Human Esophageal Epithelial Cells Research Articles

MiR-214-3p targets Ras-related protein 14 (RAB14) to inhibit cellular migration and invasion in esophageal Cancer cells

BackgroundMicroRNA (miR)-214-3p is emerging as an important tumor suppressor in esophageal cancer. In this study, we examined the interaction between miR-214-3p and RAB14, a membrane trafficking protein shown to exert oncogenic functions in other malignancies, in esophageal cancer cells.MethodsStudies were performed in a human esophageal epithelial cell line and a panel of esophageal cancer cell lines, as well in human specimens. MiR-214-3p expression was measured by digital PCR. Biotinylated RNA pull-down and luciferase reporter assays assessed binding. The xCELLigence RTCA system measured cell migration and invasion in real time. A lentiviral expression vector was used to create an esophageal cancer cell line stably expressing miR-214-3p.ResultsMiR-214-3p expression was decreased in esophageal cancer cell lines and human specimens compared to non-malignant controls. RAB14 mRNA stability and protein expression were decreased following miR-214-3p overexpression. Binding between miR-214-3p and RAB14 mRNA was observed. Either forced expression of miR-214-3p or RAB14 silencing led to a marked decrease in cellular migration and invasion. Esophageal cancer cells stably expressing miR-214-3p demonstrated decreased growth in a subcutaneous murine model.ConclusionsThese results further support the tumor-suppressive role of miR-214-3p in esophageal cancer cells by demonstrating its ability to regulate RAB14 expression.

Abstract A013: Induced pluripotent stem cells derived from normal human esophageal epithelial cells identify transcription factor networks contributing to esophageal carcinogenesis

Abstract Esophageal cancer (EsC) is the sixth leading cause of cancer-related mortality worldwide. Esophageal squamous cell cancers (ESCC) typically arise in the upper and mid-esophagus, whereas esophageal adenocarcinomas (EAC) arise in the distal esophagus and gastroesophageal junction. Presently, epigenetic mechanisms contributing to initiation, progression, and dissemination of EsC have not been fully elucidated. The present study was undertaken to determine if induced pluripotent stem cells (iPSCs) derived from normal esophageal epithelial cells (Eso-iPSCs), EsC cell lines cultured in stemness enriched conditions, and cancer stem-like cells isolated from primary EsC specimens could be used to identify unique primitive stem-like transcription factor networks associated with epigenetic plasticity, chemoresistance, and metastatic potential of EsC cells. RNA-seq analysis demonstrated that genes differentially expressed in Eso-iPSC overlapped with transcriptome signatures in lung-iPSC (Lu-iPSC) which we previously generated to identify novel therapeutic targets in lung cancers; similar to Lu-iPSC, Eso-iPSC transcriptome signatures overlapped considerably more with small cell lung cancers (SCLC) compared to non-small cell lung cancers (NSCLC), possibly reflecting greater stemness in SCLC. We next compared transcriptome signatures of Eso-iPSC with stem-like-EAC enrichment models (cell lines and PDX). 6% and 8% of differentially expressed genes in Eso-iPSC uniquely overlapped with EAC or ESCC lines, respectively, whereas 24% were common to Eso-iPSC, EAC cells, and ESCC cells. Culturing of EAC cells on matrigel or propagation of EAC as primary patient derived xenografts increased commonalities of Eso-iPSC and EAC transcriptome signatures. Lastly, we compared transcriptomes of Eso-iPSC with Barrett’s esophagus (BE; a premalignant condition in which the squamous epithelium of the distal esophagus is replaced by metaplastic columnar epithelial cells) and EAC samples. By combining and stringently filtering transcriptome, ATAC-seq, DNA methylome, and ChIP-seq datasets for tissue- and disease-specific signatures, we identified several potentially druggable, master TFs mediating stemness/plasticity, chemoresistance, and metastatic potential which are shared between EsC lines and primary tumor specimens. Enrichment techniques promoted acquisition of more stem-like states in cultured EAC cells as reflected in multi-omic signatures of these cells relative to Eso-iPSC and primary EAC specimens. Collectively, these findings establish proof of principal for our experimental approach which is now being used to further characterize epigenetic mechanisms contributing to esophageal carcinogenesis and to identify novel targets for EsC therapy. Citation Format: Haitao Wang, Ruihong Wang, Katherine P. Prothro, Sichuan Xi, Weilong Hou, Xinwei Wu, Tuana Tolunay, Vivek Shukla, Mary R. Zhang, Stephanie J. Shiffka, Sudheer Gara, David S. Schrump. Induced pluripotent stem cells derived from normal human esophageal epithelial cells identify transcription factor networks contributing to esophageal carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A013.

Esophageal Squamous Cancer from 4NQO-Induced Mice Model: CNV Alterations.

Squamous esophageal carcinoma is a common pathological type of esophageal carcinoma around the world. The prognosis of esophageal carcinoma is usually poor and diagnosed at late stages. Recently, research suggested that genomic instability occurred in esophageal cells during the development of esophageal squamous cell carcinoma (ESCC). Identifying prognostic and specific genomic characteristics, especially at the early hyperplasia stage, is critical. Mice were given 4-nitroquinoline 1-oxide (4NQO) with drinking water to induce esophageal cancer. The immortalized human esophageal epithelial cell line (NE2) was also treated with 4NQO. We performed histologic analyses, immunofluorescence, and immunohistochemical staining to detect DNA damage at different time points. Whole-exome sequencing was accomplished on the esophagus tissues at different pathological stages to detect single-nucleotide variants and copy number variation (CNV) in the genome. Our findings indicate that all mice were tumor-forming, and a series of changes from simple hyperplasia (ESSH) to intraepithelial neoplasia (IEN) to esophageal squamous cell carcinoma (ESCC) was seen at different times. The expression of γ-H2AX increased from ESSH to ESCC. In addition, mutations of the Muc4 gene were detected throughout the pathological stages. Furthermore, CNV burden appeared in the esophageal tissues from the beginning of ESSH and accumulated more in cancer with the deepening of the lesions. This study demonstrates that mutations caused by the early appearance of DNA damage may appear in the early stage of malignant tissue before the emergence of atypia. The detection of CNV and mutations of the Muc4 gene may be used as an ultra-early screening indicator for esophageal cancer.

Porphyromonas gingivalis secretion leads to dysplasia of normal esophageal epithelial cells via the Sonic hedgehog pathway.

ObjectivesTo investigate the pathogenic effect of Porphyromonas gingivalis cultured media on the esophagus and the mechanism underlying the effect.BackgroundPeriodontitis is strongly associated with esophageal squamous cell carcinoma (ESCC). The cultured media of P. gingivalis may act on healthy esophagus to trigger a malignant transformation; however, this has not been confirmed.MethodsCell migration assays and cell cycle measurements were performed on normal human esophageal epithelial cells in the presence or absence of P. gingivalis cultured media. The esophagi of healthy adult C57BL/6J mice were isolated and cultured in-vitro. Hematoxylin-eosin and immunohistochemical staining using antibodies against proliferating cell nuclear antigen (PCNA), Claudin 1 and Claudin 4 were performed to detect dysplasia in specific tissues. Total mRNA was extracted to determine transcriptional dysregulation. A specific inhibitor of Sonic hedgehog signaling, cyclopamine, was used to confirm the underlying molecular mechanism.ResultsIn the presence of P. gingivalis cultured media, proliferation and migration of normal human esophageal epithelial cells were up-regulated, and aneuploid cells appeared. Compared with control cells, the arrangement of mouse esophageal epithelial cells became disordered, the percentage of PCNA-positive cells increased, and the positive staining of Claudin 1 and Claudin 4 became weak. In addition, the expression of cancer-related pathway genes was up-regulated but tight junction-related gene expression was down-regulated. The Sonic hedgehog pathway was abnormally activated, and its inhibition reduced the pathogenic effect of P. gingivalis cultured media.ConclusionsWe revealed that the cultured media of the key periodontal pathogen, P. gingivalis, can induce the malignant transformation of normal esophageal epithelium through the Sonic hedgehog pathway.

IL-33 Participates in the Development of Esophageal Adenocarcinoma.

Background: The progression from chronic gastroesophageal reflux disease (GERD) to Barrett esophagus (BE) and esophageal adenocarcinoma (EAC) is an inflammatory-driven neoplastic change. Interleukin-33 (IL-33) has identified as a crucial factor in several inflammatory disorders and malignancies. Methods: The high-density tissue microarray of the human EAC was analyzed with IL-33 immunohistochemistry staining (IHC). By anastomosing the jejunum with the esophagus, the rat model of EAC with mixed gastroduodenal reflux was established. The expression of IL-33 was determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blot (WB), IHC and enzyme-linked immunosorbent assay (ELISA). Esophageal adenocarcinoma cells (OE19 and OE33) and human esophageal epithelial cells (HEECs) were used. Results: In the cytoplasm of human EAC tissue, IL-33 expression was substantially greater than in adjacent normal tissue. In rat model, the expression of IL-33 in the EAC group was considerably greater than in the control group, and this expression increased with the upgrade of pathological stage. In in vitro experiment, the mRNA and protein levels of IL-33 were considerably greater in OE19 and OE33 than in HEECs. The stimulation of IL-33 enhanced the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of OE19 and OE33, but soluble ST2 (sST2) inhibited these effects. IL-33 stimulated the release of IL-6 by OE19 and OE33 cells. Conclusion: This study demonstrated the overexpression of IL-33 in the transition from GERD to EAC and that IL-33 promoted carcinogenesis in EAC cells through ST2. IL-33 might be a possible preventive target for EAC.

Abstract 1486: MicroRNA (miR)199a-5p targets JUN-B to decrease cell proliferation in esophageal cancer cells

Abstract Objectives: MiR-199a-5p has been shown to be downregulated in multiple malignancies and function as a tumor suppressor. We have previously demonstrated that miR-199a-5p is markedly downregulated in esophageal cancer cell lines compared to esophageal epithelial cells. In several miR-target sequence analysis programs, miR-199a-5p is predicted to bind JUN-B mRNA with high affinity. JUN-B is an important component of the AP1 transcription factor. The objective of this study was to determine expression of JUN-B in esophageal cancer cells as well as to investigate the interaction between miR-199a-5p and JUNB in these cells and to characterize the functional implications of this interaction. Methods: Studies were performed in human esophageal epithelial (hESO) cells and in TE7, SK-GT-4 and OE21 human esophageal cancer cells. Expression of miR-199a-5p and JUNB mRNA in these cell lines was measured by real-time PCR. JUNB protein expression levels were examined by Western blot. Function of miR-199a-5p was tested through its overexpression and silencing. Cellular proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Levels of miR-199a-5p in TE7, SK-GT-4, and OE21 esophageal cancer cells are markedly reduced compared to hESO cells. JUN-B mRNA and protein expression are correspondingly elevated in these cells compared to hESO cells. JUN-B mRNA and protein expression levels were decreased following miR-199a-5p overexpression in TE7, SK-GT-4 and OE21 cells. In reciprocal experiments, silencing miR-199a-5p in hESO cells resulted in increased JUN-B protein and mRNA levels. Forced expression of miR-199a-5p in TE7, SK-GT-4 and OE21 cells led to a marked decrease in cellular proliferation. Conclusions: MiR-199a-5p expression is significantly reduced in TE7, SK-GT-4, and OE21 esophageal cancer cells relative to hESO cells. Forced expression of miR-199a-5p results in significantly decreased JUN-B mRNA and protein expression in these cells, leading to decreased cellular proliferation. These results add our understanding of the roles by which miR-199a-5p functions as a tumor suppressor in esophageal cancer cells. Further studies will investigate the association of miR-199a-5p with JUN-B mRNA using biotinylated RNA pull-down and luciferase reporter assays, in addition to determining the effect of Jun-B downregulation on AP1 promoter activity. Citation Format: Pornima Phatak, Mohan E. Tulapurkar, James M. Donahue. MicroRNA (miR)199a-5p targets JUN-B to decrease cell proliferation in esophageal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1486.

Single-cell RNA-Seq of human esophageal epithelium in homeostasis and allergic inflammation.

Inflammation of the esophageal epithelium is a hallmark of eosinophilic esophagitis (EoE), an emerging chronic allergic disease. Herein, we probed human esophageal epithelial cells at single-cell resolution during homeostasis and EoE. During allergic inflammation, the epithelial differentiation program was blocked, leading to loss of KRT6hi differentiated populations and expansion of TOP2hi proliferating, DSPhi transitioning, and SERPINB3hi transitioning populations; however, there was stability of the stem cell–enriched PDPNhi basal epithelial compartment. This differentiation program blockade was associated with dysregulation of transcription factors, including nuclear receptor signalers, in the most differentiated epithelial cells and altered NOTCH-related cell-to-cell communication. Each epithelial population expressed genes with allergic disease risk variants, supporting their functional interplay. The esophageal epithelium differed notably between EoE in histologic remission and controls, indicating that remission is a transitory state poised to relapse. Collectively, our data uncover the dynamic nature of the inflamed human esophageal epithelium and provide a framework to better understand esophageal health and disease.

Reactive oxygen species-mediated activation of NLRP3 inflammasome associated with pyroptosis in Het-1A cells induced by the co-exposure of nitrosamines.

Nitrosamines were a class of important environmental carcinogens associated with digestive tract neoplasms. As the early toxic effect of nitrosamines, inflammatory response participated in the malignant transformation of cells and promoted the occurrence and development of tumors. However, the role of NLRP3 inflammasome in the nitrosamines-induced inflammatory response was unclear. In this study, the human esophageal epithelial cells (Het-1A) were used to explore potential mechanisms of the activation of NLRP3 inflammasome under co-exposure to nine nitrosamines commonly found in drinking water at the doses of 0, 4, 20, 100, 500, and 2500 ng/mL. The results showed that nitrosamines stimulated activation of the NLRP3 inflammasome and induced cellular oxidative damage in a dose-dependent manner. Pretreatment of reactive oxygen species scavenger N-acetyl-L-cysteine (NAC), particularly mitochondrial reactive oxygen species (mtROS) scavengers Mito-TEMPO, effectively inhibited the activation of NLRP3 inflammasome, suggesting that nitrosamines could mediate the activation of NLRP3 inflammasome via mtROS. Furthermore, we found that nitrosamines co-exposure also promoted cell pyroptosis through the NLRP3/caspase-1/GSDMD pathway, which was demonstrated by adding the caspase-1 inhibitor Z-YVAD-FMK and constructing NLRP3 downregulated Het-1A cell line. This study revealed the underlying mechanism of the activation of NLRP3 inflammasome initiated by nitrosamines co-exposure and provided new perspectives on the toxic effects of nitrosamines.

Identification and function of circular RNA hsa_circ_0071106: A novel biomarker for differentiation degree of esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) has a high degree of malignancy, and there is currently no effective treatment. Circular RNA is a novel endogenous noncoding RNA with a stable loop structure. Several theories have been proposed regarding its biogenesis and usefulness as a biomarker in various cancers. Here we explore the predictive value and potential functions of a candidate circRNA (hsa_circ_0071106) in ESCC. MethodsThe altered expression of hsa_circ_0071106 was validated in clinical tissue samples from 64 patients with ESCC. The correlation between the clinical characteristics of these patients and the expression of hsa_circ_0071106 was analyzed by the chi-square test. The receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were used to evaluate the diagnostic value of hsa_circ_0071106. The human esophageal carcinoma cell lines ECA109 and ECA9706 and the normal human esophageal epithelial cell line (HET-1A) were applied for functional analysis of hsa_circ_0071106. qRT-PCR, sanger sequencing, agarose gel electrophoresis, RNase R digestion assay, Actinomycin D inhibition assay, nucleoplasm separation assay, and FISH assays were utilized to verify the circular characteristics and subcellular localization of hsa_circ_0071106. SiRNA and circRNA interference lentivirus was constructed to inhibit the expression of hsa_circ_0071106 in the ECA109 and ECA9706 cell lines. We used the EdU assay, Transwell assay, and flow cytometry to detect changes in cell proliferation, invasion, migration, and apoptosis. ResultsWe identified that hsa_circ_0071106 showed significantly decreased expression levels in ESCC cells (P < 0.001) and tissue samples (P < 0.001). The correlation analysis of clinicopathological features and gene expression revealed that low expression of hsa_circ_0071106 was related to poor differentiation grade (P < 0.05). ROC curves indicated that hsa_circ_0071106 might have a diagnostic value for ESCC (AUC = 0.826, 95% CI: 0.748–0.904) and a predictive value for a high degree of histological differentiation (AUC = 0.730, 95% CI: 0.607–0.854). Hsa_circ_0071106 has a stable circular structure and was primarily localized in the cytoplasm. A transient knockdown of hsa_circ_0071106 by siRNA promotes proliferation, migration, and invasion and inhibits apoptosis of ESCC cells. A steady knockdown of hsa_circ_0071106 by a lentivirus knockout vector significantly promoted cell migration and invasion in ECA109 and ECA9706 cell lines. ConclusionHsa_circ_0071106 was a downregulated circRNA related to ESCC and promoted ESCC progression by regulating cell migration and invasion. These findings suggest that hsa_circ_0071106 could be a promising diagnostic biomarker and potential therapeutic target and might predict ESCC’s histological differentiation degree.

Alginates for Protection Against Pepsin-Acid Induced Aerodigestive Epithelial Barrier Disruption.

Gastroesophageal reflux disease (GERD) and laryngopharyngeal reflux (LPR) are chronic conditions caused by backflow of gastric and duodenal contents into the esophagus and proximal aerodigestive tract, respectively. Mucosal barrier dysfunction resultant from the synergistic actions of chemical injury and the mucosal inflammatory response during reflux contributes to symptom perception. Alginates effectively treat symptoms of mild to moderate GERD and have recently shown benefit for LPR. In addition to forming a "raft" over gastric contents to reduce acidic reflux episodes, alginates have been found to bind the esophageal mucosa thereby preserving functional barrier integrity measured by transepithelial electrical resistance. The aim of this study was to further examine the topical protective capacity of alginate-based Gaviscon Advance (GA) and Double Action (GDA) against pepsin-acid mediated aerodigestive epithelial barrier dysfunction in vitro. Translational. Immortalized human esophageal and vocal cord epithelial cells cultured in transwells were pretreated with liquid formula GA, GDA, matched viscous placebo solution, or saline (control), then treated for 1 h with saline, acid (pH3-6) or pepsin (0.1-1mg/ml) at pH3-6. Endpoint measure was taken of horseradish peroxidase (HRP) allowed to diffuse across monolayers for 2 h. Pepsin (0.1-1mg/ml) at pH3-6 increased HRP flux through cultures pretreated with saline or placebo (p< 0.05); acid alone did not. GA and GDA prevented barrier dysfunction. GA and GDA preserved epithelial barrier function during pepsin-acid insult better than placebo suggesting that protection was due to alginate. These data support topical protection as a therapeutic approach to GERD and LPR. Laryngoscope, 132:2327-2334, 2022.

Metabolomics-based molecular signatures reveal the toxic effect of co-exposure to nitrosamines in drinking water.

Nitrosamines, a group of emerging nitrogenous pollutants, are ubiquitously found in the drinking water system. However, less is known about how systemic biological responses resist or tolerate nitrosamines, especially long-term co-exposure at low concentrations. In this study, untargeted metabolomics was used to investigate the metabolic perturbations in human esophageal epithelial Het-1A cells induced by a mixture of nine common nitrosamines in drinking water at environmentally relevant, human-internal-exposure, and genotoxic concentrations. Generally, the disrupted metabolic spectrum became complicated with nitrosamines dose increasing. Notably, two inflammation-associated pathways, namely, cysteine (Cys) and methionine (MET) metabolism, and nicotinate and nicotinamide metabolism, changed significantly under the action of nitrosamines, even at the environmentally relevant level. Furthermore, targeted metabolomics and molecular biology indicators in cells were identified in mice synchronously. For one thing, the up-regulated Cys and MET metabolism provided methyl donors for histone methylation in the context of pro-inflammatory response. For another, the down-regulated NAD+/NADH ratio inhibited the deacetylation of NF-кB p65 and eventually activated the NF-кB signaling pathway. Taken collectively, the metabolomics molecular signatures were important indicative markers for nitrosamines-induced inflammation. The potential crosstalk between the inflammatory cascade and metabolic regulation also requires further studies. These findings suggest that more attention should be paid to long-term co-exposure at low concentrations in the control of nitrosamines pollution in drinking water. Additionally, this study also highlights a good prospect of the combined metabolomic-molecular biology approach in environmental toxicology.

Chitosan/collagen layer-by-layer deposition for improving the esophageal regeneration ability of nanofibrous mats

The morbidity of a diverse array of esophageal diseases has been increasing in recent years, and postoperative bacterial infection is a common potential risk. Current research on artificial esophageal stents is lacking and less than satisfactory. Artificial materials with excellent mechanical, antimicrobial, and cytocompatible properties at the same time still have great potential. In this study, chitosan (CS) and collagen (COL) were deposited on nylon 6 (N6)/silk fibroin (SF) composite nanofibers using layer-by-layer self-assembly (LBL). The obtained nanofibrous mats were characterized, and the results indicated that the surface morphology and hydrophilicity of the composite nanofibrous mats were obviously improved by the deposition of CS and COL. The mechanical properties of the composite nanofibrous mats were also improved after LBL modification. In addition, the LBL-structured nanofibrous mats possessed excellent antibacterial activity, reaching more than 90% inhibition of gram-positive and negative bacteria. The results of cell experiments indicated that the nanofibrous mats modified by CS and COL had excellent biocompatibility for promoting human esophageal epithelial cell adhesion and proliferation. In vivo animal experiments showed that the LBL-structured nanofibrous mats were beneficial to the healing of esophageal defects compared with the control group. In summary, LBL-structured nanofibrous mats are very promising materials for esophageal replacement to reduce the incidence of postoperative complications.

Regulation of IncRNA ZNF667-AS1 in Proliferation and Invasion of Esophageal Squamous Cell Carcinoma Cells via Mediating ceRNA Network.

Esophageal squamous cell carcinoma (ESCC), classified as a primary histological subtype of esophageal cancer (EC), dominates approximately 90% of the newly diagnosed EC. Long non-coding RNAs (lncRNAs) are frequently related to the course of ESCC. The current study aimed to investigate whether lncRNA zinc finger protein 667-antisense RNA 1 (ZNF667-AS1) modulates the proliferation and invasion of ESCC cells. ESCC tissues and cell lines, para-carcinoma tissues, and human esophageal epithelial cells (HEEpiCs) were collected. lncRNA ZNF667-AS1 expression in the above tissues and cells was detected. The effect of lncRNA ZNF667-AS1 on proliferation and invasion of Eca109 cells was detected using cell counting kit-8, colony formation, and Transwell assays. lncRNA ZNF667-AS1 subcellular localization was determined via the nuclear/cytosol fractionation assay. The binding relationships between miR-18b-5p and lncRNA ZNF667-AS1 and RAS p21 protein activator 1 (RASA1) were verified using dual-luciferase reporter gene experiment and RNA immunoprecipitation experiment. The expressions of miR-18b-5p and RASA1 in the tissues and cells were identified. The roles of miR-18b-5p overexpression or silencing RASA1 in proliferation and invasion of ESCC cells were examined through rescue experiments. lncRNA ZNF667-AS1 was underexpressed in ESCC tissues and cells, and lncRNA ZNF667-AS1 overexpression hampered ESCC cell proliferation and invasiveness. miR-18b-5p targeted RASA1 while lncRNA ZNF667-AS1 promoted RASA1 transcription via binding to miR-18b-5p. Over-expression miR-18b-5p or silencing RASA1 reversed the inhibitory effects of lncRNA ZNF667-AS1 overexpression on ESCC cell proliferation and invasion. lncRNA ZNF667-AS1 overexpression accelerated RASA1 transcription by competitively binding to miR-18b-5p, thus suppressing ESCC cell proliferation and invasion.

FAT1 and PTPN14 Regulate the Malignant Progression and Chemotherapy Resistance of Esophageal Cancer through the Hippo Signaling Pathway.

Background Esophageal cancer (EC) is a common malignant tumor, which brings heavy economic burden to patients and society. Therefore, it is important to understand the molecular mechanism of recurrence, metastasis, and drug resistance of esophageal cancer. Methods Human esophageal cancer cell line TE13 (poorly differentiated squamous cell carcinoma) and normal human esophageal epithelial cell line het-1a were selected for aseptic culture. At the same time, 6 bottles of TE13 cell line were inoculated in logarithmic phase. Cell apoptosis was analyzed by flow cytometry (FCM). Cell clone formation assay was used to analyze the proliferation. Fibronectin-coated dishes were used to detect the characteristics of cell adhesion to extracellular matrix. The Transwell method was used to detect the cell invasion ability. Western blot was used to analyze the expression of Yap1, PTPN14, FAT1, and Myc. Results Results showed that FAT1 and PTPN14 were downregulated, while Yap1 was upregulated in esophageal cancer tissues. FAT1 inhibited the proliferation, adhesion, and invasion of human esophageal cancer cell lines, which might be associated with the upregulation of PTPN14 and the inhibition of Yap1 and Myc. Conclusion The results suggested that PTPN14 and FAT1 could regulate malignant progression and chemotherapy resistance of esophageal cancer based on the Hippo signaling pathway.

Association of Incidental Positron Emission Tomography Uptake in the Esophagus to Gastroesophageal Reflux Disease

The Los Angeles (LA) classification is the most accurate means of assessing esophageal injury from caustic gastric acid with focused and greater concentrations in areas of erosive disease.¹ However, data from animal models and patients have proposed that an initial diffuse inflammatory pathway contributes to injury in gastroesophageal reflux disease (GERD) mediated by interleukin (IL) 8, IL1β,^2,3 and hypoxia-inducible factors.^4,5 These observations demonstrate a lymphocyte predominant inflammatory process over course of 1-2 weeks associated with basal zone hyperplasia and dilation of intercellular spaces.⁶ In cultured human esophageal epithelial cells and patients, it is further suggested that acid causes this chronic inflammatory reaction.

Loss of grand histone H3 lysine 27 trimethylation domains mediated transcriptional activation in esophageal squamous cell carcinoma

Trimethylation of histone H3 lysine 27 trimethylation (H3K27me3) may be recruited by repressive Polycomb complexes to mediate gene silencing, which is critical for maintaining embryonic stem cell pluripotency and differentiation. However, the roles of aberrant H3K27me3 patterns in tumorigenesis are not fully understood. Here, we discovered that grand silencer domains (breadth > 50 kb) for H3K27me3 were significantly associated with epithelial cell differentiation and exhibited high gene essentiality and conservation in human esophageal epithelial cells. These grand H3K27me3 domains exhibited high modification signals involved in gene silencing, and preferentially occupied the entirety of topologically associating domains and interact with each other. We found that widespread loss of the grand H3K27me3 domains in of esophageal squamous cell carcinomas (ESCCs) were enriched in genes involved in epithelium and endothelium differentiation, which were significantly associated with overexpression with increase of active modifications of H3K4me3, H3K4me1, and H3K27ac marks, as well as DNA hypermethylation in the gene bodies. A total of 208 activated genes with loss of grand H3K27me3 domains in ESCC were identified, where the higher expression and mutation of T-box transcription factor 20 (TBX20) were associated with worse patients’ outcomes. Our results showed that knockdown of TBX20 may have led to a striking defect in esophageal cancer cell growth and carcinogenesis-related pathway, including cell cycle and homologous recombination. Together, our results reveal that loss of grand H3K27me3 domains represent a catalog of remarkable activating regulators involved in carcinogenesis.

Insilico Insight into the Association between Polycyclic Aromatic Hydrocarbons and Human Toll like Receptor in Progression of Esophageal Carcinogenesis

Cancer creates enormous burden and is a leading cause of death in most of the countries. Esophageal cancer (EC) is one of the most common cancers in the world and has the worst survival of all cancers. Esophageal Squamous Cell Carcinoma (ESCC) and adenocarcinoma are the two main types of esophageal cancer. Exposure of polycyclic aromatic hydrocarbons (PAHs) can be cited as one of the reasons for development of Esophageal Squamous Cell Carcinoma. Toll Like Receptors (TLRs) present in human esophageal epithelial cells may pose a major role in pathogen recognition, activation of innate immunity and cancer. The study attempts to predict the role of carcinogenic PAHs in TLR4 signaling pathway and how it can ultimately result in immuno suppression and development of esophageal cancer by insilico docking evaluation. For the study we retrieved the structure of sixteen PAHs from the PubChem database and the protein model of TLR4 (PDB ID: 4G8A) from protein data bank (PDB). The binding affinity of PAHs to the TLR4 receptor was calculated using molecular docking software Auto Dock1.5. The results showed that Benzo[a]pyrene showed strong interaction toward TLR4 receptor with the highest number of polar contacts along with high binding affinity values. Considering the presence of Benzo[a] pyrene in different environmental, occupational and behavioral sources globally, there is a need for future research on the risk assessment of Benzo[a]pyrene on Esophageal Squamous Cell Carcinoma.

Expression and Clinical Value of LncRNA GAPLINC in Esophageal Squamous Cell Carcinoma.

BackgroundThe long noncoding RNA (lncRNA) GAPLINC, or gastric adenocarcinoma predictive long intergenic ncRNA, plays a carcinogenic role in a variety of different tumor types. There is limited information regarding the biological function of GAPLINC in the development of esophageal squamous cell carcinoma (ESCC).MethodsSurgical tissue samples of 40 patients undergoing ESCC radical surgery were collected, including ESCC tissues and corresponding adjacent normal tissues. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of lncRNA GAPLINC in the human ESCC cell line (TE11). The function role of LncRNA GAPLINC was detected after specific siRNA interference and overexpression in the TE11 cell line. The effects of LncRNA GAPLINC on ESCC cell proliferation, migration and invasion abilities were investigated by flow cytometry, using the Cell Counting Kit-8 (CCK-8), and by Transwell migration assays, respectively.ResultsThe expression of lncRNA GAPLINC in ESCC tissues was significantly higher than that in corresponding adjacent normal tissues (P<0.05) and correlated with the degree of tumor differentiation (P<0.05). Compared with human esophageal normal epithelial cell lines, the expression of LncRNA GAPLINC was significantly higher in the human ESCC cell line (P<0.05). CCK-8 assays showed that LncRNA GAPLINC overexpression increased the growth rate of cells (P<0.05). Transwell experiments showed that LncRNA GAPLINC overexpression increased the ability of cell migration and invasion compared to control cells (P<0.05). Annexin V assay revealed that LncRNA GAPLINC silencing increased early stage apoptosis (P<0. 05).ConclusionOur results suggest that LncRNA GAPLINC may be used as a biomarker for the diagnosis and monitoring of ESCC, and may play an oncogenic role in ESCC.

Effect of Linc-POU3F3 on radiotherapy resistance and cancer stem cell markers of esophageal cancer cells.

Long non-coding RNA (LncRNA) is an important transcriptional and post-transcriptional regulatory molecule in the body. In recent years, relationship between LncRNA and malignant phenotype of tumor cells has been revealed gradually. This study aims to investigate the expression characteristics of pit-oct-unc class 3 homeobox 3 related long non-coding RNA (Linc-POU3F3) in esophageal cancer and its relationship with radiation resistance (IR) as well as the expressions of cancer stem cell (CSC) markers in esophageal cancer cells. The expression characteristics and potential interaction molecules of Linc-POU3F3 in esophageal cancer were collected from the public database via bioinformatics retrieval. Forty-two pair samples of esophageal cancer tissues and corresponding adjacent tissues were collected. Human normal esophageal epithelial cells (HEEC) and human esophageal cancer cell lines (ECA109, TE-1, TE-2, TE-13) were cultured. Real-time quantitative PCR (qPCR) was used to detect the expression level of Linc-POU3F3 in clinical tissues and cells. The formation of TE-13 IR cell line induced by different doses of radiation served as IR group cells, and the same condition treated with 0 Gy dose was set as control group (control) cells. Meanwhile, we used cell transfection technology to construct random interference sequence (siControl) cells and interference (siLinc-POU3F3) cells. In ECA109 cells, we transfected blank and over expressed Linc-POU3F3 plasmids as vector and over-expressed group (oeLinc-POU3F3). The mRNA and protein expressions of CD44, CD133 and CD90 were detected by qPCR and Western blotting, respectively. MTS [3-(4,5-dimenthylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] was used to detect the cell viability under different radiation doses, and the resistance of IR cells was verified by clone formation experiment. The expression of Linc-POU3F3 was correlated with the tumor progression and poor prognosis of esophageal cancer. The level of Linc-POU3F3 mRNA expression was significantly higher in esophageal cancer tissues and cell lines than that in normal adjacent tissues and cell lines (all P<0.01). The expressions of Linc-POU3F3 mRNA and protein expressions of CD44, CD133, and CD90 in IR cells were significantly higher than those in control cells (all P<0.01). The expression of Linc-POU3F3 in siLinc-POU3F3 cell was significantly lower than that in the siControl cells (P<0.01), and the inhibition rate was 87.21%. The mRNA and protein expressions of CD44, CD133, and CD90 in the siLinc-POU3F3 cells were significantly lower than those in the siControl cells (all P<0.05). The expressions of linc-POU3F3, CD44, CD133, and CD90 mRNA and protein in the oeLinc-POU3F3 cells were significantly higher than those in the vector cells. The relative activity and clone formation ability in the IR cells were significantly higher than those in the control cells at 2, 4, and 8 Gy doses (all P<0.01). The relative activity in the siLinc-POU3F3 cells was significantly lower than that in the siControl cells at 4 and 8 Gy doses (P<0.01). The relative activity in the oeLinc-POU3F3 cells was significantly higher than that in the vector cells at 4 and 8 Gy doses (P<0.01). Linc-POU3F3 is up-regulated in esophageal cancer and can promote IR and the expression of CSC markers in esophageal cancer cells.

SERPINE2 promotes cellular migration and invasion in esophageal squamous cell carcinoma by activating β-catenin

Objective: To clarify the function and molecular mechanisms of serpin family E member 2 (SERPINE2) in cellular migration and invasion of esophageal squamous cell carcinoma (ESCC). Methods: The expression of SERPINE2 in ESCC was analyzed by using online databases TCGA (http: //gepia.cancer-pku.cn/detail.php and http: //ualcan.path.uab. edu/index.html). The expressions of SERPINE2 mRNA in normal human esophageal epithelial cell line NE2, human ESCC cell lines KYSE30 and KYSE150 were detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). SERPINE2-konckdown or SERPINE2-overexpressed plasmid was transfected into KYSE30 cells, and the efficiencies of the knockdown and overexpression system were tested by qRT-PCR. The relationships of SERPINE2 and ESCC migration and invasion were determined by migration and invasion assays in vitro. The associations between SERPINE2 expression and β-catenin as well as its target genes including c-Myc, cyclin D1 and CD44 were analyzed by immunofluorescence, qRT-PCR and western blot, respectively. Results: The expressions of SERPINE2 were significantly upregulated in both esophageal cancer (ESCA) and ESCC tissues compared to normal tissues by analyzing 182 and 95 cases, respectively (P<0.01). SERPINE2 is highly expressed in both KYSE30 and KYSE150 cells (P<0.05). The number of migrating and invading cells in control group were (212.66±24.11)/field and (136.00±14.42)/field, while were (88.33±9.71)/field and (77.00±9.53)/field in SERPINE2-knockdown 1 group, and (66.00±8.00)/field and (45.66±3.78)/field in SERPINE2-knockdown 2 group, respectively, and the differences were dramatically significant compared with the control group (P<0.01). The number of migrating and invading cells in control group were (250.00±30.00)/field and (203.33±15.27)/field, while were (383.33±35.11)/field and (246.66±25.16)/field in SERPINE2-overpressed group, and the differences were strikingly significant compared with the control group (P<0.01). The protein expression of β-catenin was upregulated while phosphorylated β-catenin protein expression was downregulated in SERPINE2-overexpressed KYSE30 cells when compared to control cells.The transcription activity of β-catenin was significantly upregulated and the mRNA expressions of its target genes including c-Myc, cyclin D1 and CD44 were all increased. After treated with 25 μM iCRT14, the number of migrated cells in the control and SERPINE2-overpressed groups were (200.00±36.05)/field and (258.33±22.54)/field, and the number of invaded cells were (160.00±17.32)/field and (188.33±25.65)/field, respectively, the differences were dramatically significant compared with the group without iCRT14 treatment (P<0.01). Conclusion: SERPINE2 is significantly upregulated in ESCC cells and can promote cellular migration and invasion by activating β-catenin, which may provide a potential therapeutic target for patients with ESCC.