, Gajdu\u0026scaron;kov\u0026aacute; P, Ruiz de Los Mozos I, et al. CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1. Nature. 2022 Sep;609(7928):829-834.\u003c/li\u003e\n\u003cli\u003eHuang KK, Huang J, Wu JKL, et al. Long-read transcriptome sequencing reveals abundant promoter diversity in distinct molecular subtypes of gastric cancer. Genome Biol. 2021;22(1):44.\u003c/li\u003e\n\u003cli\u003eCamacho-Vanegas O, Narla G, Teixeira MS, et al. Functional inactivation of the KLF6 tumor suppressor gene by loss of heterozygosity and increased alternative splicing in glioblastoma. Int J Cancer. 2007;121(6):1390-5.\u003c/li\u003e\n\u003cli\u003eChen X, Chen J, Shao B, et al. Relationship between cancer mutations and parameter sensitivity in Rb pathway. J Theor Biol. 2016;404:120-125.\u003c/li\u003e\n\u003cli\u003eXiao W, Wang J, Wang X, et al. Therapeutic targeting of the USP2-E2F4 axis inhibits autophagic machinery essential for zinc homeostasis in cancer progression. Autophagy. 2022;18(11):2615-2635.\u003c/li\u003e\n\u003cli\u003eSu Z, Huang D. Alternative Splicing of Pre-mRNA in the Control of Immune Activity. Genes (Basel). 2021;12(4):574.\u003c/li\u003e\n\u003cli\u003eBonnal SC, L\u0026oacute;pez-Oreja I, Valc\u0026aacute;rcel J. Roles and mechanisms of alternative splicing in cancer - implications for care. Nat Rev Clin Oncol. 2020;17(8):457-474.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SNRPB2, esophageal cancer, cancer biomarker, clinical relevance, prognostic factor, tumor progression","lastPublishedDoi":"10.21203/rs.3.rs-5382410/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5382410/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction:\u003c/h2\u003e \u003cp\u003eEsophageal cancer (ESCA) has poor prognosis. Small nuclear ribonucleoprotein polypeptide B2 (SNRPB2) involves in \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003epre-mRNA splicing a component of the spliceosome.\u003c/span\u003e However, the potential role of SNRPB2 in tumors remain\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003es\u003c/span\u003e poorly understood. This study aimed to determine the clinical relevance and prognostic value of SNRPB2 in \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003epatients with\u003c/span\u003e ESCA.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSNRPB2 mRNA expression levels and genetic alterations were analyzed using \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eGEPIA2 and\u003c/span\u003e cBioPortal. The SNRPB2 protein were detected by immunohistochemical staining using paraffin-embedded tissue specimens of esophageal squamous cell carcinoma (ESCC). Cell experiments were performed to verify the role of SNRPB2 in ESCC cells. TIMER, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eGO\u003c/span\u003e, and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eGSEA analyses were performed to investigate potential biological functions of\u003c/span\u003e SNRPB2. Cycloheximide (CHX) chase was used to test protein stability.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSNRPB2 mRNA was highly expressed in ESCA and associated with tumor progression and clinical prognosis. SNRPB2 protein was highly expressed in ESCC and significantly correlated with vessel carcinoma embolus, lymph node metastasis, clinical stage, and tumor grade. \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eexperiments\u003c/span\u003e showed \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethat the\u003c/span\u003e knockdown of SNRPB2 significantly suppressed proliferation, migration, and invasion. GSEA showed that SNRPB2 inhibits \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe Rb/E2F pathway\u003c/span\u003e. SNRPB2 positively correlates with E2F4 by increasing E2F4 protein \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003estability\u003c/span\u003e. E2F4 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eoverexpression dramatically eliminated the effects of\u003c/span\u003e SNRPB2 knockdown on ESCC tumor progression.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eSNRPB2 promotes \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe proliferation, migration, and invasion\u003c/span\u003e of ESCC cells by increasing E2F4 protein \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003estability.\u003c/span\u003e SNRPB2 has a prognostic role and immunoregulatory potential in ESCA, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand is\u003c/span\u003e an important factor for prediction, evaluation, and individualized treatment.\u003c/p\u003e","manuscriptTitle":"SNRPB2 facilitates esophageal squamous cell carcinoma oncogenesis and progression via E2F4 stabilization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-02 16:12:47","doi":"10.21203/rs.3.rs-5382410/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1d992f20-f7a3-43d0-85d2-ac15a34b692a","owner":[],"postedDate":"December 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-04T15:53:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-02 16:12:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5382410","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5382410","identity":"rs-5382410","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}