Molecular mechanism and clinical significance of Notch3 negative regulation of BRD4 on the growth and survival of esophageal squamous cell carcinoma

Molecular mechanism and clinical significance of Notch3 negative regulation of BRD4 on the growth and survival of esophageal squamous cell carcinoma | 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 Article Molecular mechanism and clinical significance of Notch3 negative regulation of BRD4 on the growth and survival of esophageal squamous cell carcinoma Xiaoke Qin, Hena Yang, Yue Wang, Hong Zhou, Tianyu Zhu, Chao Shi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7554734/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 Objective The aim of this study was to investigate the expression of bromodomain-containing protein 4 (BRD4) and Notch receptor protein 3 (Notch3) in esophageal squamous cell carcinoma (ESCC) and the effect of their interaction on the growth and survival of ESCC cells. Methods The expression of target genes in ESCC and the binding sites between genes were analyzed by TCGA and ChIP-Atlas databases. The expression levels, interactions and effects of target genes on tumor cell activity in ESCC were detected by immunohistochemical staining methods, WB, qRT-PCR, ChIP-qPCR and CCK-8 assays. Dual luciferase reporter gene assays were performed to investigate the regulation between target genes and the signaling mechanism. In vivo experiments were performed to further explore the regulatory effects of target genes on ESCC cell growth. Results BRD4 was highly expressed in ESCC. BRD4 regulated ESCC cell apoptosis by modulating MCL1 and Survivin. In ESCC cell lines, Notch3 silenced BRD4. Notch3 mediated apoptosis in ESCC cells partly by antagonizing BRD4. Notch3 was highly expressed in ESCC tissues. Notch3 expression correlated with the degree of tumor differentiation. When BRD4 was inhibited in ESCC cells, Notch3 expression was also reduced. In vivo, inhibition of BRD4 combined with overexpression of Notch3 better inhibited ESCC cell growth. Conclusion Notch3 negatively feedback regulates BRD4 thereby inhibiting ESCC cell growth and survival. The use of BRD4 inhibitors in combination with overexpression of Notch3 may be better highlight the clinical implication. Biological sciences/Cancer Biological sciences/Cell biology BRD4 Notch3 esophageal squamous cell carcinoma growth and survival Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Surgery was the first rational treatment for cancer. With the advancement of science and technology, surgical techniques have continuously improved, making surgery a powerful tool in cancer treatment. However, new cancer treatment methods such as drug hormone therapy have also been under constant development [ 1 ] .Latest research indicates that directly targeting tumor cells remains the cornerstone of cancer treatment, with traditional methods such as surgery, radiotherapy, and chemotherapy forming the pillars of therapy. Advances in gene-targeted therapies, including treatments for specific genetic mutations, can further refine this strategy [ 2 ] . According to the website Global Cancer Observatory (iarc.fr), there will be 511,000 new cases of esophageal cancer in 2022, ranking 11th among the world's most prevalent cancers, and 445,000 deaths, making it the seventh leading cause of cancer-related deaths [ 3 ] . Two main histologic types of esophageal cancer exist: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), with squamous cell carcinoma being the most prevalent, accounting for approximately 90% of esophageal cancers [ 4 ] , and ESCC is more prevalent in East Asia [ 5 ] . ESCC is difficult to treat because of its complications, high morbidity and mortality, grim prognosis and high risk of recurrence [ 6 ] . Recent advances have shifted the management of ESCC towards precision medicine, immunotherapy and molecularly targeted therapies, breaking the traditional triad of surgery, radiotherapy and chemotherapy and offering new hope for ESCC patients [ 7 ] . Notch is a transmembrane receptor for developmental signaling, and four immediate homologs of Notch (Notch1-4) have evolved in the course of human genetic evolution. The large extracellular domain (ECD) consists mainly of tightly linked tandem repeat sequences of epidermal growth factor (EGF), the number of which varies among Notch homologs. These EGF modules are characterized by six highly conserved cysteines that form three stereoisomeric disulfide bonds and thus contribute to the folding stability of the EGF modules [ 8 ] . Protein hydrolytic cleavage is an important feature of the Notch activation mechanism, forming processing heterodimers that are cleaved at the S1 site and bind delta-like ligands (DLL 1, 3, 4) and Jagged ligands (JAG 1, 2) [ 9 ] . Upon ligand binding, mechanical forces cause hydrolytic cleavage of sequence proteins in the intracellular portion of the receptor, ultimately leading to release of the Notch intracellular domain (NICD) into the cytoplasm. NICD then migrates to the nucleus and binds to other transcriptional coactivators, including recombination signaling protein-binding immunoglobulin kappa J (RBPJ) and MAML1-3 [ 9 – 11 ] , driving transcription of several downstream Notch target genes, including the Hey and His gene families encoding the main helix-loop-helix transcription factors, which normally act as transcription repressors [ 11 ] . Notch3, originally discovered in proliferating neuroepithelium [ 12 ] on chromosome 19p13.12 (19:15159632–15200980), is a heterotrimeric single-channel membrane receptor protein of 2321 amino acids encoded by 33 exons, whose NICD3 functions in the same way as the above-mentioned NICD. Its NICD3 also functions like the above-mentioned NICD by entering the nucleus and binding to other transcriptional promoters to activate the transcription of Hey and His1 [ 13 ] . Notch3 is mainly expressed in vascular smooth muscle, the central nervous system and thymocyte subpopulations. However, studies have shown that dysregulation of Notch3 is associated with a number of cancers [ 13 , 14 ] , such as head and neck cancer [ 15 ] , cystourethroepithelial carcinoma [ 16 ] , ovarian carcinoma [ 17 , 18 ] and colorectal carcinoma [ 19 ] , affecting tumor invasiveness and resistance to chemotherapy [ 13 ] . However, the literature has also shown that Notch3 plays a role in stimulating cost and can trigger apoptosis in thyroid cancer [ 20 , 21 ] , providing a new therapeutic target. BRD4 belongs to the bromodomain and ectodomain (BET) family, which also includes BRD2, BRD3 and BRDT. Each member of the BET family contains two tandem bromodomain domains that bind with high affinity to acetylated lysine in histone tails, and an ET domain that interacts with various transcription factors. The former binds to acetylated lysine in histone tails with high affinity, while the latter interacts with a number of transcription factors [ 22 ] . Studies have shown that BRD4 is involved in a number of tumor development processes, for example, BRD4 can promote the growth of breast tumors [ 23 , 24 ] ; BRD4 regulates self-renewal, self-propagation and tumorigenicity of glioma cells by enriching the Notch1 promoter region [ 25 ] ; BRD4 inhibition synergizes with PD-1 blockade to improve radiotherapy of non-small cell lung cancer [ 26 ] . As BRD4 is expressed in a variety of cancers, inhibition of BRD4 has proved to be a promising therapeutic approach for the treatment of cancer. JQ1, a BET protein inhibitor, is an effective growth inhibitor in many cancers, but some studies have also shown that JQ1 can activate other oncogenic pathways, for example, in ovarian cancer, JQ1 can induce cancer cells to respond to different oncogenic pathways [ 27 ] . BET proteins exhibit transcriptional and functional oppositions during epithelial-mesenchymal transition (EMT) [ 28 ] , suggesting that JQ1 may contribute to EMT and cancer metastasis [ 29 ] . Consequently, BRD4 inhibition by JQ1 alone is not a very reliable therapeutic approach. Alejandro's study showed that BRD4 is located in the Notch3 promoter and that inhibition of BRD4 can directly reduce Notch3 expression and affect the expression of Notch3 factors (e.g. Hes1) [ 30 ] ; Fabiano's study also showed that Notch3 genes are localized next to BRD4 and that BRD4-Notch3 gene fusion is associated for the first time with clinical symptoms [ 31 ] , opening up a new avenue for the targeted treatment of cancer patients. Given that there are no reports exploring whether BRD4 and Notch3 interact in ESCC, and the molecular mechanisms of both in ESCC development have not been explored, this thesis aims to investigate the molecular mechanisms and clinical significance of the interaction of BRD4 and Notch3 in regulating the growth and survival of ESCC cells. Materials and Methods Tissue chip production The hospitalized cases diagnosed as esophageal cancer in the Department of Pathology of the Second Affiliated Hospital of Nantong University from January 2010 to December 2017 were selected as the study subjects. The corresponding HE-stained sections were read and sampling marks were made on the corresponding tumor tissue wax blocks and paracancerous tissue wax blocks of the sections. Tissue chip wax blocks were made by resampling and fusion. Immunohistochemical staining After dewaxing, hydration, antigen repair, and endogenous peroxidase blocking, sections were blocked with goat serum, incubated with primary (Proteintech, China) and secondary antibodies (Proteintech, China) for DAB staining, and then restained with hematoxylin. Sections were photographed using an image analysis system with a Leica microscope. The stained sections were independently evaluated by two professionally qualified pathologists using a double-blind method. The criteria for determining the intensity of staining were as follows: dark brown was strongly positive (3 points), brownish yellow was moderately positive (2 points), light yellow was weakly positive (1 point), and no staining was negative (0 points). Four 400x fields of view were randomly selected for each sample for scoring, and the final score was calculated according to the following formula: total score = (proportion of strongly positive cells × 3 + proportion of moderately positive cells × 2 + proportion of weakly positive cells × 1 + proportion of negative cells × 0) × 100, with a scoring interval of 0-300 points. Cell culture Normal esophageal epithelial cells (Het-1A) and esophageal squamous cell carcinoma cell lines (KYSE510, KYSE150, Eca109, TE-1) were provided by the cell bank of the Medical Research Center of the Second Affiliated Hospital of Nantong University. Human esophageal cancer cells were grown in RPMI 1640 medium (Gibco, USA), supplemented with 10% FBS (fetal bovine serum), 10,000units/ml penicillin plus 10,000 ug/ml streptomycin (NCM Biotech, China). All human cell lines were cultured at 37°C with 5% carbon dioxide. Cell transfection Transfection was performed when the cell density reached 60%-70% and was in good condition. 100µL of basal medium and 2µg of plasmid were added to tube A, and 100µL of basal medium and Lipofectamine 2000 were added to tube B. The tubes were allowed to stand for 5 minutes. The liquids in both tubes were mixed and allowed to stand for 20 minutes. After 4 hours, the old medium was discarded, complete medium was added, and the incubator was placed in the incubator for further incubation. 48 hours later, the protein could be extracted. Western blotting analysis Protein uploading buffer was added to the samples and boiled at 100°C for 5 minutes. The samples were upsampled onto a 10% PAGE gel (EpiZyme, China), electrophoresed, and then transferred to a PVDF membrane and immunoblotted with different antibodies (Proteintech, China). After incubation with HRP-coupled secondary antibodies (Proteintech, China) for 2 hours at room temperature, the immunoreactivity was observed using ECL Plus (NCM Biotech, China). RNA isolation and qRT-PCR RNA was extracted from cells using Trizol reagent according to instructions. Reverse transcription. Reverse transcription was performed using the PrimeScript RT Reagent kit (Takara, Japan). After reverse transcription, cDNA samples were diluted in a 1:20 ratio for gene expression analysis by qRT-PCR. qRT-PCR was performed with TB Green Premix Ex Taq II (Takara, Japan) on a StepOnePlus real-time PCR system (Thermo Scientific, USA). Each sample was repeated three times and relative mRNA expression was calculated using β-actin as internal control. Immunofluorescence Spread cells by placing a crawler in a 24-well plate 24 hours in advance. When the cell growth was fused to 70%-80%, 4% paraformaldehyde was added and fixed for 15 minutes, then add osmotic solution and fix for 10 minutes. After sealing, the primary antibody was incubated at 4°C overnight. After incubating the secondary antibody the next day, 20µL of DAPI-containing sealer was added dropwise on the slide, and the cell crawler was inverted on the sealer. CCK-8 CCK-8 is important for detecting tumor cell viability and proliferation levels in vitro [ 32 – 34 ] . The cells with good growth status were digested, centrifuged, counted, and spread to 96-well plates after adjusting the cell concentration. 100µL of cell suspension was added to each well, the number of cells was around 3000-4000cells/well, 3 replicate wells per group, and the edge wells were replenished with PBS to reduce the evaporation of culture medium. Add 10µL of CCK8 solution to each well according to the time point. The 96-well plate was incubated in an incubator at 37°C, 5% CO 2 for 1 hour protected from light. The absorbance value at 450nm was determined and recorded by an enzyme meter. Dual-luciferase reporter assay The promoter sequence of Hes1 gene in the interval from 2000bp upstream of the transcription start site to the start site was amplified and inserted into the pGL3-Basic vector polyclonal site to construct the recombinant plasmid pGL3-Basic-Hes1. The cell suspension was inoculated in a 96-well culture plate, and when the cells grew to 50% fusion, 500 ng of pGL3-Basic-Hes1 recombinant plasmid was introduced using liposome transfection. After 48 hours of transfection, JQ1 compound was added for treatment. The relative luciferase activity was measured using Promega's Dual Luciferase Reporter Gene Assay System following the instructions procedure to assess Hes1 promoter transcriptional activity. ChIP-qPCR Add 270 µL of 37% formaldehyde solution to the cells to complete the protein-DNA cross-linking reaction. 1.1mL of 10× glycine solution was added and left at room temperature for 5 minutes. After sonication of the DNA, the supernatant was collected by centrifugation and mixed with ChIP dilution buffer taken as 1.8mL 1mM PMSF. 20 µL of the sample was retained as an Input control for subsequent analysis. To the remaining sample, 70 µL of Protein A + G agarose/salmon sperm DNA was added, incubated for 30 min at 4°C, and the supernatant was collected by centrifugation. The primary antibody was incubated overnight at 4°C, and 60 µL of Protein A + G agarose/salmon sperm DNA beads were added and mixed with Low Salt Immune Complex Wash Buffer, High Salt Immune Complex Wash Buffer, High Salt Immune Complex Wash Buffer, LiCl Immune Complex Wash Buffer once, TE Buffer twice, and centrifuged to obtain the precipitate, which was used for subsequent PCR amplification of the target gene. DNA agarose gel electrophoresis Dissolve agarose powder and add GeneGreen nucleic acid dye to prepare an agarose gel. Take 5 µL of DNA sample and 1 µL of 6×loading buffer and mix well, use a pipette gun to add the mixture slowly into the spiking well, set a constant voltage of 120 V, and electrophoresis time 30–60 minutes. After electrophoresis, the gel was transferred to the observation platform of the UV transilluminator, and the UV light source was activated after opening the shield. Record the fluorescence signal of DNA bands by gel imaging system and analyze the bands with professional software. Tumor formation experiments in nude mice Fifteen female thymus-free nude mice, aged 8 weeks (body-weight range 21–25 g), were provided by the Laboratory Animal Centre of Nantong University and housed in an SPF-grade environment. They were selected and randomly divided into three groups, the first group was the control group of pCDNA5 + DMSO, the second group was the experimental group of pCDNA5 + JQ1, and the third group was the experimental group of pCDNA5-3×flag-NICD3 + JQ1. Firstly, KYSE510 cells were injected subcutaneously into the back of mice 10 days in advance. During these 10 days, tumor growth was monitored daily using digital calipers, and the experiment was initiated when tumors reached the predefined initial size: 8–10 mm in long diameter and 100–120 mm³ in volume (calculated by the formula V = 0.5 × L × W², where V = volume, L = length, W = width). After 10 days, mice were anesthetized with 4% isoflurane in 100% O₂ for induction (≈ 60 s) and then maintained with 1.5% isoflurane delivered via a nose-cone at 1 L min⁻¹; adequate anesthesia was verified by loss of the toe-pinch reflex and stable respiratory rate. While under anesthesia, mice received an intratumoral injection of pCDNA5 or pCDNA5-3×flag-NICD3 (500µg/kg) and an intraperitoneal injection of DMSO or JQ1 (500mg/kg). Tumor volume was measured daily with digital calipers and calculated as V = 0.5 × L × W²(V = volume, L = length, W = width). The tumors were resected after 12 days, and the tumor size was measured to compare the proliferation ability of ESCC cells in different groups. Mice were executed using cervical dislocation. Statistical analysis Statistical analysis and graphing were performed using SPSS 26.0, GraphPad Prism 8. Expressions in clinicopathological parameters were analyzed by χ 2 test. WB bands were analyzed by applying ImageJ software. Differences between experimental and control groups were analyzed by t-test. Biological repetitions were performed ≥ 3 times. p < 0.05 was considered as statistically significant difference. Results BRD4 is highly expressed in ESCC First, we found that BRD4 was significantly higher expressed in tumor tissues than in normal tissues in esophageal cancer in the TCGA database (Fig. 1 A). In addition, high BRD4 expression was correlated with advanced tumor grading and cancer stage as well as lymph node metastasis in esophageal cancer (Fig. 1 B- 1 D). And the expression of BRD4 in ESCC was higher than that in ECA (Fig. 1 E), and by analyzing the dataset GSE20347 from the GEO database [ 35 ] , the results showed that BRD4 was highly expressed in ESCC (Fig. 1 F, 1 G), which was consistent with previous findings by our group [ 36 ] . BRD4 can regulate apoptosis in ESCC cells There are eight members of the inhibitor of apoptosis (IAP) family, including baculovirus IAP repeat 5 (BIRC5), also known as survivin. Unlike other IAPs, survivin has a unique inhibitor structure containing a baculovirus inhibitor of apoptosis (BIR) repeat domain and an alpha helix in the carboxyl-terminal region [ 37 ] . Myeloid cell leukaemia 1 (MCL1) is a member of the B-cell lymphoma 2 (BCL2) protein family, which contains three structural domains specific to BCL2 homologs (BH, BH1-BH3) and a putative BH4 structural domain. MCL1 regulates cell death and other related processes such as cell cycle progression and mitochondrial homeostasis. In cancer, MCL1 overexpression promotes cell survival, suppresses apoptosis and increases resistance to chemotherapeutic drugs [ 38 ] . Analysis through the ChIP-Atlas database revealed that BRD4 has binding sites on both MCL1 and Survivin promoter sequences (Fig. 2 A, 2 B). WB results showed that the expression level of BRD4 was higher than that of normal esophageal epithelial cells in ESCC cell lines TE-1, Eca109, KYSE150, and KYSE510, with Eca109 having the highest expression and KYSE510 having the lowest expression (Figure. 2C), and thus these two cell lines were selected for subsequent studies. To explore the relationship between BRD4 and MCL1 and Survivin, ChIP-qPCR experiments were performed. The results showed that BRD4 could bind to MCL1 and Survivin gene promoter fragments with statistically significant differences when BRD4 antibody was added to Eca109 and KYSE510 cells, while the copy numbers of MCL1 and Survivin gene promoter fragments bound to control IgG were much lower than those of MCL1 and Survivin bound to BRD4 gene promoter copy numbers, precluding the effect of non-specific binding of antibodies on this experiment (Fig. 2 D- 2 F). The results indicated that BRD4 could regulate ESCC cell apoptosis by regulating MCL1 and Survivin. ESCC cell lines Eca109 and KYSE510 were transfected with shBRD4 to knock down BRD4 gene expression. Transfection of shScramble was used as the control group, and transfection of shBRD4 was used as the experimental group. qRT-PCR results showed that the transcript levels of anti-apoptotic genes MCL1 and Survivin were down-regulated in the experimental group with statistically significant differences in the experimental group of KYSE510 cells compared with the control group, and the lack of statistically significant differences in the Eca109 cells (Figure. 2G). WB results showed that the BRD4 expression was down-regulated in the experimental group of Eca109 and KYSE510 cells, indicating that BRD4 was successfully knocked down. Meanwhile, the expression levels of MCL1 and Survivin were also down-regulated in the experimental group (Fig. 2 H). The results suggested that BRD4 interacted with the anti-apoptotic genes MCL1 and Survivin, and when BRD4 expression was down-regulated, MCL1 and Survivin expression levels were also down-regulated. In order to investigate the regulatory role of BRD4 in ESCC cell proliferation, two cell lines, Eca109 and KYSE510, were selected for this study. The cell proliferation activity was detected by setting three time points, 24 h, 48 h and 72 h, using the CCK8 method. The experimental data showed that the difference in cell activity between the BRD4 knockdown group and the control group gradually increased with the extension of the culture time. In particular, at the 72-hour time point, the proliferative activity of both Eca109 and KYSE510 cell experimental groups showed a significant decrease compared with that of the control group, and statistical analysis showed that the difference was significant (Fig. 2 I, 2 J). These results confirmed that down-regulation of BRD4 gene expression could significantly inhibit the proliferative ability of ESCC cells. In ESCC cell lines, Notch3 is a silencer of BRD4 Analysis through the ChIP-Atlas database revealed two binding sites for RBPJ in the BRD4 intronic region (Fig. 3 A) and one binding site for BRD4 on the Notch3 sequence (Fig. 3 B). In order to investigate the regulatory relationship between BRD4 and Notch3, ChIP-qPCR experiments were performed in this study, and the results showed that Notch3 could bind to the DNA fragment of BRD4 gene with a statistically significant difference; while the copy number of the DNA fragment of the BRD4 gene bound to the control IgG was much lower than the copy number of the DNA fragment of the BRD4 gene bound to Notch3, excluding the effect of non-specific binding of antibodies on this experiment (Fig. 3 C- 3 E). Next, ESCC cell lines Eca109 and KYSE510 were transfected with shNotch3 to knock down the Notch3 gene expression. Transfection of shScramble was used as the control group, and transfection of shNotch3 was used as the experimental group. The qRT-PCR results showed that the transcript level of Notch3 was down-regulated and that of BRD4 was up-regulated in Eca109 and KYSE510 cells of the experimental group compared with the control group (Fig. 3 F). Eca109 and KYSE510 were transfected with the intracellular activation fragment of Notch3, NICD3, to overexpress the Notch3 gene. qRT-PCR results showed that the transcript level of Notch3 was significantly enhanced in Eca109 and KYSE510 cells, while the transcript level of BRD4 was significantly decreased (Fig. 3 G). The results of WB showed that the Notch3 and NICD3 protein blots were thickened in Eca109 and KYSE510 cells of the experimental group, indicating successful overexpression of Notch3. Meanwhile, BRD4 expression level was down-regulated in the experimental group (Fig. 3 H). The results suggested that there was an interaction between BRD4 and Notch3, and when Notch3 was knocked down, the expression level of BRD4 was up-regulated; while when Notch3 was overexpressed, the expression level of BRD4 was down-regulated, which might suggest the existence of a negative feedback mechanism between BRD4 and Notch3. Notch3 regulates ESCC apoptosis by silencing BRD4 In order to further investigate the regulatory mechanism between BRD4 and Notch3, ChIP-qPCR experiments were performed again in this study, and it was found that when Notch3 was overexpressed, the copy number of BRD4 bound to the promoters of the MCL1 and Survivin genes was significantly reduced with statistically significant differences in Eca109 and KYSE510 cells, and the copy number of the control IgG bound to the MCL1 and Survivin gene promoter fragments had much lower copy numbers than those of MCL1 and Survivin gene promoters bound to BRD4, ruling out the effect of non-specific binding of antibodies on this experiment (Fig. 4 A, 4 B). The results suggested that Notch3 could regulate ESCC cell apoptosis by silencing BRD4. Subsequently, in this study, the two cell lines, Eca109 and KYSE510, were inoculated in 96-well plates, and three time points, 24 h, 48 h and 72 h, were set for culture. At each predetermined time point, cell proliferation activity was detected according to the kit manual procedure. The results showed that when Notch3 was overexpressed the activity of Eca109 and KYSE510 cells in the experimental group would be decreased, and the difference was significant at 72 h. When overexpressed BRD4 was added, the previous inhibitory effect of Notch3 on ESCC cells would be alleviated to a certain extent, and the difference was statistically significant (Fig. 4 C). The results suggested that Notch3 mediated ESCC cell apoptosis partly by antagonizing BRD4. Notch3 is highly expressed in ESCC Next, the expression of Notch3 in ESCC was explored in this section. qRT-PCR results showed that the transcript levels of Notch1-4 were higher than those of normal esophageal mucosal epithelium in the four cell lines of ESCC, but only Notch3 was statistically significant in the ESCC cell lines (Fig. 5 A). Subsequently, in this section, paraffin specimens of 179 ESCC patients from the Second Affiliated Hospital of Nantong University were collected, and a total of 179 ESCC tissues and 52 paracancerous normal esophageal tissues were obtained, which were fabricated into tissue microarrays and stained with immunohistochemistry. By comparing and analyzing the expression levels of Notch3 in ESCC tissues and normal esophageal mucosal epithelial tissues, it was found that Notch3 showed significantly up-regulated expression in cancerous tissues, whereas the expression level was significantly reduced in normal tissues next to the cancer (Fig. 5 B). Immunohistochemical staining showed that the positive signals were mainly manifested as brownish-yellow to blackish-brown granular deposits in the cytoplasm, and positive signals were also seen in the nucleus of a few cells. The results indicated that Notch3 protein was mainly localized in the cytoplasm, and only a small amount was expressed in the nuclear region. The results of the biochemical analysis also yielded the same results as immunohistochemistry. Analysis of the TCGA database revealed that Notch3 expression levels were highly expressed in esophageal cancer (Fig. 5 C). In addition, high Notch3 expression was correlated with advanced tumor grade and cancer stage (Fig. 5 D, 5 E). And Notch3 expression was higher in ESCC than in ECA (Fig. 5 F). In WB experiments, the expression level of Notch3 in four ESCC cell lines was much higher than that in normal esophageal mucosal epithelial cells (Fig. 5 G). The results indicated that Notch3 was highly expressed in ESCC. Relationship between Notch3 expression and clinicopathologic parameters in ESCC patients Next, this section analyzed the relationship between Notch3 expression and clinicopathological parameters in ESCC patients. The expression of Notch3 was classified into low and high expression groups according to the optimal cutoff value using X-tile,and statistical analysis was performed using χ 2 test. The results showed that the expression of Notch3 was only correlated with the degree of tumor differentiation (χ 2 = 12.666, P = 0.000), while it was not correlated with age, gender, tumor site, TNM stage, clinical stage, nerve invasion and intravascular cancer thrombus(Table 1 ). Table 1 Relationship between Notch3 expression and clinicopathologic parameters in ESCC patients Chatacteristic Patients (n = 179) Notch3 Low (n = 95) High (n = 84) χ 2 P value Age(years) 0.300 0.584 ≤ 66 92(51.40%) 4(51.08%) 45(48.91%) >66 87(48.60%) 48(55.17%) 39(44.83%) Gender 0.171 0.679 Female 43(24.02%) 24(55.81%) 19(44.19%) Male 136(75.98%) 71(52.21%) 65(47.79%) Location 1.470 0.225 Upper 13(7.26%) 9(69.23%) 4(30.77%) Middle and lower 166(92.74%) 86(51.81%) 80(48.19%) T stage 1.161 0.281 T1 + T2 63(35.20%) 30(47.62%) 33(52.38%) T3 + T4 116(64.80%) 65(56.03%) 51(43.97%) N stage 0.821 0.365 N0 98(54.75%) 49(50.00%) 49(50.00%) N1 + N2 + N3 81(45.25%) 46(56.79%) 35(43.21%) M stage 0.283 0.595 M0 170(94.97%) 91(53.53%) 79(46.47%) M1 9(5.03%) 4(44.44%) 5(55.56%) Clinical stage 1.683 0.195 Ⅰ+Ⅱ 106(59.22%) 52(49.06%) 54(50.94%) Ⅲ+Ⅳ 73(40.78%) 43(58.90%) 30(41.10%) Differentiation 12.666 0.000 High 35(19.55%) 28(80.00%) 7(20.00%) Medium and low 144(80.45%) 67(46.53%) 77(53.47%) Perineural invasion 0.619 0.431 No 143(79.89%) 78(54.55%) 65(45.45%) Yes 36(20.11%) 17(47.22%) 19(52.78%) Intravascular tumor thrombus 0.329 0.566 No 146(81.56%) 76(52.05%) 70(47.95%) Yes 33(18.44%) 19(57.58%) 14(42.42%) In ESCC cell lines, JQ1 treatment inhibits Notch3 expression Next, this section explored the effect of BRD4 inhibitor JQ1 on Notch3. JQ1 was added to Eca109 and KYSE510 cells, and the transcript levels of Notch3 were measured at 0 h, 3 h, 6 h, 12 h, and 24 h. The qRT-PCR results showed that in Eca109 cells, the transcript level of Notch3 was decreased after 3 h; in KYSE510 cells, the transcript level of Notch3 was decreased, while by 24 h Notch3 transcript levels were up-regulated(Fig. 6 A). Subsequent immunofluorescence experiments in this study revealed that Notch3 was mainly localized in the cytoplasm and exhibited green fluorescence, which was significantly attenuated in the presence of JQ1 (Fig. 6 B). In order to further explore how JQ1 regulates Notch3 expression in ESCC, the dual luciferase reporter gene system was used in this study. Two ESCC cell lines, Eca109 and KYSE510, were selected for the experiment, first transfected with pGL3-Basic-Hes1 recombinant plasmid, and then cultured for 48 hours before adding JQ1 treatment. Seventy-two hours after transfection, the fluorescence intensity of firefly luciferase and sea kidney luciferase was measured separately using an enzyme marker and the ratio was calculated. The experimental data showed that the relative luciferase activity of the JQ1-treated group was significantly down-regulated compared with the control group transfected with pGL3-Basic-Hes1 alone, suggesting that JQ1 can effectively inhibit the transcriptional activity of the Hes1 promoter (Fig. 6 C). Hes1 is a downstream target gene of Notch3, and the decrease in his activity indicates a decrease in the activity of the upstream signaling pathway, suggesting that JQ1 will inhibit the Notch3 expression in ESCC when BRD4 is inhibited. In vivo JQ1 co-overexpression of Notch3 inhibits ESCC cell growth In order to further investigate the effect of the regulatory interaction between BRD4 and Notch3 on the proliferative effects of ESCC cells, nude mice tumorigenic experiments were performed in this study. The mice were randomly divided into three groups, one was the control group of pCDNA5 + DMSO, and the other two groups were the experimental groups of pCDNA5 + JQ1 and pCDNA5-3×flag-NICD3 + JQ1, respectively (Fig. 7 A). The experimental results showed that compared with the control group, the tumor volume growth of the pCDNA5 + JQ1 experimental group was slowed down, and the size of the tumor could be observed to be smaller than that of the control group on day 5, and all of them were smaller than that of the control group after 12 days; whereas, the tumor volume growth of the pCDNA5-3×flag-NICD3 + JQ1 experimental group was slowed down significantly, and the tumor size could be observed to be smaller than that of the control group on day 3, and all of them were smaller than that of the control group after 12 days later were smaller than the control group (Fig. 7 B, 7 C). The results suggest that in vivo JQ1 co-overexpression of Notch3 can inhibit the proliferation of ESCC cells, which provides a new direction for targeted therapy for ESCC patients. Discussion Esophageal cancer is the seventh leading cause of death from malignancy worldwide, and will continue to cause a high number of new cases and deaths in 2022, representing a considerable burden on global health. ESCC is the leading histological form of esophageal cancer in China, accounting for around 90% of all forms. High-risk groups for ESCC include people aged 40 and over who live in high-incidence areas, or who have a history of cancer, a family history of esophageal cancer, or other risk factors such as smoking, alcohol abuse, a history of squamous cell carcinoma of the head and neck, or a preference for spicy and canned foods. For these individuals, endoscopy combined with biopsy of the suspected area is the recommended screening method to reduce mortality from ESCC. Thanks to revolutionary advances in screening, surgery and new treatments, the prognosis of ESCC has improved considerably, and combination therapies (e.g. surgery, chemotherapy, radiotherapy) have proved effective in treating locally advanced esophageal cancer, with immunotherapy now an important therapeutic option [ 39 ] . Despite some therapeutic advances, we still know little about the mechanisms of ESCC development. As ESCC is a highly lethal type of cancer, its development involves complex molecular mechanisms. BRD4, a member of the BET family, plays a key role in ESCC. Numerous studies have shown that BRD4 transcript and protein expression levels are significantly elevated in the tissues of ESCC patients. In Zhang's study, we found that inhibition of BRD4 function by genetic means (e.g. RNA interference) or by specific BRD4 inhibitors (e.g. JQ1) significantly suppressed ESCC cell proliferation in vitro and tumor growth in vivo. Mechanistically, BRD4 can recruit transcriptional complexes to the RCC2 promoter region by interacting with the key transcription factor TP73 and regulate its transcription, which in turn affects the biological behavior of ESCC cells [ 40 ] . In this study, we found that BRD4 expression was significantly higher in tumor tissue than in normal esophageal cancer tissue by analyzing the TCGA database, and that high BRD4 expression correlated with high tumor grade and cancer stage, as well as metastasis to esophageal cancer lymph nodes. We also found that BRD4 expression was significantly higher in ESCC than in EAC, consistent with Zhang's study. After analyzing the database, we decided to investigate how BRD4 regulates ESCC cell growth and survival, and whether BRD4 might be linked to proteins that inhibit apoptosis. To test this hypothesis, we analyzed the ChIP-Atlas database and discovered that BRD4 has a binding site on the promoter sequences of MCL1 and survivin, respectively. The results of subsequent ChIP-qPCR experiments showed that BRD4 does indeed bind to the promoters of MCL1 and survivin, and when BRD4 was inactivated, MCL1 and survivin expression also decreased, and the growth activity of ESCC cells was suppressed. The results suggest that BRD4 may promote tumor growth by regulating MCL1 and Survivin and thus inhibiting apoptosis in ESCC cells. One of the reasons for the poor treatment and prognosis of ESCC, one of the most aggressive and lethal malignancies, is that the phenotypic transformation of epithelial cells in cancer can lead to resistance to chemotherapy. Epithelial-mesenchymal transformation in cancer is linked to tumor formation, invasion, proliferation, metastasis and resistance to stressors such as anticancer drugs, radiation and hypoxia. The Notch signaling pathway regulates cell growth and differentiation processes in response to the environment. When the Notch receptor binds its ligand, it triggers translocation of the Notch intracellular activating fragment, NICD, into the nucleus, where it forms a transcriptional activator complex with the transcription factor RBPJ and regulates expression of target genes of the Hes/HEY family [ 41 ] . NICD is an important part of Notch signaling pathway activation. Liu's article suggests that Icariin (ICA) suppresses Notch2 mRNA expression by inhibiting the action of NICD1 [ 42 ] . This provides us with new ideas as to whether some conventional drugs can also enhance Notch3 expression by acting on NICD3 and thus achieve therapeutic effects for ESCC, while largely reducing the side effects of using JQ1 [ 43 ] . The Notch signaling pathway is thought to be important for the differentiation of esophageal epithelium, as the intracellular activated part of Notch1, NICD1, directly activates Notch3 transcription, resulting in squamous differentiation of epithelial cells. In addition to squamous cell differentiation, Notch1 also regulates the cell cycle, senescence and phenotypic transformation of epithelial cells. Notch1 is a positive EMT effector [ 44 ] , but Notch3 limited the ability of EMT to expand esophageal keratinocytes. Thus, although Notch1 and Notch3 cooperatively stimulate squamous cell differentiation, these Notch homologs may play opposing roles in the phenotypic transformation of squamous cells and, unlike Notch1, Notch3 can limit the phenotypic transformation of squamous cells, allowing them to differentiate normally [ 45 ] , suggesting that Notch3 may play an oncogenic role in ESCC. By analyzing through the ChIP-Atlas database, it was found that RBPJ has two binding sites in the intronic region of BRD4, and BRD4 has one binding site on the sequence of Notch3, suggesting that there may be a regulatory relationship between BRD4 and Notch3. To investigate this relationship, ChIP-qPCR experiments were performed in this study, and the results showed that Notch3 could bind to the DNA fragment of the BRD4 gene. When Notch3 was knocked down, qRT-PCR showed that the transcript level of BRD4 was up-regulated; while when NICD3 was overexpressed, the transcript level of BRD4 was down-regulated compared with the control group. WB results also showed that the expression level of BRD4 was down-regulated. The results suggested that BRD4 interacted with Notch3, and when Notch3 was knocked down, the expression level of BRD4 was up-regulated; whereas when Notch3 was overexpressed, the expression level of BRD4 was down-regulated, suggesting that Notch3 may have a repressive effect on BRD4. In order to further investigate the regulatory mechanism between BRD4 and Notch3, ChIP-qPCR experiments were performed again in this study, and it was found that when Notch3 was overexpressed, the copy number of BRD4 with the promoters of the MCL1 and Survivin genes was significantly reduced. CCK8 experiments showed that the activity of the tumor cells would be decreased when Notch3 was overexpressed; and when overexpressed BRD4, the previous inhibitory effect of Notch3 on ESCC cells would be alleviated to some extent, but the cell activity was still lower than normal. These results suggest that Notch3 can mediate apoptosis in ESCC cells by inhibiting BRD4 and thus affecting the expression of MCL1 and Survivin. We next found that Notch3 was highly expressed in ESCC by database analysis, immunohistochemistry, and WB experiments, and that clinicopathological features showed a significant correlation between the expression level of Notch3 and the degree of differentiation of the tumor tissue only. However, this index did not show statistically significant associations with clinicopathological characteristics such as patient age, gender, site of tumor occurrence, TNM stage, clinical stage, nerve infiltration, and vascular cancer embolism. Previous findings have shown that Notch3 inhibits BRD4, so next we wanted to explore how BRD4 acts on Notch3 and whether there is a negative feedback mechanism between the two. By qRT-PCR, immunofluorescence and dual-luciferase reporter gene assay, the results suggested that JQ1 inhibits the expression of Notch3 in ESCC, and when BRD4 is inhibited, the expression of Notch3 in ESCC also decreases, which, combined with the results of the previous studies, suggests that there is a negative feedback mechanism between BRD4 and Notch3, and Notch3 inhibits ESCC cells by negatively regulating the expression of BRD4. BRD4 to inhibit ESCC cell growth and survival. Alejandro has shown that BRD4 mRNA expression is highly upregulated in ovarian cancer, and TCGA analysis has also shown that increased BRD4 expression in ovarian cancer is associated with a significant reduction in survival. Notch3 has been shown to be highly upregulated in ovarian cancer and associated with increased tumor cell proliferation, increased resistance to chemotherapy and decreased survival. Alejandro found that Notch3 expression and BRD4 expression are indeed closely linked in ovarian cancer, and that treatment with BRD4 inhibitors reduces Notch3 expression in vitro and in vivo, while BRD4 inhibition also reduces the expression of Notch3 targets, including Hes1 [ 30 ] . This is consistent with our findings that JQ1 can inhibit Notch3 expression. Subsequently, we conducted in vivo experiments and constructed patient-derived xenograft (PDX) models, which are widely used for evaluating tumor behavior, therapeutic response, and metastatic potential in vivo. PDX models offer a more physiologically relevant tumor microenvironment compared to traditional cell line xenografts, preserving patient-specific histological and molecular characteristics. Prior research has successfully leveraged such models to explore diverse aspects of cancer biology [ 46 , 47 ] . The results showed that the tumor volume growth was slowed down by adding JQ1 alone; however, the tumor volume growth was significantly slowed down by combining with NICD3. The results suggest that in vivo JQ1 combined with overexpression of Notch3 can more effectively inhibit the proliferative ability of ESCC cells, which provides a new direction for targeted therapy for ESCC patients. The following shortcomings exist in this study: first, this study failed to collect fresh tissue specimens to explore the expression of BRD4 and Notch3 in ESCC; second, the specific molecular mechanism of Notch3 negative feedback regulation of BRD4 has not been investigated, and further refinement is needed next. In summary, this study initially revealed the expression of BRD4 and Notch3 in ESCC and the molecular mechanism regulating the growth and survival of ESCC cells. Notch3 negatively feedback regulates BRD4 and thus inhibits the growth and survival of ESCC cells. Therefore, the use of BRD4 inhibitors in combination with overexpression of Notch3 may become a new option for the treatment of ESCC, bringing new hope for the treatment of ESCC patients. Declarations Author's contribution HL designed the study. XQ and HY performed the study and wrote the paper. YW and TZ analyzed the data. HZ and CS staged the patients for pTNM and scored the immunohistochemical staining results. Funding This study was supported by Nantong Science and Technology Project (JC2021001) and Nantong Social Livelihood Science and Technology Program (MSZ2023099) Data Availability All data generated or analyzed during this study are included in this published article. Conflict of interest The Authors declare that they have no conflict of interest. Ethical approval This study and included experimental procedures were approved by the institutional animal care and use committee of Nantong University. All animal housing and experiments were conducted in strict accordance with the institutional guidelines for care and use of laboratory animals. The study is reported in accordance with ARRIVE guidelines. The study confirmed that informed consent was obtained from all subjects and/or their legal guardians. The study was conducted with the approval of the appropriate ethics committee of the Second Affiliated Hospital of Nantong University(IRB No. 2021KT057). The investigation conformed to the principles outlined in the Declaration of Helsinki. Open Access This article is available under a Creative Commons Attribution-NonCommercial 4.0 International License, which permits any non-commercial use, sharing, adaptation, distribution, and reproduction in any medium or format, provided the author and source are credited, a Creative Commons license is cited, and changes are indicated. 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18:40:43","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24015,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/b9c424163653a7994b2b1f2c.png"},{"id":93074244,"identity":"21aaff80-1dc4-467d-ae6d-b12ea5049468","added_by":"auto","created_at":"2025-10-08 18:40:39","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98734,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/08921f15c57c3155c48d8df7.png"},{"id":93074331,"identity":"6a31952a-78b1-4e6b-9fd4-45ba57c46d58","added_by":"auto","created_at":"2025-10-08 18:40:42","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":93087,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/4559ccc90f19e2385652bfc4.png"},{"id":93074294,"identity":"65bb74da-edda-4403-b31d-5679ebcdcc88","added_by":"auto","created_at":"2025-10-08 18:40:41","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":314876,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/fbfbd21040ecfbffcda70268.png"},{"id":93074296,"identity":"f75e7784-8370-4a27-8f02-f2fafb9690f9","added_by":"auto","created_at":"2025-10-08 18:40:41","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":60653,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/2810cb12e708c3bcf8b5e6e4.png"},{"id":93074456,"identity":"834e436d-23ea-4b80-8bec-a163a2da5454","added_by":"auto","created_at":"2025-10-08 18:40:50","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99469,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/54ef220797d473629cf4110e.png"},{"id":93074413,"identity":"1865e204-ea18-4cc1-a820-2198d1af6e2a","added_by":"auto","created_at":"2025-10-08 18:40:44","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48785,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/ee81f0fd4434cdc39b54eeb0.png"},{"id":93074369,"identity":"0d4acb2f-be72-494d-8049-e053c7fb2e2a","added_by":"auto","created_at":"2025-10-08 18:40:42","extension":"xml","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":118870,"visible":true,"origin":"","legend":"","description":"","filename":"33343b3cb05147b1aa330e8aa96e27e41structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/a9000ba0a89caad875df9ef1.xml"},{"id":93074285,"identity":"b4c3d610-c4bc-4be2-a103-dad2aa045c93","added_by":"auto","created_at":"2025-10-08 18:40:40","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":129623,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/e0c2b263ebe95b5dc27356b4.html"},{"id":93074292,"identity":"7ad4b8f7-1683-412d-9433-87d5e106cd3f","added_by":"auto","created_at":"2025-10-08 18:40:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":149905,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBRD4 is highly expressed in ESCC. \u003c/strong\u003e(A) Expression of BRD4 in esophageal cancer versus normal tissues in the TCGA database. (B) Relationship between BRD4 expression and cancer stage of esophageal cancer in the TCGA database. (C) Relationship between BRD4 expression and cancer grading of esophageal cancer in the TCGA database. (D) Relationship between BRD4 expression and lymph node metastasis of esophageal cancer in the TCGA database. (E) Expression of BRD4 in EAC and ESCC in the TCGA database. (F) Expression of BRD4 in ESCC dataset GSE20347. (G) Expression of BRD4 in normal esophageal epithelial mucosal tissues and tumor tissues in ESCC dataset GSE20347.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/a49bbb39e95c47c8e915846e.png"},{"id":93074460,"identity":"4b779980-0fa1-4f55-bf12-bc76e786517c","added_by":"auto","created_at":"2025-10-08 18:40:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":368631,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBRD4 can regulate apoptosis in ESCC cells. \u003c/strong\u003e(A) BRD4 binding site on the MCL1 promoter sequence. (B) BRD4 binding site on the BIRC5 promoter sequence. (C) BRD4 expression in ESCC cell lines TE-1, Eca109, KYSE150, and KYSE510. (D) ChIP-qPCR experiments were performed in Eca109 cells and KYSE510 cells after BRD4 pull-down in the promoter region of the MCL1 and Survivin genes. (E) ChIP-qPCR was applied to detect the binding efficiency of BRD4 to the promoter fragments of MCL1 and Survivin gene in Eca109 cells. (F) ChIP-qPCR was applied to detect the binding efficiency of BRD4 to MCL1 and Survivin gene promoter fragments in KYSE510 cells. (G) Application of qRT-PCR to detect the transcript levels of MCL1 and Survivin after knockdown of BRD4 in Eca109 cells and KYSE510 cells. (H) WB assay was applied to detect the protein expression levels of MCL1 and Survivin after knockdown of BRD4 in Eca109 cells and KYSE510 cells. (I) Application of CCK8 assay to detect Eca109 cell activity after knockdown of BRD4. (J) Application of CCK8 assay to detect KYSE510 cell activity after knockdown of BRD4.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/00d4ad83fc0bfac81c1e7235.png"},{"id":93075087,"identity":"44f04a0c-5453-4747-bf1a-19fad7ab1d59","added_by":"auto","created_at":"2025-10-08 19:01:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":189102,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn ESCC cell lines, Notch3 is a silencer of BRD4. \u003c/strong\u003e(A) Application of the ChIP-Atlas database for analysis revealed two binding sites for the transcription factor RBPJ of Notch3 in the intronic region of BRD4. (B) Application of the ChIP-Atlas database for analysis revealed one binding site for BRD4 in the NOTCH3 sequence. (C) ChIP-qPCR experiments were performed in Eca109 cells and KYSE510 cells in the promoter region of the BRD4 gene after 3×flag-NICD3 pull-down. (D) ChIP-qPCR was applied to detect the binding efficiency of NICD3 to the promoter fragment of BRD4 gene in Eca109 cells. (E) ChIP-qPCR was applied to detect the binding efficiency of NICD3 with BRD4 gene promoter fragment in KYSE510 cells. (F) Application of qRT-PCR to detect the transcript level of BRD4 after knockdown of Notch3 in Eca109 cells and KYSE510 cells. (G) Application of qRT-PCR to detect the transcript level of BRD4 after overexpression of NICD3 in Eca109 cells and KYSE510 cells. (H) WB method was applied to detect the protein expression level of BRD4 after overexpression of NICD3 in Eca109 cells and KYSE510 cells.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/0c8be08462d2d6ae40e4fd95.png"},{"id":93074420,"identity":"70138eae-3767-4272-a764-44093937433f","added_by":"auto","created_at":"2025-10-08 18:40:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":201578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNotch3 regulates ESCC apoptosis by silencing BRD4. \u003c/strong\u003e(A) ChIP-qPCR experiments were performed in Eca109 cells and KYSE510 cells when NICD3 was overexpressed and BRD4 was pulled down in the promoter region of MCL1 and Survivin genes. (B) ChIP-qPCR assay was applied to detect the binding efficiency of BRD4 to the promoter fragments of MCL1 and Survivin gene in Eca109 cells and KYSE510 cells when NICD3 was overexpressed. (C) CCK8 assay was applied to detect Eca109 and KYSE510 cell activity after overexpression of NICD3, and Eca109 and KYSE510 cell activity after co-overexpression of BRD4.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/f9270afc8a3cc21a52e8d752.png"},{"id":93074410,"identity":"28f590f5-6a84-4a3b-8043-26805ab3e9c2","added_by":"auto","created_at":"2025-10-08 18:40:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1168934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNotch3 is highly expressed in ESCC. \u003c/strong\u003e(A) qRT-PCR was applied to detect the transcript levels of Notch1-4 in Het-1A, TE-1, Eca109, KYSE150 and KYSE510 cells, and only Notch3 was statistically significant. (B) Immunohistochemistry was applied to detect Notch3 expression in ESCC tissues. (C) Expression of Notch3 in esophageal cancer versus normal tissues in the TCGA database. (D) Relationship between Notch3 expression and cancer grade of esophageal cancer in the TCGA database. (E) Relationship between Notch3 expression and cancer stage of esophageal cancer in the TCGA database. (F) Expression of BRD4 in EAC and ESCC in the TCGA database. (G) Application of WB to detect the expression of Notch3 and NICD3 in Het-1A, TE-1, Eca109, KYSE150 and KYSE510 cells.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/e42e5484d5eaf9fad7616df2.png"},{"id":93074246,"identity":"f4b60475-e36d-4c48-8d10-37b2bed56ce4","added_by":"auto","created_at":"2025-10-08 18:40:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":340605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn ESCC cell lines, JQ1 treatment inhibits Notch3 expression. \u003c/strong\u003e(A)The qRT-PCR method was applied to detect the transcript levels of Notch3 after 3 h, 6 h, 12 h and 24 h of JQ1 treatment in Eca109 cells and KYSE510 cells. (B) Immunofluorescence was applied to detect the expression of Notch3 after JQ1 treatment in Eca109 cells and KYSE510 cells. (C) Dual luciferase reporter gene assay was applied to compare the relative luciferase activities of pGL3-Basic-Hes1 and pGL3-Basic-Hes1+JQ1 groups in Eca109 cells and KYSE510 cells.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/b5921637687646aff78ae5d0.png"},{"id":93074431,"identity":"83c3e2fb-d208-4956-9f97-4bc910bc3a51","added_by":"auto","created_at":"2025-10-08 18:40:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":193382,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo JQ1 co-overexpression of Notch3 inhibits ESCC cell growth. \u003c/strong\u003e(A) Tumor model construction scheme. (B) Harvesting of tumor bodies generated under different conditions. (C) Changes in tumor volume over 12 days.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/04a5cdd82c137991be28651f.png"},{"id":103232384,"identity":"84f2128b-18dd-4acb-b4af-8c844077ed76","added_by":"auto","created_at":"2026-02-23 12:27:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3669137,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/ddd03050-2578-4781-88eb-60de87b4b11f.pdf"},{"id":93074376,"identity":"cd424f44-d812-4056-83a8-918770a883bf","added_by":"auto","created_at":"2025-10-08 18:40:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2798579,"visible":true,"origin":"","legend":"","description":"","filename":"MainfiguresrawWB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7554734/v1/517b578360e94b0e30c63021.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular mechanism and clinical significance of Notch3 negative regulation of BRD4 on the growth and survival of esophageal squamous cell carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSurgery was the first rational treatment for cancer. With the advancement of science and technology, surgical techniques have continuously improved, making surgery a powerful tool in cancer treatment. However, new cancer treatment methods such as drug hormone therapy have also been under constant development\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e.Latest research indicates that directly targeting tumor cells remains the cornerstone of cancer treatment, with traditional methods such as surgery, radiotherapy, and chemotherapy forming the pillars of therapy. Advances in gene-targeted therapies, including treatments for specific genetic mutations, can further refine this strategy\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAccording to the website Global Cancer Observatory (iarc.fr), there will be 511,000 new cases of esophageal cancer in 2022, ranking 11th among the world's most prevalent cancers, and 445,000 deaths, making it the seventh leading cause of cancer-related deaths\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Two main histologic types of esophageal cancer exist: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), with squamous cell carcinoma being the most prevalent, accounting for approximately 90% of esophageal cancers\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, and ESCC is more prevalent in East Asia\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. ESCC is difficult to treat because of its complications, high morbidity and mortality, grim prognosis and high risk of recurrence\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Recent advances have shifted the management of ESCC towards precision medicine, immunotherapy and molecularly targeted therapies, breaking the traditional triad of surgery, radiotherapy and chemotherapy and offering new hope for ESCC patients\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNotch is a transmembrane receptor for developmental signaling, and four immediate homologs of Notch (Notch1-4) have evolved in the course of human genetic evolution. The large extracellular domain (ECD) consists mainly of tightly linked tandem repeat sequences of epidermal growth factor (EGF), the number of which varies among Notch homologs. These EGF modules are characterized by six highly conserved cysteines that form three stereoisomeric disulfide bonds and thus contribute to the folding stability of the EGF modules\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Protein hydrolytic cleavage is an important feature of the Notch activation mechanism, forming processing heterodimers that are cleaved at the S1 site and bind delta-like ligands (DLL 1, 3, 4) and Jagged ligands (JAG 1, 2)\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Upon ligand binding, mechanical forces cause hydrolytic cleavage of sequence proteins in the intracellular portion of the receptor, ultimately leading to release of the Notch intracellular domain (NICD) into the cytoplasm. NICD then migrates to the nucleus and binds to other transcriptional coactivators, including recombination signaling protein-binding immunoglobulin kappa J (RBPJ) and MAML1-3\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, driving transcription of several downstream Notch target genes, including the Hey and His gene families encoding the main helix-loop-helix transcription factors, which normally act as transcription repressors\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNotch3, originally discovered in proliferating neuroepithelium\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e on chromosome 19p13.12 (19:15159632\u0026ndash;15200980), is a heterotrimeric single-channel membrane receptor protein of 2321 amino acids encoded by 33 exons, whose NICD3 functions in the same way as the above-mentioned NICD. Its NICD3 also functions like the above-mentioned NICD by entering the nucleus and binding to other transcriptional promoters to activate the transcription of Hey and His1\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Notch3 is mainly expressed in vascular smooth muscle, the central nervous system and thymocyte subpopulations. However, studies have shown that dysregulation of Notch3 is associated with a number of cancers\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, such as head and neck cancer\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, cystourethroepithelial carcinoma\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, ovarian carcinoma\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003eand colorectal carcinoma\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, affecting tumor invasiveness and resistance to chemotherapy\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, the literature has also shown that Notch3 plays a role in stimulating cost and can trigger apoptosis in thyroid cancer\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, providing a new therapeutic target.\u003c/p\u003e\u003cp\u003eBRD4 belongs to the bromodomain and ectodomain (BET) family, which also includes BRD2, BRD3 and BRDT. Each member of the BET family contains two tandem bromodomain domains that bind with high affinity to acetylated lysine in histone tails, and an ET domain that interacts with various transcription factors. The former binds to acetylated lysine in histone tails with high affinity, while the latter interacts with a number of transcription factors\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that BRD4 is involved in a number of tumor development processes, for example, BRD4 can promote the growth of breast tumors\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e; BRD4 regulates self-renewal, self-propagation and tumorigenicity of glioma cells by enriching the Notch1 promoter region\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e; BRD4 inhibition synergizes with PD-1 blockade to improve radiotherapy of non-small cell lung cancer\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. As BRD4 is expressed in a variety of cancers, inhibition of BRD4 has proved to be a promising therapeutic approach for the treatment of cancer. JQ1, a BET protein inhibitor, is an effective growth inhibitor in many cancers, but some studies have also shown that JQ1 can activate other oncogenic pathways, for example, in ovarian cancer, JQ1 can induce cancer cells to respond to different oncogenic pathways\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. BET proteins exhibit transcriptional and functional oppositions during epithelial-mesenchymal transition (EMT)\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, suggesting that JQ1 may contribute to EMT and cancer metastasis\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Consequently, BRD4 inhibition by JQ1 alone is not a very reliable therapeutic approach.\u003c/p\u003e\u003cp\u003eAlejandro's study showed that BRD4 is located in the Notch3 promoter and that inhibition of BRD4 can directly reduce Notch3 expression and affect the expression of Notch3 factors (e.g. Hes1)\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e; Fabiano's study also showed that Notch3 genes are localized next to BRD4 and that BRD4-Notch3 gene fusion is associated for the first time with clinical symptoms\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, opening up a new avenue for the targeted treatment of cancer patients.\u003c/p\u003e\u003cp\u003eGiven that there are no reports exploring whether BRD4 and Notch3 interact in ESCC, and the molecular mechanisms of both in ESCC development have not been explored, this thesis aims to investigate the molecular mechanisms and clinical significance of the interaction of BRD4 and Notch3 in regulating the growth and survival of ESCC cells.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eTissue chip production\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;The hospitalized cases diagnosed as esophageal cancer in the Department of Pathology of the Second Affiliated Hospital of Nantong University from January 2010 to December 2017 were selected as the study subjects. The corresponding HE-stained sections were read and sampling marks were made on the corresponding tumor tissue wax blocks and paracancerous tissue wax blocks of the sections. Tissue chip wax blocks were made by resampling and fusion.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eImmunohistochemical staining\u003c/h3\u003e\n\u003cp\u003eAfter dewaxing, hydration, antigen repair, and endogenous peroxidase blocking, sections were blocked with goat serum, incubated with primary (Proteintech, China) and secondary antibodies (Proteintech, China) for DAB staining, and then restained with hematoxylin. Sections were photographed using an image analysis system with a Leica microscope. The stained sections were independently evaluated by two professionally qualified pathologists using a double-blind method. The criteria for determining the intensity of staining were as follows: dark brown was strongly positive (3 points), brownish yellow was moderately positive (2 points), light yellow was weakly positive (1 point), and no staining was negative (0 points). Four 400x fields of view were randomly selected for each sample for scoring, and the final score was calculated according to the following formula: total score = (proportion of strongly positive cells \u0026times; 3\u0026thinsp;+\u0026thinsp;proportion of moderately positive cells \u0026times; 2\u0026thinsp;+\u0026thinsp;proportion of weakly positive cells \u0026times; 1\u0026thinsp;+\u0026thinsp;proportion of negative cells \u0026times; 0) \u0026times; 100, with a scoring interval of 0-300 points.\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eNormal esophageal epithelial cells (Het-1A) and esophageal squamous cell carcinoma cell lines (KYSE510, KYSE150, Eca109, TE-1) were provided by the cell bank of the Medical Research Center of the Second Affiliated Hospital of Nantong University. Human esophageal cancer cells were grown in RPMI 1640 medium (Gibco, USA), supplemented with 10% FBS (fetal bovine serum), 10,000units/ml penicillin plus 10,000 ug/ml streptomycin (NCM Biotech, China). All human cell lines were cultured at 37\u0026deg;C with 5% carbon dioxide.\u003c/p\u003e\n\u003ch3\u003eCell transfection\u003c/h3\u003e\n\u003cp\u003eTransfection was performed when the cell density reached 60%-70% and was in good condition. 100\u0026micro;L of basal medium and 2\u0026micro;g of plasmid were added to tube A, and 100\u0026micro;L of basal medium and Lipofectamine 2000 were added to tube B. The tubes were allowed to stand for 5 minutes. The liquids in both tubes were mixed and allowed to stand for 20 minutes. After 4 hours, the old medium was discarded, complete medium was added, and the incubator was placed in the incubator for further incubation. 48 hours later, the protein could be extracted.\u003c/p\u003e\n\u003ch3\u003eWestern blotting analysis\u003c/h3\u003e\n\u003cp\u003eProtein uploading buffer was added to the samples and boiled at 100\u0026deg;C for 5 minutes. The samples were upsampled onto a 10% PAGE gel (EpiZyme, China), electrophoresed, and then transferred to a PVDF membrane and immunoblotted with different antibodies (Proteintech, China). After incubation with HRP-coupled secondary antibodies (Proteintech, China) for 2 hours at room temperature, the immunoreactivity was observed using ECL Plus (NCM Biotech, China).\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eRNA isolation and qRT-PCR\u003c/h2\u003e\n\u003cp\u003eRNA was extracted from cells using Trizol reagent according to instructions. Reverse transcription. Reverse transcription was performed using the PrimeScript RT Reagent kit (Takara, Japan). After reverse transcription, cDNA samples were diluted in a 1:20 ratio for gene expression analysis by qRT-PCR. qRT-PCR was performed with TB Green Premix Ex Taq II (Takara, Japan) on a StepOnePlus real-time PCR system (Thermo Scientific, USA). Each sample was repeated three times and relative mRNA expression was calculated using \u0026beta;-actin as internal control.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eSpread cells by placing a crawler in a 24-well plate 24 hours in advance. When the cell growth was fused to 70%-80%, 4% paraformaldehyde was added and fixed for 15 minutes, then add osmotic solution and fix for 10 minutes. After sealing, the primary antibody was incubated at 4\u0026deg;C overnight. After incubating the secondary antibody the next day, 20\u0026micro;L of DAPI-containing sealer was added dropwise on the slide, and the cell crawler was inverted on the sealer.\u003c/p\u003e\n\u003ch3\u003eCCK-8\u003c/h3\u003e\n\u003cp\u003eCCK-8 is important for detecting tumor cell viability and proliferation levels in vitro\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. The cells with good growth status were digested, centrifuged, counted, and spread to 96-well plates after adjusting the cell concentration. 100\u0026micro;L of cell suspension was added to each well, the number of cells was around 3000-4000cells/well, 3 replicate wells per group, and the edge wells were replenished with PBS to reduce the evaporation of culture medium. Add 10\u0026micro;L of CCK8 solution to each well according to the time point. The 96-well plate was incubated in an incubator at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e for 1 hour protected from light. The absorbance value at 450nm was determined and recorded by an enzyme meter.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eDual-luciferase reporter assay\u003c/h2\u003e\n\u003cp\u003eThe promoter sequence of Hes1 gene in the interval from 2000bp upstream of the transcription start site to the start site was amplified and inserted into the pGL3-Basic vector polyclonal site to construct the recombinant plasmid pGL3-Basic-Hes1. The cell suspension was inoculated in a 96-well culture plate, and when the cells grew to 50% fusion, 500 ng of pGL3-Basic-Hes1 recombinant plasmid was introduced using liposome transfection. After 48 hours of transfection, JQ1 compound was added for treatment. The relative luciferase activity was measured using Promega's Dual Luciferase Reporter Gene Assay System following the instructions procedure to assess Hes1 promoter transcriptional activity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eChIP-qPCR\u003c/h2\u003e\n\u003cp\u003eAdd 270 \u0026micro;L of 37% formaldehyde solution to the cells to complete the protein-DNA cross-linking reaction. 1.1mL of 10\u0026times; glycine solution was added and left at room temperature for 5 minutes. After sonication of the DNA, the supernatant was collected by centrifugation and mixed with ChIP dilution buffer taken as 1.8mL 1mM PMSF. 20 \u0026micro;L of the sample was retained as an Input control for subsequent analysis. To the remaining sample, 70 \u0026micro;L of Protein A\u0026thinsp;+\u0026thinsp;G agarose/salmon sperm DNA was added, incubated for 30 min at 4\u0026deg;C, and the supernatant was collected by centrifugation. The primary antibody was incubated overnight at 4\u0026deg;C, and 60 \u0026micro;L of Protein A\u0026thinsp;+\u0026thinsp;G agarose/salmon sperm DNA beads were added and mixed with Low Salt Immune Complex Wash Buffer, High Salt Immune Complex Wash Buffer, High Salt Immune Complex Wash Buffer, LiCl Immune Complex Wash Buffer once, TE Buffer twice, and centrifuged to obtain the precipitate, which was used for subsequent PCR amplification of the target gene.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eDNA agarose gel electrophoresis\u003c/h2\u003e\n\u003cp\u003eDissolve agarose powder and add GeneGreen nucleic acid dye to prepare an agarose gel. Take 5 \u0026micro;L of DNA sample and 1 \u0026micro;L of 6\u0026times;loading buffer and mix well, use a pipette gun to add the mixture slowly into the spiking well, set a constant voltage of 120 V, and electrophoresis time 30\u0026ndash;60 minutes. After electrophoresis, the gel was transferred to the observation platform of the UV transilluminator, and the UV light source was activated after opening the shield. Record the fluorescence signal of DNA bands by gel imaging system and analyze the bands with professional software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eTumor formation experiments in nude mice\u003c/h2\u003e\n\u003cp\u003eFifteen female thymus-free nude mice, aged 8 weeks (body-weight range 21\u0026ndash;25 g), were provided by the Laboratory Animal Centre of Nantong University and housed in an SPF-grade environment. They were selected and randomly divided into three groups, the first group was the control group of pCDNA5\u0026thinsp;+\u0026thinsp;DMSO, the second group was the experimental group of pCDNA5\u0026thinsp;+\u0026thinsp;JQ1, and the third group was the experimental group of pCDNA5-3\u0026times;flag-NICD3\u0026thinsp;+\u0026thinsp;JQ1. Firstly, KYSE510 cells were injected subcutaneously into the back of mice 10 days in advance. During these 10 days, tumor growth was monitored daily using digital calipers, and the experiment was initiated when tumors reached the predefined initial size: 8\u0026ndash;10 mm in long diameter and 100\u0026ndash;120 mm\u0026sup3; in volume (calculated by the formula V\u0026thinsp;=\u0026thinsp;0.5 \u0026times; L \u0026times; W\u0026sup2;, where V\u0026thinsp;=\u0026thinsp;volume, L\u0026thinsp;=\u0026thinsp;length, W\u0026thinsp;=\u0026thinsp;width). After 10 days, mice were anesthetized with 4% isoflurane in 100% O₂ for induction (\u0026asymp;\u0026thinsp;60 s) and then maintained with 1.5% isoflurane delivered via a nose-cone at 1 L min⁻\u0026sup1;; adequate anesthesia was verified by loss of the toe-pinch reflex and stable respiratory rate. While under anesthesia, mice received an intratumoral injection of pCDNA5 or pCDNA5-3\u0026times;flag-NICD3 (500\u0026micro;g/kg) and an intraperitoneal injection of DMSO or JQ1 (500mg/kg). Tumor volume was measured daily with digital calipers and calculated as V\u0026thinsp;=\u0026thinsp;0.5 \u0026times; L \u0026times; W\u0026sup2;(V\u0026thinsp;=\u0026thinsp;volume, L\u0026thinsp;=\u0026thinsp;length, W\u0026thinsp;=\u0026thinsp;width). The tumors were resected after 12 days, and the tumor size was measured to compare the proliferation ability of ESCC cells in different groups. Mice were executed using cervical dislocation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eStatistical analysis and graphing were performed using SPSS 26.0, GraphPad Prism 8. Expressions in clinicopathological parameters were analyzed by \u0026chi;\u003csup\u003e2\u003c/sup\u003e test. WB bands were analyzed by applying ImageJ software. Differences between experimental and control groups were analyzed by t-test. Biological repetitions were performed\u0026thinsp;\u0026ge;\u0026thinsp;3 times. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant difference.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eBRD4 is highly expressed in ESCC\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;First, we found that BRD4 was significantly higher expressed in tumor tissues than in normal tissues in esophageal cancer in the TCGA database (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). In addition, high BRD4 expression was correlated with advanced tumor grading and cancer stage as well as lymph node metastasis in esophageal cancer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB-\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). And the expression of BRD4 in ESCC was higher than that in ECA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE), and by analyzing the dataset GSE20347 from the GEO database\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, the results showed that BRD4 was highly expressed in ESCC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF, \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG), which was consistent with previous findings by our group\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eBRD4 can regulate apoptosis in ESCC cells\u003c/h2\u003e\n\u003cp\u003eThere are eight members of the inhibitor of apoptosis (IAP) family, including baculovirus IAP repeat 5 (BIRC5), also known as survivin. Unlike other IAPs, survivin has a unique inhibitor structure containing a baculovirus inhibitor of apoptosis (BIR) repeat domain and an alpha helix in the carboxyl-terminal region\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMyeloid cell leukaemia 1 (MCL1) is a member of the B-cell lymphoma 2 (BCL2) protein family, which contains three structural domains specific to BCL2 homologs (BH, BH1-BH3) and a putative BH4 structural domain. MCL1 regulates cell death and other related processes such as cell cycle progression and mitochondrial homeostasis. In cancer, MCL1 overexpression promotes cell survival, suppresses apoptosis and increases resistance to chemotherapeutic drugs\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAnalysis through the ChIP-Atlas database revealed that BRD4 has binding sites on both MCL1 and Survivin promoter sequences (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). WB results showed that the expression level of BRD4 was higher than that of normal esophageal epithelial cells in ESCC cell lines TE-1, Eca109, KYSE150, and KYSE510, with Eca109 having the highest expression and KYSE510 having the lowest expression (Figure. 2C), and thus these two cell lines were selected for subsequent studies. To explore the relationship between BRD4 and MCL1 and Survivin, ChIP-qPCR experiments were performed. The results showed that BRD4 could bind to MCL1 and Survivin gene promoter fragments with statistically significant differences when BRD4 antibody was added to Eca109 and KYSE510 cells, while the copy numbers of MCL1 and Survivin gene promoter fragments bound to control IgG were much lower than those of MCL1 and Survivin bound to BRD4 gene promoter copy numbers, precluding the effect of non-specific binding of antibodies on this experiment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD-\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). The results indicated that BRD4 could regulate ESCC cell apoptosis by regulating MCL1 and Survivin. ESCC cell lines Eca109 and KYSE510 were transfected with shBRD4 to knock down BRD4 gene expression. Transfection of shScramble was used as the control group, and transfection of shBRD4 was used as the experimental group. qRT-PCR results showed that the transcript levels of anti-apoptotic genes MCL1 and Survivin were down-regulated in the experimental group with statistically significant differences in the experimental group of KYSE510 cells compared with the control group, and the lack of statistically significant differences in the Eca109 cells (Figure. 2G). WB results showed that the BRD4 expression was down-regulated in the experimental group of Eca109 and KYSE510 cells, indicating that BRD4 was successfully knocked down. Meanwhile, the expression levels of MCL1 and Survivin were also down-regulated in the experimental group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH). The results suggested that BRD4 interacted with the anti-apoptotic genes MCL1 and Survivin, and when BRD4 expression was down-regulated, MCL1 and Survivin expression levels were also down-regulated.\u003c/p\u003e\n\u003cp\u003eIn order to investigate the regulatory role of BRD4 in ESCC cell proliferation, two cell lines, Eca109 and KYSE510, were selected for this study. The cell proliferation activity was detected by setting three time points, 24 h, 48 h and 72 h, using the CCK8 method. The experimental data showed that the difference in cell activity between the BRD4 knockdown group and the control group gradually increased with the extension of the culture time. In particular, at the 72-hour time point, the proliferative activity of both Eca109 and KYSE510 cell experimental groups showed a significant decrease compared with that of the control group, and statistical analysis showed that the difference was significant (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eJ). These results confirmed that down-regulation of BRD4 gene expression could significantly inhibit the proliferative ability of ESCC cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eIn ESCC cell lines, Notch3 is a silencer of BRD4\u003c/h2\u003e\n\u003cp\u003eAnalysis through the ChIP-Atlas database revealed two binding sites for RBPJ in the BRD4 intronic region (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA) and one binding site for BRD4 on the Notch3 sequence (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). In order to investigate the regulatory relationship between BRD4 and Notch3, ChIP-qPCR experiments were performed in this study, and the results showed that Notch3 could bind to the DNA fragment of BRD4 gene with a statistically significant difference; while the copy number of the DNA fragment of the BRD4 gene bound to the control IgG was much lower than the copy number of the DNA fragment of the BRD4 gene bound to Notch3, excluding the effect of non-specific binding of antibodies on this experiment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC-\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\n\u003cp\u003eNext, ESCC cell lines Eca109 and KYSE510 were transfected with shNotch3 to knock down the Notch3 gene expression. Transfection of shScramble was used as the control group, and transfection of shNotch3 was used as the experimental group. The qRT-PCR results showed that the transcript level of Notch3 was down-regulated and that of BRD4 was up-regulated in Eca109 and KYSE510 cells of the experimental group compared with the control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). Eca109 and KYSE510 were transfected with the intracellular activation fragment of Notch3, NICD3, to overexpress the Notch3 gene. qRT-PCR results showed that the transcript level of Notch3 was significantly enhanced in Eca109 and KYSE510 cells, while the transcript level of BRD4 was significantly decreased (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG). The results of WB showed that the Notch3 and NICD3 protein blots were thickened in Eca109 and KYSE510 cells of the experimental group, indicating successful overexpression of Notch3. Meanwhile, BRD4 expression level was down-regulated in the experimental group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH). The results suggested that there was an interaction between BRD4 and Notch3, and when Notch3 was knocked down, the expression level of BRD4 was up-regulated; while when Notch3 was overexpressed, the expression level of BRD4 was down-regulated, which might suggest the existence of a negative feedback mechanism between BRD4 and Notch3.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003eNotch3 regulates ESCC apoptosis by silencing BRD4\u003c/h2\u003e\n\u003cp\u003eIn order to further investigate the regulatory mechanism between BRD4 and Notch3, ChIP-qPCR experiments were performed again in this study, and it was found that when Notch3 was overexpressed, the copy number of BRD4 bound to the promoters of the MCL1 and Survivin genes was significantly reduced with statistically significant differences in Eca109 and KYSE510 cells, and the copy number of the control IgG bound to the MCL1 and Survivin gene promoter fragments had much lower copy numbers than those of MCL1 and Survivin gene promoters bound to BRD4, ruling out the effect of non-specific binding of antibodies on this experiment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). The results suggested that Notch3 could regulate ESCC cell apoptosis by silencing BRD4.\u003c/p\u003e\n\u003cp\u003eSubsequently, in this study, the two cell lines, Eca109 and KYSE510, were inoculated in 96-well plates, and three time points, 24 h, 48 h and 72 h, were set for culture. At each predetermined time point, cell proliferation activity was detected according to the kit manual procedure. The results showed that when Notch3 was overexpressed the activity of Eca109 and KYSE510 cells in the experimental group would be decreased, and the difference was significant at 72 h. When overexpressed BRD4 was added, the previous inhibitory effect of Notch3 on ESCC cells would be alleviated to a certain extent, and the difference was statistically significant (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). The results suggested that Notch3 mediated ESCC cell apoptosis partly by antagonizing BRD4.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eNotch3 is highly expressed in ESCC\u003c/h2\u003e\n\u003cp\u003eNext, the expression of Notch3 in ESCC was explored in this section. qRT-PCR results showed that the transcript levels of Notch1-4 were higher than those of normal esophageal mucosal epithelium in the four cell lines of ESCC, but only Notch3 was statistically significant in the ESCC cell lines (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Subsequently, in this section, paraffin specimens of 179 ESCC patients from the Second Affiliated Hospital of Nantong University were collected, and a total of 179 ESCC tissues and 52 paracancerous normal esophageal tissues were obtained, which were fabricated into tissue microarrays and stained with immunohistochemistry. By comparing and analyzing the expression levels of Notch3 in ESCC tissues and normal esophageal mucosal epithelial tissues, it was found that Notch3 showed significantly up-regulated expression in cancerous tissues, whereas the expression level was significantly reduced in normal tissues next to the cancer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Immunohistochemical staining showed that the positive signals were mainly manifested as brownish-yellow to blackish-brown granular deposits in the cytoplasm, and positive signals were also seen in the nucleus of a few cells. The results indicated that Notch3 protein was mainly localized in the cytoplasm, and only a small amount was expressed in the nuclear region. The results of the biochemical analysis also yielded the same results as immunohistochemistry. Analysis of the TCGA database revealed that Notch3 expression levels were highly expressed in esophageal cancer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). In addition, high Notch3 expression was correlated with advanced tumor grade and cancer stage (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). And Notch3 expression was higher in ESCC than in ECA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF). In WB experiments, the expression level of Notch3 in four ESCC cell lines was much higher than that in normal esophageal mucosal epithelial cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG). The results indicated that Notch3 was highly expressed in ESCC.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003eRelationship between Notch3 expression and clinicopathologic parameters in ESCC patients\u003c/h2\u003e\n\u003cp\u003eNext, this section analyzed the relationship between Notch3 expression and clinicopathological parameters in ESCC patients. The expression of Notch3 was classified into low and high expression groups according to the optimal cutoff value using X-tile,and statistical analysis was performed using \u0026chi;\u003csup\u003e2\u003c/sup\u003e test. The results showed that the expression of Notch3 was only correlated with the degree of tumor differentiation (\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;12.666, P\u0026thinsp;=\u0026thinsp;0.000), while it was not correlated with age, gender, tumor site, TNM stage, clinical stage, nerve invasion and intravascular cancer thrombus(Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eRelationship between Notch3 expression and clinicopathologic parameters in ESCC patients\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth colspan=\"2\" rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eChatacteristic\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ePatients\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;179)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eNotch3\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLow\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;95)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eHigh\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;84)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e\u0026chi;\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eP value\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" 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\" char=\".\"\u003e\n\u003cp\u003e0.300\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.584\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;66\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e92(51.40%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4(51.08%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e45(48.91%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;66\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e87(48.60%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e48(55.17%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e39(44.83%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eGender\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\" char=\".\"\u003e\n\u003cp\u003e0.171\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.679\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFemale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e43(24.02%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e24(55.81%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19(44.19%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e136(75.98%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e71(52.21%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e65(47.79%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eLocation\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\" char=\".\"\u003e\n\u003cp\u003e1.470\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.225\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUpper\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e13(7.26%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9(69.23%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4(30.77%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMiddle and lower\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e166(92.74%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e86(51.81%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e80(48.19%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eT stage\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\" char=\".\"\u003e\n\u003cp\u003e1.161\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.281\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT1\u0026thinsp;+\u0026thinsp;T2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e63(35.20%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30(47.62%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e33(52.38%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT3\u0026thinsp;+\u0026thinsp;T4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e116(64.80%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e65(56.03%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51(43.97%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eN stage\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\" char=\".\"\u003e\n\u003cp\u003e0.821\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.365\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eN0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98(54.75%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e49(50.00%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e49(50.00%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eN1\u0026thinsp;+\u0026thinsp;N2\u0026thinsp;+\u0026thinsp;N3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e81(45.25%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e46(56.79%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35(43.21%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eM stage\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\" char=\".\"\u003e\n\u003cp\u003e0.283\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.595\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e170(94.97%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e91(53.53%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e79(46.47%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9(5.03%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4(44.44%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5(55.56%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eClinical stage\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\" char=\".\"\u003e\n\u003cp\u003e1.683\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.195\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eⅠ+Ⅱ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e106(59.22%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e52(49.06%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e54(50.94%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eⅢ+Ⅳ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e73(40.78%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e43(58.90%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30(41.10%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eDifferentiation\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\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e12.666\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.000\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHigh\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35(19.55%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e28(80.00%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7(20.00%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMedium and low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e144(80.45%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e67(46.53%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e77(53.47%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ePerineural 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\" char=\".\"\u003e\n\u003cp\u003e0.619\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.431\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e143(79.89%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e78(54.55%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e65(45.45%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e36(20.11%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e17(47.22%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19(52.78%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIntravascular tumor thrombus\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\" char=\".\"\u003e\n\u003cp\u003e0.329\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.566\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e146(81.56%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e76(52.05%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e70(47.95%)\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\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e33(18.44%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19(57.58%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14(42.42%)\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\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n\u003ch2\u003eIn ESCC cell lines, JQ1 treatment inhibits Notch3 expression\u003c/h2\u003e\n\u003cp\u003eNext, this section explored the effect of BRD4 inhibitor JQ1 on Notch3. JQ1 was added to Eca109 and KYSE510 cells, and the transcript levels of Notch3 were measured at 0 h, 3 h, 6 h, 12 h, and 24 h. The qRT-PCR results showed that in Eca109 cells, the transcript level of Notch3 was decreased after 3 h; in KYSE510 cells, the transcript level of Notch3 was decreased, while by 24 h Notch3 transcript levels were up-regulated(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Subsequent immunofluorescence experiments in this study revealed that Notch3 was mainly localized in the cytoplasm and exhibited green fluorescence, which was significantly attenuated in the presence of JQ1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eIn order to further explore how JQ1 regulates Notch3 expression in ESCC, the dual luciferase reporter gene system was used in this study. Two ESCC cell lines, Eca109 and KYSE510, were selected for the experiment, first transfected with pGL3-Basic-Hes1 recombinant plasmid, and then cultured for 48 hours before adding JQ1 treatment. Seventy-two hours after transfection, the fluorescence intensity of firefly luciferase and sea kidney luciferase was measured separately using an enzyme marker and the ratio was calculated. The experimental data showed that the relative luciferase activity of the JQ1-treated group was significantly down-regulated compared with the control group transfected with pGL3-Basic-Hes1 alone, suggesting that JQ1 can effectively inhibit the transcriptional activity of the Hes1 promoter (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). Hes1 is a downstream target gene of Notch3, and the decrease in his activity indicates a decrease in the activity of the upstream signaling pathway, suggesting that JQ1 will inhibit the Notch3 expression in ESCC when BRD4 is inhibited.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n\u003ch2\u003eIn vivo JQ1 co-overexpression of Notch3 inhibits ESCC cell growth\u003c/h2\u003e\n\u003cp\u003eIn order to further investigate the effect of the regulatory interaction between BRD4 and Notch3 on the proliferative effects of ESCC cells, nude mice tumorigenic experiments were performed in this study. The mice were randomly divided into three groups, one was the control group of pCDNA5\u0026thinsp;+\u0026thinsp;DMSO, and the other two groups were the experimental groups of pCDNA5\u0026thinsp;+\u0026thinsp;JQ1 and pCDNA5-3\u0026times;flag-NICD3\u0026thinsp;+\u0026thinsp;JQ1, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). The experimental results showed that compared with the control group, the tumor volume growth of the pCDNA5\u0026thinsp;+\u0026thinsp;JQ1 experimental group was slowed down, and the size of the tumor could be observed to be smaller than that of the control group on day 5, and all of them were smaller than that of the control group after 12 days; whereas, the tumor volume growth of the pCDNA5-3\u0026times;flag-NICD3\u0026thinsp;+\u0026thinsp;JQ1 experimental group was slowed down significantly, and the tumor size could be observed to be smaller than that of the control group on day 3, and all of them were smaller than that of the control group after 12 days later were smaller than the control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). The results suggest that in vivo JQ1 co-overexpression of Notch3 can inhibit the proliferation of ESCC cells, which provides a new direction for targeted therapy for ESCC patients.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEsophageal cancer is the seventh leading cause of death from malignancy worldwide, and will continue to cause a high number of new cases and deaths in 2022, representing a considerable burden on global health. ESCC is the leading histological form of esophageal cancer in China, accounting for around 90% of all forms. High-risk groups for ESCC include people aged 40 and over who live in high-incidence areas, or who have a history of cancer, a family history of esophageal cancer, or other risk factors such as smoking, alcohol abuse, a history of squamous cell carcinoma of the head and neck, or a preference for spicy and canned foods. For these individuals, endoscopy combined with biopsy of the suspected area is the recommended screening method to reduce mortality from ESCC. Thanks to revolutionary advances in screening, surgery and new treatments, the prognosis of ESCC has improved considerably, and combination therapies (e.g. surgery, chemotherapy, radiotherapy) have proved effective in treating locally advanced esophageal cancer, with immunotherapy now an important therapeutic option\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Despite some therapeutic advances, we still know little about the mechanisms of ESCC development.\u003c/p\u003e\u003cp\u003eAs ESCC is a highly lethal type of cancer, its development involves complex molecular mechanisms. BRD4, a member of the BET family, plays a key role in ESCC. Numerous studies have shown that BRD4 transcript and protein expression levels are significantly elevated in the tissues of ESCC patients. In Zhang's study, we found that inhibition of BRD4 function by genetic means (e.g. RNA interference) or by specific BRD4 inhibitors (e.g. JQ1) significantly suppressed ESCC cell proliferation in vitro and tumor growth in vivo. Mechanistically, BRD4 can recruit transcriptional complexes to the RCC2 promoter region by interacting with the key transcription factor TP73 and regulate its transcription, which in turn affects the biological behavior of ESCC cells\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we found that BRD4 expression was significantly higher in tumor tissue than in normal esophageal cancer tissue by analyzing the TCGA database, and that high BRD4 expression correlated with high tumor grade and cancer stage, as well as metastasis to esophageal cancer lymph nodes. We also found that BRD4 expression was significantly higher in ESCC than in EAC, consistent with Zhang's study. After analyzing the database, we decided to investigate how BRD4 regulates ESCC cell growth and survival, and whether BRD4 might be linked to proteins that inhibit apoptosis. To test this hypothesis, we analyzed the ChIP-Atlas database and discovered that BRD4 has a binding site on the promoter sequences of MCL1 and survivin, respectively. The results of subsequent ChIP-qPCR experiments showed that BRD4 does indeed bind to the promoters of MCL1 and survivin, and when BRD4 was inactivated, MCL1 and survivin expression also decreased, and the growth activity of ESCC cells was suppressed. The results suggest that BRD4 may promote tumor growth by regulating MCL1 and Survivin and thus inhibiting apoptosis in ESCC cells.\u003c/p\u003e\u003cp\u003eOne of the reasons for the poor treatment and prognosis of ESCC, one of the most aggressive and lethal malignancies, is that the phenotypic transformation of epithelial cells in cancer can lead to resistance to chemotherapy. Epithelial-mesenchymal transformation in cancer is linked to tumor formation, invasion, proliferation, metastasis and resistance to stressors such as anticancer drugs, radiation and hypoxia. The Notch signaling pathway regulates cell growth and differentiation processes in response to the environment. When the Notch receptor binds its ligand, it triggers translocation of the Notch intracellular activating fragment, NICD, into the nucleus, where it forms a transcriptional activator complex with the transcription factor RBPJ and regulates expression of target genes of the Hes/HEY family\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. NICD is an important part of Notch signaling pathway activation. Liu's article suggests that Icariin (ICA) suppresses Notch2 mRNA expression by inhibiting the action of NICD1\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. This provides us with new ideas as to whether some conventional drugs can also enhance Notch3 expression by acting on NICD3 and thus achieve therapeutic effects for ESCC, while largely reducing the side effects of using JQ1\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe Notch signaling pathway is thought to be important for the differentiation of esophageal epithelium, as the intracellular activated part of Notch1, NICD1, directly activates Notch3 transcription, resulting in squamous differentiation of epithelial cells. In addition to squamous cell differentiation, Notch1 also regulates the cell cycle, senescence and phenotypic transformation of epithelial cells. Notch1 is a positive EMT effector\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e, but Notch3 limited the ability of EMT to expand esophageal keratinocytes. Thus, although Notch1 and Notch3 cooperatively stimulate squamous cell differentiation, these Notch homologs may play opposing roles in the phenotypic transformation of squamous cells and, unlike Notch1, Notch3 can limit the phenotypic transformation of squamous cells, allowing them to differentiate normally\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e, suggesting that Notch3 may play an oncogenic role in ESCC.\u003c/p\u003e\u003cp\u003eBy analyzing through the ChIP-Atlas database, it was found that RBPJ has two binding sites in the intronic region of BRD4, and BRD4 has one binding site on the sequence of Notch3, suggesting that there may be a regulatory relationship between BRD4 and Notch3. To investigate this relationship, ChIP-qPCR experiments were performed in this study, and the results showed that Notch3 could bind to the DNA fragment of the BRD4 gene. When Notch3 was knocked down, qRT-PCR showed that the transcript level of BRD4 was up-regulated; while when NICD3 was overexpressed, the transcript level of BRD4 was down-regulated compared with the control group. WB results also showed that the expression level of BRD4 was down-regulated. The results suggested that BRD4 interacted with Notch3, and when Notch3 was knocked down, the expression level of BRD4 was up-regulated; whereas when Notch3 was overexpressed, the expression level of BRD4 was down-regulated, suggesting that Notch3 may have a repressive effect on BRD4. In order to further investigate the regulatory mechanism between BRD4 and Notch3, ChIP-qPCR experiments were performed again in this study, and it was found that when Notch3 was overexpressed, the copy number of BRD4 with the promoters of the MCL1 and Survivin genes was significantly reduced. CCK8 experiments showed that the activity of the tumor cells would be decreased when Notch3 was overexpressed; and when overexpressed BRD4, the previous inhibitory effect of Notch3 on ESCC cells would be alleviated to some extent, but the cell activity was still lower than normal. These results suggest that Notch3 can mediate apoptosis in ESCC cells by inhibiting BRD4 and thus affecting the expression of MCL1 and Survivin.\u003c/p\u003e\u003cp\u003eWe next found that Notch3 was highly expressed in ESCC by database analysis, immunohistochemistry, and WB experiments, and that clinicopathological features showed a significant correlation between the expression level of Notch3 and the degree of differentiation of the tumor tissue only. However, this index did not show statistically significant associations with clinicopathological characteristics such as patient age, gender, site of tumor occurrence, TNM stage, clinical stage, nerve infiltration, and vascular cancer embolism. Previous findings have shown that Notch3 inhibits BRD4, so next we wanted to explore how BRD4 acts on Notch3 and whether there is a negative feedback mechanism between the two. By qRT-PCR, immunofluorescence and dual-luciferase reporter gene assay, the results suggested that JQ1 inhibits the expression of Notch3 in ESCC, and when BRD4 is inhibited, the expression of Notch3 in ESCC also decreases, which, combined with the results of the previous studies, suggests that there is a negative feedback mechanism between BRD4 and Notch3, and Notch3 inhibits ESCC cells by negatively regulating the expression of BRD4. BRD4 to inhibit ESCC cell growth and survival.\u003c/p\u003e\u003cp\u003eAlejandro has shown that BRD4 mRNA expression is highly upregulated in ovarian cancer, and TCGA analysis has also shown that increased BRD4 expression in ovarian cancer is associated with a significant reduction in survival. Notch3 has been shown to be highly upregulated in ovarian cancer and associated with increased tumor cell proliferation, increased resistance to chemotherapy and decreased survival. Alejandro found that Notch3 expression and BRD4 expression are indeed closely linked in ovarian cancer, and that treatment with BRD4 inhibitors reduces Notch3 expression in vitro and in vivo, while BRD4 inhibition also reduces the expression of Notch3 targets, including Hes1\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. This is consistent with our findings that JQ1 can inhibit Notch3 expression. Subsequently, we conducted in vivo experiments and constructed patient-derived xenograft (PDX) models, which are widely used for evaluating tumor behavior, therapeutic response, and metastatic potential in vivo. PDX models offer a more physiologically relevant tumor microenvironment compared to traditional cell line xenografts, preserving patient-specific histological and molecular characteristics. Prior research has successfully leveraged such models to explore diverse aspects of cancer biology\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. The results showed that the tumor volume growth was slowed down by adding JQ1 alone; however, the tumor volume growth was significantly slowed down by combining with NICD3. The results suggest that in vivo JQ1 combined with overexpression of Notch3 can more effectively inhibit the proliferative ability of ESCC cells, which provides a new direction for targeted therapy for ESCC patients.\u003c/p\u003e\u003cp\u003eThe following shortcomings exist in this study: first, this study failed to collect fresh tissue specimens to explore the expression of BRD4 and Notch3 in ESCC; second, the specific molecular mechanism of Notch3 negative feedback regulation of BRD4 has not been investigated, and further refinement is needed next.\u003c/p\u003e\u003cp\u003eIn summary, this study initially revealed the expression of BRD4 and Notch3 in ESCC and the molecular mechanism regulating the growth and survival of ESCC cells. Notch3 negatively feedback regulates BRD4 and thus inhibits the growth and survival of ESCC cells. Therefore, the use of BRD4 inhibitors in combination with overexpression of Notch3 may become a new option for the treatment of ESCC, bringing new hope for the treatment of ESCC patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor\u0026apos;s contribution\u003c/strong\u003e HL designed the study. XQ and HY performed the study and wrote the paper. YW and TZ analyzed the data. HZ and CS staged the patients for pTNM and scored the immunohistochemical staining results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This study was supported by Nantong Science and Technology Project (JC2021001) and Nantong Social Livelihood Science and Technology Program (MSZ2023099)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e All data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The Authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eThis study and included experimental procedures were approved by the institutional animal care and use committee of Nantong University. All animal housing and experiments were conducted in strict accordance with the institutional guidelines for care and use of laboratory animals. The study is reported in accordance with ARRIVE guidelines. The study confirmed that informed consent was obtained from all subjects and/or their legal guardians. The study was conducted with the approval of the appropriate ethics committee of the Second Affiliated Hospital of Nantong University(IRB No. 2021KT057). The investigation conformed to the principles outlined in the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u003c/strong\u003e This article is available under a Creative Commons Attribution-NonCommercial 4.0 International License, which permits any non-commercial use, sharing, adaptation, distribution, and reproduction in any medium or format, provided the author and source are credited, a Creative Commons license is cited, and changes are indicated. Images or other third-party material in this article are subject to a Creative Commons license for the article unless otherwise stated in the article credits. If the material is not covered by a Creative Commons license for an article and the intended use is not permitted by law or regulation or beyond fair use, a license must be obtained directly from the copyright holder.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSonkin, D., Thomas, A. \u0026amp; Teicher, B. A. Cancer treatments: Past, present, and future[J]. Cancer Genet, 286\u0026ndash;287: pp. 18-2410.1016/j.cancergen.2024.06.002. (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu, H. \u0026amp; Dilger, J. P. Different strategies for cancer treatment: Targeting cancer cells or their neighbors?[J]. Chin J Cancer Res, 37(2): pp. 289-29210.21147/j.issn.1000-9604.2025.02.12. (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBray, F. et al. 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(2024).\u003c/span\u003e\u003c/li\u003e\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":"BRD4, Notch3, esophageal squamous cell carcinoma, growth and survival","lastPublishedDoi":"10.21203/rs.3.rs-7554734/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7554734/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective \u003c/strong\u003eThe aim of this study was to investigate the expression of bromodomain-containing protein 4 (BRD4) and Notch receptor protein 3 (Notch3) in esophageal squamous cell carcinoma (ESCC) and the effect of their interaction on the growth and survival of ESCC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e The expression of target genes in ESCC and the binding sites between genes were analyzed by TCGA and ChIP-Atlas databases. The expression levels, interactions and effects of target genes on tumor cell activity in ESCC were detected by immunohistochemical staining methods, WB, qRT-PCR, ChIP-qPCR and CCK-8 assays. Dual luciferase reporter gene assays were performed to investigate the regulation between target genes and the signaling mechanism. In vivo experiments were performed to further explore the regulatory effects of target genes on ESCC cell growth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e BRD4 was highly expressed in ESCC. BRD4 regulated ESCC cell apoptosis by modulating MCL1 and Survivin. In ESCC cell lines, Notch3 silenced BRD4. Notch3 mediated apoptosis in ESCC cells partly by antagonizing BRD4. Notch3 was highly expressed in ESCC tissues. Notch3 expression correlated with the degree of tumor differentiation. When BRD4 was inhibited in ESCC cells, Notch3 expression was also reduced. In vivo, inhibition of BRD4 combined with overexpression of Notch3 better inhibited ESCC cell growth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e Notch3 negatively feedback regulates BRD4 thereby inhibiting ESCC cell growth and survival. The use of BRD4 inhibitors in combination with overexpression of Notch3 may be better highlight the clinical implication.\u003c/p\u003e","manuscriptTitle":"Molecular mechanism and clinical significance of Notch3 negative regulation of BRD4 on the growth and survival of esophageal squamous cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 18:37:39","doi":"10.21203/rs.3.rs-7554734/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":"f3caa0b2-3a46-42ff-b764-10e2f6c6a163","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55798022,"name":"Biological sciences/Cancer"},{"id":55798023,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2026-04-09T09:09:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 18:37:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7554734","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7554734","identity":"rs-7554734","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}