Objective To screen antigen targets for immunotherapy by analyzing over-expressed genes, and to identify significant pathways and molecular mechanisms in esophageal cancer by using bioinformatic methods such as enrichment analysis, protein-protein interaction (PPI) network, and survival analysis based on the Gene Expression Omnibus (GEO) database.Methods By screening with highly expressed genes, we mainly analyzed proteins MUC13 and EPCAM with transmembrane domain and antigen epitope from TMHMM and IEDB websites. Significant genes and pathways associated with the pathogenesis of esophageal cancer were identified using enrichment analysis, PPI network, and survival analysis. Several software and platforms including Prism 8, R language, Cytoscape, DAVID, STRING, and GEPIA platform were used in the search and/or figure creation.Results Genes MUC13 and EPCAM were over-expressed with several antigen epitopes in esophageal squamous cell carcinoma (ESCC) tissue. Enrichment analysis revealed that the process of keratinization was focused and a series of genes were related with the development of esophageal cancer. Four genes including ALDH3A1, C2, SLC6A1,and ZBTB7C were screened with significant P value of survival curve.Conclusions Genes MUC13 and EPCAM may be promising antigen targets or biomarkers for esophageal cancer. Keratinization may greatly impact the pathogenesis of esophageal cancer. Genes ALDH3A1, C2, SLC6A1,and ZBTB7C may play important roles in the development of esophageal cancer.
Humans ; Esophageal Neoplasms/metabolism* ; Esophageal Squamous Cell Carcinoma/metabolism* ; Epithelial Cell Adhesion Molecule/metabolism* ; Gene Expression Profiling/methods* ; Gene Regulatory Networks ; Gene Expression ; Gene Expression Regulation, Neoplastic ; Intracellular Signaling Peptides and Proteins

Objective To investigate the effects of natural killer (NK)-cell-derived miR-30e-3p-containing exosomes (Exo) on esophageal squamous cell carcinoma (ESCC) cell proliferation, apoptosis and invasion. Methods NK cells were isolated and amplified from the peripheral blood of healthy donors, and NK cell-derived Exo was isolated and identified, which were further co-cultured with NEC cells and were randomly grouped into Exo1 and Exo2 groups. Transmission electron microscopy (TEM) was used to observe the morphology and size of exosomes. Western blot analysis was used to detect the expression levels of exosome markers apoptosis related gene 2- interacting protein X(ALIX), tumor susceptibility gene 101(TSG101), CD81 and calnexin. The NC plasmids, mimics and inhibitors of miR030e-3p were respectively delivered into the NK cells, and the corresponding NK cells-derived Exo were co-cultured with NEC cells, which were divided into NC, Exo, mimic and inhibitor groups. CCK-8 assay was used to evaluate cell proliferation, flow cytometry was conducted to determine cell cycle, annexin V-FITC/PI double staining was employed to detect cell apoptosis, and Transwell^TM assay was performed to detect cell invasion abilities. Real-time quantitative PCR was used to detect the expression of miR-23b, miR-422a, miR-133b, miR-124, miR-30e-3p and miR-99a in NCE cells and exosomes. Results The percentages of CD56⁺CD3⁺ cells and CD56⁺CD16⁺ cells in NK cells were (0.071±0.008)% and (90.6±10.6)%, respectively. Exosome isolated from NK cells ranged from 30 nm to 150 nm, and was positive for ALIX, TSG101 and CD81, while negative for calnexin. NK cell-derived Exos inhibited the proliferation, reduced the proportion of S-phase cells and the number of invaded cells of NEC cells, and promoted the apoptosis and the proportion of G1 phase cells. Overexpression of miR-30E-3p in NK cell-derived exosome inhibited the proliferation and invasion of NEC cells, and blocked cell cycle and promoted apoptosis, while knockdown miR-30e-3p in NK cell-derived exosomes did the opposite. Conclusion miR-30e-3p in NK cell-derived exosomes can inhibit the proliferation and invasion of ESCC cells, block their cell cycle and induce their apoptosis.
Humans ; Esophageal Squamous Cell Carcinoma/genetics* ; Esophageal Neoplasms/genetics* ; Exosomes/metabolism* ; Calnexin/metabolism* ; Cell Movement/genetics* ; MicroRNAs/metabolism* ; Cell Proliferation/genetics* ; Killer Cells, Natural ; Cell Line, Tumor ; Apoptosis/genetics*

Treating patients with esophageal squamous cell carcinoma (ESCC) is challenging due to the high chemoresistance. Growth differentiation factor 15 (GDF15) is crucial in the development of various types of tumors and negatively related to the prognosis of ESCC patients according to our previous research. In this study, the link between GDF15 and chemotherapy resistance in ESCC was further explored. The relationship between GDF15 and the chemotherapy response was investigated through in vitro and in vivo studies. ESCC patients with high levels of GDF15 expression showed an inferior chemotherapeutic response. GDF15 improved the tolerance of ESCC cell lines to low-dose cisplatin by regulating AKT phosphorylation via TGFBR2. Through an in vivo study, we further validated that the anti-GDF15 antibody improved the tumor inhibition effect of cisplatin. Metabolomics showed that GDF15 could alter cellular metabolism and enhance the expression of UGT1A. AKT and TGFBR2 inhibition resulted in the reversal of the GDF15-induced expression of UGT1A, indicating that TGFBR2-AKT pathway-dependent metabolic pathways were involved in the resistance of ESCC cells to cisplatin. The present investigation suggests that a high level of GDF15 expression leads to ESCC chemoresistance and that GDF15 can be targeted during chemotherapy, resulting in beneficial therapeutic outcomes.
Humans ; Esophageal Squamous Cell Carcinoma/drug therapy* ; Cisplatin/metabolism* ; Esophageal Neoplasms/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Carcinoma, Squamous Cell/genetics* ; Growth Differentiation Factor 15/therapeutic use* ; Receptor, Transforming Growth Factor-beta Type II/therapeutic use* ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic

Objective: To investigate the effect of acetyl-CoA carboxylase 1 (ACC1) knockdown on the migration of esophageal squamous cell carcinoma (ESCC) KYSE-450 cell and underlying mechanism. Methods: Lentiviral transfection was conducted to establish sh-NC control cell and ACC1 knocking down cell (sh-ACC1). Human siRNA HSP27 and control were transfected by Lipo2000 to get si-HSP27 and si-NC. The selective acetyltransferase P300/CBP inhibitor C646 was used to inhibit histone acetylation and DMSO was used as vehicle control. Transwell assay was performed to detect cell migration. The expression of HSP27 mRNA was examined by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and the expressions of ACC1, H3K9ac, HSP27 and epithelial-mesenchymal transition-related proteins E-cadherin and Vimentin were detected by western blot. Results: The expression level of ACC1 in sh-NC group was higher than that in sh-ACC1 group (P<0.01). The number of cell migration in sh-NC group was (159.00±24.38), lower than (361.80±26.81) in sh-ACC1 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC group were statistically significant compared with sh-AAC1 group (P<0.05). The migrated cell number in sh-NC+ si-NC group was (189.20±16.02), lower than (371.60±38.40) in sh-ACC1+ si-NC group (P<0.01). The migrated cell number in sh-NC+ si-NC group was higher than that in sh-NC+ si-HSP27 group (152.40±24.30, P<0.01), and the migrated cell number in sh-ACC1+ si-NC group was higher than that in sh-ACC1+ si-HSP27 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC+ si-NC group were significantly different from those in sh-ACC1+ si-NC and sh-NC+ si-HSP27 groups (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-ACC1+ si-NC group were significantly different from those in sh-ACC1+ si-HSP27 group (P<0.01). After 24 h treatment with C646 at 20 μmmo/L, the migrated cell number in sh-NC+ DMSO group was (190.80±11.95), lower than (395.80±17.10) in sh-ACC1+ DMSO group (P<0.01). The migrated cell number in sh-NC+ DMSO group was lower than that in sh-NC+ C646 group (256.20±23.32, P<0.01). The migrated cell number in sh-ACC1+ DMSO group was higher than that in sh-ACC1+ C646 group (87.80±11.23, P<0.01). The protein expressions of H3K9ac, HSP27, E-cadherin and Vimentin in sh-NC+ DMSO group were significantly different from those in sh-ACC1+ DMSO group and sh-NC+ C646 group (P<0.01). The protein expression levels of H3K9ac, HSP27, E-cadherin and Vimentin in sh-ACC1+ DMSO group were significantly different from those in sh-ACC1+ C646 group (P<0.01). Conclusion: Knockdown of ACC1 promotes the migration of KYSE-450 cell by up-regulating HSP27 and increasing histone acetylation.
Humans ; Esophageal Neoplasms/pathology* ; Esophageal Squamous Cell Carcinoma/genetics* ; Vimentin/metabolism* ; Dimethyl Sulfoxide ; HSP27 Heat-Shock Proteins/metabolism* ; Histones/metabolism* ; Cadherins/metabolism* ; Cell Movement ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic

Objective: To explore the function and mechanism of long non-coding RNA MIR503HG in esophageal squamous cell carcinoma (ESCC). Methods: The MIR503HG expression data in 60, 119 and 23 cases of ESCC and their paired adjacent tissues were chosen from three ESCC datasets GSE53622, GSE53624 and GSE130078, respectively. The expression data of MIR503HG in 81 ESCC tissues and 271 unpaired normal esophageal tissues were screened from the combined dataset of Cancer Genome Atlas and Genotype-Tissue Expression Database (TCGA+ GTEx). The MIR503HG knockdown plasmid was constructed, packaged into lentivirus. The lentivirus was used to infect with esophageal squamous cell carcinoma cell lines KYSE30 and KYSE510 to screen out the stable MIR503HG knockdown cell lines. ESCC cell line KYSE30 was transiently transfected with miRNA mimics to overexpress hsa-miR-503-3p and hsa-miR-503-5p.The expression levels of MIR503HG, hsa-miR-503-3p and hsa-miR-503-5p were detected by quantitative real-time polymerase chain reaction. The proliferation ability of the cells was detected by cell counting kit 8 and clone formation assay. The invasion and migration ability of the cells were detected by Transwell assay. Cell cycle was detected by flow cytometry. The effect of MIR503HG on the proliferation of ESCC was detected by xenograft experiment in BALB/c-nu/nu mice. Results: Both GEO and TCGA+ GTEx databases showed that the expression of MIR503HG in ESCC tissues was higher than that in adjacent tissues and normal esophageal tissues (P<0.01). Compared with shNC group, the proliferation rates of KYSE30 and KYSE510 cells after knockdown of MIR503HGwere significantly inhibited (P<0.001). The colony formation numbers of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were (2.00±1.41) and (1.33±0.47), respectively, significantly lower than that of the shNC group (P=0.002). The clone formation numbers of KYSE510 cells in shMIR503HG1 group and shMIR503HG2 group were (174.67±15.97) and (80.33±6.34), respectively, significantly lower than that of the shNC group (P<0.001). The invasive numbers of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were 75.33±6.02 and 45.67±7.59, significantly lower than that of the shNC group(P<0.001). The migrating number of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were 244.00±10.23 and 210.67±13.52, significantly lower than that of the shNC group(P<0.001), and the cell cycle was arrested in G(0)/G(1) phase. The xenograft experiment showed that the subcutaneous tumor in shMIR503HG group was significantly smaller than that in shNC group, and the tumor weight in shMIR503HG group was (0.097±0.026) g, which was lower than (0.166±0.021) g in shNC group (P<0.001). After knockdown of MIR503HG, the relative expression levels of hsa-miR-503-3p in KYSE30 cells of shMIR503HG1 group and shMIR503HG2 group were 0.66±0.02 and 0.58±0.00, respectively, the relative expression levels of hsa-miR-503-5p were 0.64±0.00 and 0.68±0.03, respectively, which were all lower than those in shNC group (P<0.01). After knockdown of MIR503HG, overexpression of hsa-miR-503-3p and hsa-miR-503-5p attenuated the inhibitory effects of knockdown of MIR503HG on proliferation (P<0.001), invasion (P<0.01) and migration (P<0.001) of KYSE30 cells. Conclusions: MIR503HG promotes the proliferation, invasion and migration of ESCC cells by regulating hsa-miR-503 pathway and can be used as a new potential target for targeted therapy of ESCC.
Animals ; Humans ; Mice ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Esophageal Neoplasms/pathology* ; Esophageal Squamous Cell Carcinoma/pathology* ; Gene Expression Regulation, Neoplastic ; Mice, Nude ; MicroRNAs/metabolism*

OBJECTIVE: To investigate the expression of MC1R in esophageal squamous cell carcinoma and its correlation with the clinicopathological parameters. METHODS: We analyzed the expression of MC1R in esophageal cancer based on data from TCGA databse and examined its expression levels using RT-PCR and Western blotting in a human esophageal epithelial cell line BAr-T, human esophageal squamous cell carcinoma cell lines ECA109, KYSE30, KYSE150, KYSE510, TE-1, TE-13, and EC9706, a human gastric cancer cell line SGC7901 and 19 pairs of esophageal squamous cell carcinoma tissues and adjacent tissues.Immunohistochemistry was used to detect MC1R expression levels in 32 pairs of paraffin-embedded sections of esophageal squamous cell carcinoma and adjacent tissues, and the correlation of MC1R expression and the patients'clinicopathological characteristics was analyzed. RESULTS: Bioinformatics analysis showed that MC1R was significantly overexpressed in esophageal cancer tissues (P < 0.05).MC1R expression was also increased in 5 esophageal squamous cell carcinoma cell lines ECA109, KYSE30, KYSE510, TE-13, EC9706 and the gastric cancer cell line SGC7901 as compared with that in esophageal epithelial cells (P < 0.05).Immunohistochemistry revealed significantly increased MC1R expression in esophageal squamous cell carcinoma tissue sections in comparison with the adjacent tissue sections (P < 0.05).In patients with esophageal squamous cell carcinoma, a high MC1R expression was detected mainly in those with an old age, positive for middle-thoracic involvement, and with moderately differentiated tumor cells, and showed a correlation with T stage of tumor (P < 0.05), but not with the other clinicopathological parameters such as gender, age, degree of cell differentiation, primary tumor site, or TNM stage (P>0.05). CONCLUSION MC1R is highly expressed in esophageal squamous cell carcinoma and may serve as a molecular biomarker to assist in the diagnosis of esophageal squamous cell carcinoma.
Humans ; Esophageal Squamous Cell Carcinoma ; Esophageal Neoplasms/metabolism* ; Stomach Neoplasms ; Cell Line, Tumor ; Immunohistochemistry ; Cell Proliferation ; Gene Expression Regulation, Neoplastic

Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Esophageal Neoplasms/pathology* ; Esophageal Squamous Cell Carcinoma/genetics* ; Gene Expression Regulation, Neoplastic ; Humans ; MicroRNAs/metabolism* ; RNA, Long Noncoding/metabolism* ; Wnt Signaling Pathway/genetics*

Objective: To study the effects of dihydromyricetin (DMY) on the proliferation, apoptosis and epithelial mesenchymal transition (EMT) of esophageal squamous cell carcinoma (ESCC) cell KYSE150 and KYSE410. Methods: KYSE150 and KYSE410 cells were treated with different concentrations of DMY (0, 25, 50, 100, 150, 200 μmol/L) for 24 hours. The median inhibition concentration (IC₅₀) values of KYSE150 and KYSE410 were detected by cell counting kit-8 (CCK-8) method. Then 0.5‰ dimethyl sulfoxide (DMSO) was used as control group, dihydromyricetin (DMY), dihydromyricetin and transforming growth factor-β1 (DMY+ TGF-β1), transforming growth factor-β1 (TGF-β1) were used as experimental group. Cell proliferation and apoptosis rates were measured by clonal formation and flow cytometry. Transwell invasion and wound healing assay were used to detect cell invasion and migration. The protein expression levels of Caspase-3, Caspase-9, Bcl-2, Bax, Smad2/3, phosphorylation-Smad2/3 (p-Smad2/3) and Vimentin were detected by western blot. Results: The IC₅₀ values of DMY on KYSE410 and KYSE150 cells were 100.51 and 101.27 μmol/L. The clone formation numbers of KYSE150 and KYSE410 in DMY group [(0.53±0.03) and (0.31±0.03)] were lower than those in DMSO group [(1.00±0.10) and (1.00±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in DMY group [(1.84±0.22)% and (2.80±0.07)%] were higher than those in DMSO group [(1.00±0.18)% and (1.00±0.07)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in DMY group [(0.42±0.03) and (0.29±0.05)] were lower than those in DMSO group [(1.00±0.08) and (1.00±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in DMY group [(0.65±0.14)% and (0.40±0.17)%] were lower than those in DMSO group [(1.00±0.10)% and (1.00±0.08)%, P<0.05]. The clone formation numbers of KYSE150 and KYSE410 in TGF-β1 group [(1.01±0.08) and (0.99±0.25)] were higher than those in DMY+ TGF-β1 group [(0.73±0.10) and (0.58±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in TGF-β1 group [(0.81±0.14)% and (1.18±0.10)%] were lower than those in DMY+ TGF-β1 group [(1.38±0.22)% and (1.85±0.04)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in TGF-β1 group [(1.19±0.11) and (1.39±0.11)] were higher than those in DMY+ TGF-β1 group [(0.93±0.09) and (0.93±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in TGF-β1 group [(1.87±0.19)% and (1.32±0.04)%] were higher than those in DMY+ TGF-β1 group [(0.86±0.16)% and (0.77±0.12)%, P<0.05]. The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY group were higher than those in DMSO group, while the protein expression level of Bcl-2 was lower than that in DMSO group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in DMY group were lower than those in DMSO group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in TGF-β1 group were lower than those in DMY+ TGF-β1 group, and the protein expression level of Bcl-2 was higher than that in DMY+ TGF-β1 group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY+ TGF-β1 group were lower than those in DMY group, and the protein expression level of Bcl-2 was higher than that in DMY group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in TGF-β1 group were higher than those in DMY+ TGF-β1 group (P<0.05). Conclusion: DMY can inhibit the proliferation and EMT of ESCC mediated by TGF-β1 and promote cell apoptosis.
Apoptosis ; Caspase 3/metabolism* ; Caspase 9/metabolism* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Dimethyl Sulfoxide/pharmacology* ; Epithelial-Mesenchymal Transition ; Esophageal Neoplasms/metabolism* ; Esophageal Squamous Cell Carcinoma ; Flavonols ; Humans ; Signal Transduction ; Transforming Growth Factor beta1/pharmacology* ; Vimentin/metabolism* ; bcl-2-Associated X Protein/pharmacology*

BACKGROUND: Gene promoter methylation is a major epigenetic change in cancers, which plays critical roles in carcinogenesis. As a crucial regulator in the early stages of B-cell differentiation and embryonic neurodevelopment, the paired box 5 (PAX5) gene is downregulated by methylation in several kinds of tumors and the role of this downregulation in esophageal squamous cell carcinoma (ESCC) pathogenesis remains unclear. METHODS: To elucidate the role of PAX5 in ESCC, eight ESCC cell lines, 51 primary ESCC tissue samples, and eight normal esophageal mucosa samples were studied and The Cancer Genome Atlas (TCGA) was queried. PAX5 expression was examined by reverse transcription-polymerase chain reaction and western blotting. Cell apoptosis, proliferation, and chemosensitivity were detected by flow cytometry, colony formation assays, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays in ESCC cell lines with PAX5 overexpression or silencing. Tumor xenograft models were established for in vivo verification. RESULTS: PAX5 methylation was found in 37.3% (19/51) of primary ESCC samples, which was significantly associated with age (P = 0.007) and tumor-node-metastasis stage (P = 0.014). TCGA data analysis indicated that PAX5 expression was inversely correlated with promoter region methylation (r = -0.189, P = 0.011 for cg00464519 and r = -0.228, P = 0.002 for cg02538199). Restoration of PAX5 expression suppressed cell proliferation, promoted apoptosis, and inhibited tumor growth of ESCC cell lines, which was verified in xenografted mice. Ectopic PAX5 expression significantly increased p53 reporter luciferase activity and increased p53 messenger RNA and protein levels. A direct interaction of PAX5 with the p53 promoter region was confirmed by chromatin immunoprecipitation assays. Re-expression of PAX5 sensitized ESCC cell lines KYSE150 and KYSE30 to fluorouracil and docetaxel. Silencing of PAX5 induced resistance of KYSE450 cells to these drugs. CONCLUSIONS As a tumor suppressor gene regulated by promoter region methylation in human ESCC, PAX5 inhibits proliferation, promotes apoptosis, and induces activation of p53 signaling. PAX5 may serve as a chemosensitive marker of ESCC.
Animals ; Carcinoma, Squamous Cell/genetics* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Epithelial Cells/metabolism* ; Esophageal Neoplasms/genetics* ; Esophageal Squamous Cell Carcinoma/genetics* ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; PAX5 Transcription Factor/genetics* ; Tumor Suppressor Protein p53/genetics* ; Xenograft Model Antitumor Assays

Esophageal squamous cell carcinoma (ESCC) is a deadly malignancy with regard to mortality and prognosis, and the 5-year survival rate for all patients diagnosed with ESCC remains poor. A better understanding of the biological mechanisms of ESCC tumorigenesis and progression is of great importance to improve treatment of this disease. In this study, we demonstrated that the glutathione metabolism pathway is highly enriched in ESCC cells compared with normal esophageal epithelial cells in an in vivo mouse model. In addition, treatment with L-buthionine-sulfoximine (BSO) to deplete glutathione decreased the ESCC tumor burden in mice, thus demonstrating the critical role of glutathione metabolism in ESCC progression. BSO treatment also led to decreased cell proliferation and activation of cell apoptosis in ESCC. Finally, BSO treatment blocked NF-κB pathway activation in ESCC. Our study reveals a new pathway that regulates ESCC progression and suggests that inhibition of glutathione metabolism may be a potential strategy for ESCC treatment.
Animals ; Apoptosis ; Carcinogenesis ; Carcinoma, Squamous Cell ; Cell Proliferation ; Epithelial Cells ; Esophageal Neoplasms* ; Glutathione* ; Humans ; Metabolism* ; Mice ; Mortality ; Prognosis ; Survival Rate ; Tumor Burden