Carcinogenesis Of Esophageal Squamous Cell Carcinoma Research Articles

An effective two-stage NMBzA-induced rat esophageal tumor model revealing that the FAT-Hippo-YAP1 axis drives the progression of ESCC

Rat model of N-nitrosomethylbenzylamine (NMBzA)-induced esophageal squamous cell carcinoma (ESCC) is routinely used to study ESCC initiation, progression and new therapeutic strategies. However, the model is time-consuming and malignant tumor incidences are low. Here, we report the usage of multi-kinase inhibitor sorafenib as a tumor promoter to establish an efficient two-stage NMBzA-induced rat ESCC carcinogenesis model, resulting in increments of tumor incidences and shortened tumor formation times. By establishing the model and applying whole-genome sequencing, we discover that benign papillomas and malignant ESCCs harbor most of the “driver” events found in rat ESCCs (e.g. recurrent mutations in Ras family, the Hippo and Notch pathways and histone modifier genes) and the mutational landscapes of rat and human ESCCs overlap extensively. We generate tumor cell lines derived from NMBzA-induced papillomas and ESCCs, showing that papilloma cells retain more characteristics of normal epithelial cells than carcinoma cells, especially their exhibitions of normal rat cell karyotypes and inabilities of forming tumors in immunodeficient mice. Three-dimensional (3-D) organoid cultures and single cell RNA sequencing (scRNA-seq) indicate that, when compared to control- and papilloma-organoids, ESCC-organoids display salient abnormalities at tissue and single-cell levels. Multi-omic analyses indicate that NMBzA-induced rat ESCCs are accompanied by progressive hyperactivations of the FAT-Hippo-YAP1 axis and siRNA or inhibitors of YAP1 block the growth of rat ESCCs. Taken together, these studies provide a framework of using an effective rat ESCC model to investigate multilevel functional genomics of ESCC carcinogenesis, which justify targeting YAP1 as a therapeutic strategy for ESCC.

DUSP4 maintains the survival and LSD1 protein stability in esophageal squamous cell carcinoma cells by inhibiting JNK signaling-dependent autophagy.

DUSP4 is a biomarker of esophageal squamous cell carcinoma (ESCC), which is responsible for the prognosis in ESCC. However, the underlying mechanism of DUSP4-regulated ESCC carcinogenesis is unknown. As a negative regulator of JNK, DUSP4 can inhibit autophagy, which contributes to tumorigenesis. This study aimed to explore the role of autophagy in DUSP4-regulated ESCC carcinogenesis. Our results showed that DUSP4 overexpression inhibited autophagy and promoted LSD1 protein expression in ESCC cells, while DUSP4 silencing showed the opposite effects. However, DUSP4 overexpression and silencing did not affect LSD1 mRNA expression. But the regulatory ability of DUSP4 overexpression on autophagy, death level, and LSD1 protein was reversed by rapamycin. In addition, DUSP4 overexpression inhibited JNK and Bcl2 phosphorylation and the dissociation of Bcl2-Beclin1 complex, while DUSP4 silencing promoted JNK and Bcl2 phosphorylation. Moreover, the regulatory ability of DUSP4 overexpression on autophagy, death, and LSD1 protein was reversed by JNK activator anisomycin. The xenograft assays also showed that DUSP4 overexpression-promoted ESCC tumor growth in vivo and LC3II and LSD1 protein expression in tumor tissues were reversed by rapamycin or anisomycin. Overall, DUSP4 inhibits Bcl2-Beclin1-autophagy signal transduction through the negative regulation of JNK, thus suppressing autophagic death and the autophagic degradation of LSD1 in ESCC, by which DUSP4 promotes ESCC carcinogenesis.

The role of microbiota in esophageal squamous cell carcinoma: A review of the literature.

Esophageal squamous cell carcinoma (ESCC) exhibits high incidence with poor prognosis. Alcohol drinking, cigarette smoking, and betel nut chewing are well-known risk factors. Dysbiosis, an imbalance of the microbiota residing in a local environment, is known to be associated with human diseases, especially cancer. This article reviews the current evidence of esophageal microbiota in ESCC carcinogenesis, including initiation, progression, and drug resistance. Articles involving the esophageal microbiota, diagnosis, treatment, and the progression of esophageal cancer were acquired using a comprehensive literature search in PubMed in recent 10 years. Based on 16S rRNA sequencing of human samples, cell, and animal studies, current evidence suggests dysbiosis of the esophagus promotes ESCC progression and chemotherapy resistance, leading to a poor prognosis. Smoking and drinking are associated with esophageal dysbiosis. Specific bacteria have been reported to promote carcinogenesis, involving either progression or drug resistance in ESCC, for example Porphyromonas gingivalis and Fusobacterium nucleatum. These bacteria promote ESCC cell proliferation and migration via the TLR4/NF-κB and IL-6/STAT3 pathways. F. nucleatum induces cisplatin resistance via the enrichment of immunosuppressive myeloid-derived suppressor cells (MDSCs). Correcting the dysbiosis and reducing the abundance of specific esophageal pathogens may help in suppressing cancer progression. In conclusion, esophageal dysbiosis is associated with ESCC progression and chemoresistance. Screening the oral and esophageal microbiota is a potential diagnostic tool for predicting ESCC development or drug-resistance. Repairing esophageal dysbiosis is a novel treatment for ESCC. Clinical trials with probiotics in addition to current chemotherapy are warranted to study the therapeutic effects.

319. SIGNIFICATIONS OF EXPRESSION OF TOLL-LIKE RECEPTORS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA CELLS

Abstract Well known risk factors of Esophageal squamous cell carcinoma (ESCC) are habit of alcoholic drinking, smoking and poor diet. Moreover, recent reports showed that poor oral health is associated with upper aerodigestive tract malignancies including ESCC. We previously reported that among 232 ESCC patients who had preoperative assessment of oral function by dentist, about 70% of patients were diagnosed with periodontitis and about half of those patients were needed dental extraction. Toll-like receptors (TLRs), a family of pattern recognition receptors, are able to recognize pathogen-associated molecular patterns (PAMPs) associated with a wide range of viruses, bacteria, fungi and parasites and thus play key roles in innate immune responses. Among these TLRs, TLR4 recognizes LPS, which construct the cell wall of gram-negative bacteria. Based on these findings, it is supposed that continuous exposure of LPS from gram-negative periodontal bacteria induces chronic inflammation caused by inflammatory cytokines from TLR4 signals, then carcinogenesis and progression of ESCC occurs. Among these TLRs, TLR4 recognizes LPS, which construct the cell wall of gram-negative bacteria. We reported that ESCC patients who had higher TLR4 expression status in resected main tumor specimen showed significantly poor overall survival compared to lower patients after esophagectomy. (Sato Y, et al. Esophagus 2020) On the other hand, TLR3 recognizes dsRNA released form cell invading viruses. We previously reported that ESCC patients who had higher TLR3 expression status in resected main tumor specimen showed significantly better overall survival compared to lower patients after esophagectomy. (Sato Y, et al. Am J Surg. 2018). Though TLR3 and TLR4 are in the same TLR family, those functional mechanism and relationships to prognosis in ESCC are totally different. Because TLR4 signaling is stimulatory on proliferative ability of ESCC, it is supposed that improvement of oral health inhibits carcinogenesis and progression of ESCC. On the other hand, because TLR3 signaling is suppressive, TLR3 agonist should be a possible candidate of therapeutic agent.

Comparison of tumor-associated and nontumor-associated esophageal mucosa microbiota in patients with esophageal squamous cell carcinoma.

Esophageal microbiota plays important roles in esophageal squamous cell carcinoma (ESCC). The aims of this study were to clarify the changes in the bacterial community during ESCC development and identify latent pathogenic bacteria which may contribute to esophageal carcinogenesis and progression. Fresh tumor and nontumor esophageal mucosal samples were collected from 31 men with ESCC. High-throughput 16s rRNA sequencing was performed, and the operational taxonomic unit data and bacterial classification annotation were obtained and analyzed. The Ace, Chao, Shannon, Simpson indexes, and operational taxonomic unit numbers were higher in nontumor tissues than in tumor tissues, although without statistical significance. There were 4 phyla and 28 genera found to show significant differences between tumor and nontumor samples. The general probiotic Lactobacillus was 1.98-fold higher in nontumor tissues, while the general pathogenic genera Fusobacterium was 4.35-fold higher in tumor tissues. For tumor tissue samples, the genera Treponema and Brevibacillus were significantly higher in N1 and N2 stages, respectively, and Acinetobacter was significantly higher in T3 stage. For nontumor tissues, the genus Fusicatenibacter was significantly higher in T2 stage, and Corynebacterium, Aggregatibacter, Saccharimonadaceae-TM7x, and Cupriavidus were significantly higher in T4 stage. Additionally, bacteria related to nitrotoluene degradation were enriched in nontumor tissues, while bacteria related to base excision repair were enriched in tumor tissues. The relative abundance of several phyla and genera are different between tumor and nontumor tissue samples. The altered bacterial microbiota is correlated with different tumor stages and some microbes may take part in the carcinogenesis and development of ESCC.

Circular RNA hsa_circ_0000277 promotes tumor progression and DDP resistance in esophageal squamous cell carcinoma

BackgroundCircular RNAs (circRNAs) are well-known regulators of cancer progression and chemoresistance in various types of cancers. This study was performed to investigate the function of hsa_circ_0000277 in esophageal squamous cell carcinoma (ESCC).MethodsRNA levels were analyzed via the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell Counting Kit-8 (CCK-8) assay was applied to determine cell proliferation and half maximal inhibitory concentration (IC50) of cisplatin (DDP). Colony formation ability was evaluated by colony formation assay. Cell cycle and apoptosis were measured using flow cytometry. RNA immunoprecipitation (RIP), pull-down assay and dual-luciferase reporter assays were performed for target interaction analysis. The protein levels were determined through western blot. Xenograft models were established for researching hsa_circ_0000277 function in vivo.ResultsHsa_circ_0000277 expression was increased in ESCC cells and tissues, and it had important clinical significance. Downregulation of hsa_circ_0000277 repressed ESCC cell proliferation, colony formation, cell cycle, and DDP resistance. Hsa_circ_0000277 acted as a microRNA-873-5p (miR-873-5p) sponge and Sry-related high-mobility group box 4 (SOX4) was validated as a target of miR-873-5p. Moreover, hsa_circ_0000277/miR-873-5p axis and miR-873-5p/SOX4 axis regulated ESCC cell progression and DDP resistance. Hsa_circ_0000277/miR-873-5p axis activated SOX4/Wnt/β-catenin signaling pathway. Hsa_circ_0000277 facilitated tumorigenesis and DDP resistance by miR-873-5p/SOX4 axis in vivo.ConclusionThese findings unraveled that hsa_circ_0000277 promoted ESCC progression and DDP resistance via miR-873-5p/SOX4/Wnt/β-catenin axis, showing a specific molecular mechanism of carcinogenesis and chemoresistance in ESCC.

Expression profile, clinical significance and biological functions of IGF2BP2 in esophageal squamous cell carcinoma.

The ectopic expression of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) has been demonstrated to facilitate tumorigenesis and induce proliferation in a various types of cancer. However, the role of IGF2BP2 in esophageal squamous cell carcinoma (ESCC) has yet been fully elucidated. In this regard, the current study assessed the expression patterns and clinical significance of IGF2BP2 in 94 Chinese patients diagnosed with ESCC. Immunohistochemistry and reverse transcription-quantitative PCR assays were employed to assess IGF2BP2 expression in ESCC tissues compared with adjacent healthy tissues. The results revealed that the protein expression of IGF2BP2 was substantially upregulated in ESCC tissues compared with adjacent ESCC tissues. More specifically, higher IGF2BP2 expression strongly associated with tumor node metastasis stage, lymphatic infiltration and lymph node metastasis. Using two ESCC cell lines (TE-1 and TE-10), the inhibition of IGF2BP2 expression by small interfering RNA was proven to induce apoptosis and suppress proliferation, migration and cell cycle progression in vitro. Collectively, the present findings indicated that IGF2BP2 may serve a major role in the development of ESCC carcinogenesis. The present study may be helpful in the design of potential drug targets in the treatment of ESCC.

The Role of DBR1 as a Candidate Prognosis Biomarker in Esophageal Squamous Cell Carcinoma.

Aims: Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent malignancies with unfavorable clinical outcomes and limited therapeutic methods. As a key enzyme in RNA metabolism, debranching RNA Lariats 1 (DBR1) is involved in intron turnover and biogenesis of noncoding RNA. Although cancer cells often show disorder of nucleic acid metabolism, it is unclear whether DBR1 has any effect on the carcinogenesis and progression of ESCC. Methods: Here we detected DBR1 expression in 112 ESCC samples by immunohistochemistry and analyzed its correlation with clinical parameters and survival. Results: DBR1 is mainly located in the nucleus of ESCC tissue. And DBR1 was associated with several malignant clinical features in patients, including tumor location (χ2 = 9.687, P = .021), pathologic T stage (χ2 = 5.771, P = .016), lymph node metastasis (χ2 = 8.215, P = .004) and N classification (χ2 = 10.066, P = .018). Moreover, Kaplan-Meier analysis showed that ESCC patients harboring lower DBR1 expression had a worse prognosis in comparison with those with higher DBR1 expression (P = .005). Univariate and multivariate Cox proportional hazards regression analyses indicated that decreased DBR1 might act as an independent predictor of poor prognosis for ESCC patients. Conclusion: Abnormal RNA metabolism might play a critical role in promoting the progression of ESCC, and DBR1 may be a promising potential biomarker for predicting the prognosis of ESCC patients.

IL6 and BCL3 Expression Are Potential Biomarkers in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) ranks among the most lethal tumors worldwide, as a consequence of late detection and poor treatment response, evidencing the need for diagnosis anticipation and new therapeutic targets. First, we investigated the IL6 gene and protein expression in the esophagus of individuals without esophageal disorders (healthy), ESCC, and non-tumoral surrounding tissue (NTST). Our results showed that IL6 mRNA and protein expression is upregulated in tumor cells relative to NTST. In the TCGA dataset, we identified a set of genes whose expression was correlated with IL6 mRNA levels, including the antiapoptotic gene BCL3. By using an immortalized esophageal cell line, we confirmed that IL6 was capable of inducing BCL3 expression in esophageal cells. BCL3 mRNA and protein are overexpressed in ESCC and NTST compared to healthy esophagus, and BCL3 mRNA could distinguish the morphologically normal samples (healthy and NTST) with 100% sensitivity and 95.12% specificity. The spatial intratumoral heterogeneity of both IL6 and BCL3 expression was evaluated, corroborating IL6 upregulation throughout the tumor, while tumor and NTST showed a consistent increase of BCL3 expression relative to the healthy esophagus. Our study shows that IL6 overexpression seems to be a key event in ESCC carcinogenesis, contributing to ESCC through a homogeneous antiapoptotic signalling via BCL3 overexpression, thus suggesting anti-IL6 therapies to be further considered for ESCC treatment. Finally, our data support the use of BCL3 mRNA expression as a potential biomarker for ESCC detection.

Non-Acid Fluid Exposure and Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) accounts for the large majority of esophageal cancer cases worldwide. In this review, we examine the potential role of non-acidic fluid (NAF) exposure in ESCC carcinogenesis. Esophageal NAF consists of a mixture of salivary, esophageal, gastric, and duodenal fluids, containing inflammatory constituents such as digestive enzymes and bile acids that induce DNA damage, as well as known carcinogens such as acetaldehyde and N-nitrosamines. Exposure to NAF can occur in the setting of increased non-acid reflux, decreased gastric acidity, and decreased esophageal fluid clearance. Non-acid reflux has been associated with ESCC in small observational studies, and in animal models bile reflux can promote the development of ESCC. Associations have been found between increased ESCC risk and atrophic gastritis, a history of partial gastrectomy, and proton pump inhibitor use, all of which raise the pH of refluxate. Additionally, a minimally or non-acidic gastric environment contains an altered microbiome that can increase the production of acetaldehyde and N-nitrosamines. Esophageal motility disorders such as achalasia and opioid-induced esophageal dysfunction result in increased stasis and exposure to these potentially proinflammatory constituents of NAF. NAF may promote the development of ESCC via multiple mechanisms and is an understudied area of research.

Comprehensive mutational analysis of background mucosa in patients with Lugol-voiding lesions.

Somatic mutations including the background mucosa in patients with Lugol‐voiding lesions (LVLs) are still not well known. The aim of this study was to evaluate the somatic mutations of the background mucosa in patients with LVLs (Squamous cell carcinoma (SCC), intraepithelial neoplasia (IN), and hyperplasia). Twenty‐five patients with LVLs (9 with SCC, 6 with IN, and 10 with hyperplasia) were included. A targeted sequence was performed for LVLs and background mucosa using an esophageal cancer panel. Each mutation was checked whether it was oncogenic or not concerning OncoKB. In LVLs, TP53 was the most dominant mutation (80%). Furthermore, 72% of TP53 mutations was putative drivers. In background mucosa, NOTCH1 was the most dominant mutation (88%) and TP53 was the second most dominant mutation (48%). Furthermore, 73% of TP53 mutations and 8% of NOTCH1 mutations were putative drivers. Putative driver mutations of TP53 had significantly higher allele frequency (AF) in SCC than in IN and hyperplasia. Conversely, putative driver mutations of NOTCH1 did not have a significant accumulation of AF in the progression of carcinogenesis. Furthermore, in SCC, AF of TP53 mutations was significantly higher in LVLs than in background mucosa, but not in IN and hyperplasia. Regarding NOTCH1, a significant difference was not observed between LVLs and background mucosa in each group. The background mucosa in patients with LVLs already had putative driver mutations such as TP53 and NOTCH1. Of these two genes, TP53 mutation could be the main target gene of carcinogenesis in esophageal SCC. Clinical Trials registry: UMIN000034247.

Tumor-Associated Microbiota in Esophageal Squamous Cell Carcinoma.

Important evidence indicates the microbiota plays a key role in esophageal squamous cell carcinoma (ESCC). The esophageal microbiota was prospectively investigated in 18 patients with ESCC and 11 patients with physiological normal (PN) esophagus by 16S rRNA gene profiling, using next-generation sequencing. The microbiota composition in tumor tissues of ESCC patients were significantly different from that of patients with PN tissues. The ESCC microbiota was characterized by reduced microbial diversity, by decreased abundance of Bacteroidetes, Fusobacteria, and Spirochaetes. Employing these taxa into a microbial dysbiosis index demonstrated that dysbiosis microbiota had good capacity to discriminate between ESCC and PN esophagus. Functional analysis characterized that ESCC microbiota had altered nitrate reductase and nitrite reductase functions compared with PN group. These results suggest that specific microbes and the microbiota may drive or mitigate ESCC carcinogenesis, and this study will facilitate assigning causal roles in ESCC development to certain microbes and microbiota.

SOX2 enhances cell survival and induces resistance to apoptosis under serum starvation conditions through the AKT/GSK-3β signaling pathway in esophageal squamous cell carcinoma.

The human SOX2 gene was recently identified as a novel major oncogene, recurrently amplified and overexpressed in esophageal squamous cell carcinoma (ESCC). However, the role and molecular mechanism of SOX2 in the carcinogenesis of ESCC remain to be elucidated. The present study investigated the effect of SOX2 on ESCC cell survival and resistance to apoptosis under serum starvation conditions. An adenoviral vector-mediated expression system and RNA interference were used to study the effect of SOX2. The present results revealed that SOX2 promoted ESCC cell survival and enhanced resistance to apoptosis under serum starvation conditions, but not in culture conditions with serum. Mechanistically, SOX2 increased the expression levels of phosphorylated AKT and glycogen synthase kinase-3β (GSK-3β), a downstream factor of AKT, under serum starvation conditions, leading to the promotion of ESCC cell survival. Additionally, SOX2 activated AKT through the PTEN/PI3K/phosphoinositide-dependent protein kinase 1 and mammalian target of rapamycin complex 2 signaling pathways. Therefore, SOX2 may facilitate the survival of ESCC cells under poor nutrient conditions by activating the AKT/GSK-3β signaling pathway.

Identification and Validation of Potential Pathogenic Genes and Prognostic Markers in ESCC by Integrated Bioinformatics Analysis.

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal malignancies of the digestive tract, but its underlying molecular mechanisms are not known. We aim to identify the genes involved in ESCC carcinogenesis and discover potential prognostic markers using integrated bioinformatics analysis. Three pairs of ESCC tissues and paired normal tissues were sequenced by high-throughput RNA sequencing (RNA-seq). Integrated bioinformatics analysis was used to identify differentially expressed coding genes (DECGs) and differentially expressed long non-coding RNA (lncRNA) genes (DELGs). A protein–protein interaction (PPI) network of DECGs was established using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) website and visualized with Cytoscape. Survival analysis was conducted by log-rank tests to identify “hub” genes with potential prognostic value, and real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was conducted to assess expression of these genes in ESCC tissues. TranswellTM assays were employed to examine the migration ability of cells after knockdown of LINC01614 expression, followed by investigation of epithelial–mesenchymal transition (EMT) by western blotting (WB). A total of 106 upregulated genes and 42 downregulated genes were screened out from the ESCC data sets. Survival analysis showed two hub protein-coding genes with higher expression in module 1 of the PPI network (SPP1 and BGN) and another three upregulated lncRNAs (LINC01614, LINC01415, NKILA) that were associated with a poor prognosis. High expression of SPP1, BGN, LINC01614, and LINC01415 in tumor samples was validated further by RT-qPCR. In vitro experiments show that knockdown of LINC01614 expression could significantly inhibit the migration of ESCC cells by regulating EMT, which was confirmed by WB. These results indicate that BGN, SPP1, LINC01614, and LINC01415 might be critical genes in ESCC and potential prognostic biomarkers.

Circ0120816 acts as an oncogene of esophageal squamous cell carcinoma by inhibiting miR-1305 and releasing TXNRD1

BackgroundCircular RNAs (circRNAs) have been discovered to participate in the carcinogenesis of multiple cancers. However, the role of circRNAs in esophageal squamous cell carcinoma (ESCC) progression is yet to be properly understood. This research aimed to investigate and understand the mechanism used by circRNAs to regulate ESCC progression.MethodsBioinformatics analysis was first performed to screen dysregulated circRNAs and differentially expressed genes in ESCC. The ESCC tissue samples and adjacent normal tissue samples utilized in this study were obtained from 36 ESCC patients. All the samples were subjected to qRT-PCR analysis to identify the expression of TXNRD1, circRNAs, and miR-1305. Luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay were later conducted to verify the existing relationship among circ0120816, miR-1305 and TXNRD1. CCK-8, BrdU, cell adhesion, cell cycle, western blot and caspase 3 activity assays were also employed to evaluate the regulation of these three biological molecules in ESCC carcinogenesis. To evaluate the effect of circ0120816 on ESCC tumor growth and metastasis, the xenograft mice model was constructed.ResultsExperimental investigations revealed that circ0120816 was the highest upregulated circRNA in ESCC tissues and that this non-coding RNA acted as a miR-1305 sponge in enhancing cell viability, cell proliferation, and cell adhesion as well as repressing cell apoptosis in ESCC cell lines. Moreover, miR-1305 was observed to exert a tumor-suppressive effect in ESCC cells by directly targeting and repressing TXNRD1. It was also noticed that TXNRD1 could regulate cyclin, cell adhesion molecule, and apoptosis-related proteins. Furthermore, silencing circ0120816 was found to repress ESCC tumor growth and metastasis in vivo.ConclusionsThis research confirmed that circ0120816 played an active role in promoting ESCC development by targeting miR-1305 and upregulating oncogene TXNRD1.

Common gene signatures and key pathways in hypopharyngeal and esophageal squamous cell carcinoma

Background:Hypopharyngeal and esophageal squamous cell carcinoma (ESCC) are the most common double primary tumors with poor prognosis. Intensive work has been made to illuminate the etiology, but the common carcinogenic mechanism remains unclear. Thus, we conducted the study to seek to find the common gene signatures and key functional pathways associated with oncogenesis and treatment in hypopharyngeal squamous cell carcinoma (HSCC) and ESCC by bioinformatic analysis.Methods:Three independent datasets (GSE2379, GSE20347, and GSE75241) were screened out from the Gene Expression Omnibus (GEO) database and the overlapping differentially expressed genes (DEGs) were identified using GEO2R online platform. Subsequently, the Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis of DEGs were conducted using database for annotation, visualization and integrated discovery (DAVID). Meanwhile, the protein–protein interaction network (PPI) constructed by search tool for the retrieval of interacting genes (STRING) was visualized using Cytoscape. Afterwards, the most key module and hub genes were extracted from the PPI network using the Molecular Complex Detection plugin. Moreover, the gene expression profiling interactive analysis (GEPIA) was applied to verify the expression differences and conduct the survival analyses of hub genes. Finally, the interaction network of miRNAs and hub genes constructed by encyclopedia of RNA interactomes (ENCORI) was visualized using Cytoscape.Results:A total of 43 DEGs were identified, comprising 25 upregulated genes and 18 downregulated genes, which were mainly involved in the extracellular matrix-receptor interaction, collagen metabolic, epidermis development, cell adhesion, and PI3K/Akt signaling pathways. Subsequently, 12 hub genes were obtained and survival analysis demonstrated SERPINE1 and SPP1 were closely related to poor prognosis of patients with HSCC and ESCC. Finally, hsa-miR-29c-3p, hsa-miR-29a-3p, and hsa-miR-29b-3p were confirmed as the top 3 interactive miRNAs that target the most hub genes according to the interaction network of miRNAs and hub genes.Conclusion:The common gene signatures and functional pathways identified in the study may contribute to understanding the molecular mechanisms involved in the carcinogenesis and progression of HSCC and ESCC, and provide potential diagnostic and therapeutic targets.

ADAMTS9-AS2 and CADM2 expression and association with the prognosis in esophageal squamous cell carcinoma.

Background: We investigated whether ADAMTS9-AS2 and CADM2 were related to esophageal squamous cell carcinoma (ESCC). Methodology: ESCC microarray datasets and reverse transcriptase qualitativePCR were used to analyze ADAMTS9-AS2 and CADM2 expression. Results: TheGSE120356 and GSE33810 datasets identified ADAMTS9-AS2 and CADM2 as the candidates and ADAMTS9-AS2 and CADM2 expression was downregulated in ESCC. ADAMTS9-AS2 and CADM2 were positively correlated with ESCC. ADAMTS9-AS2 and CADM2 expression coulddiscriminate ESCC from normal tissue. Five-year overall survivalwas shorter in underexpressed ADAMTS9-AS2 patients, and CADM2 expression level was related to 5-yearoverall survival. ADAMTS9-AS2 and CADM2 expression were independentprognosis indicators in ESCC patients. Conclusion: Our findings shed new light on the clinical significance of ADAMTS9-AS2 and CADM2 in ESCC carcinogenesis.

Effects of microRNA-182-5p on cell proliferation and invasion of esophageal squamous cell carcinoma and related molecular mechanisms

Objective: To investigate the effects of microRNA-182-5p (miR-182-5p) on cell proliferation and invasion of esophageal squamous cell carcinoma (ESCC) and its related molecular mechanisms. Methods: Real-time quantitative polymerase chain reaction (RT-qPCR) was employed to detect the miR-182-5p expression in ESCC tissues and cells. MiR-182-5p inhibitor, miR-182-5p mimic and negative control (NC) were transfected into ESCC Eca109 and TE1 cells, and miR-182-5p expression after transfection was examined using RT-qPCR. Cell counting kit-8 (CCK-8) was utilized to investigate the cell proliferation and Transwell chamber was used to detect the cell invasion ability. Dual-luciferase reporter assay was used to detect the direct interaction of miR-182-5p and cell adhesion molecule 2 (CADM2), RT-qPCR was employed to detect CADM2 expression in ESCC tissues, the correlation between CADM2 and miR-182-5p was also examined. Finally, western blot was used to detect the protein expressions of CADM2, focal adhesion kinase (FAK), p-Akt and Akt after transfection. Results: The miR-182-5p level in ESCC tissues was (2.180±1.295), significantly higher than (0.890±0.284) in normal esophageal epithelial tissues (P<0.001). The survival ratio of ESCC patients with high miR-182-5p level was evidently lower than that of ESCC patients with low miR-182-5p level (P<0.05). MiR-182-5p expression was significantly associated with TNM staging and lymph node metastasis (P<0.05). The expressions of miR-182-5p in ESCC cells including EC9706, Eca109, TE1, KYSE450 and KYSE70 were (2.449±0.082), (2.965±0.088), (4.873±0.258), (1.338±0.045) and (1.999±0.082), respectively, obviously higher than (0.989±0.087) in normal esophageal epithelial cell Het-1A (all P<0.01). Besides, miR-182-5p inhibitor significantly downregulated the miR-182-5p expression in Eca109 and TE1, and suppressed cell proliferation and invasion ability. Conversely, miR-182-5p mimic significantly upregulated the miR-182-5p expression in Eca109 and TE1, and promoted cell proliferation and invasion ability. Dual-luciferase reporter assay revealed that co-transfection of CADM2-3'UTR-WT and miR-182-5p mimic significantly reduced the luciferase activities in Eca109 and TE1 cells (P<0.01), and CADM2 was the direct target of miR-182-5p. The expression of CADM2 in ESCC tissues was (0.190±0.143), markedly lower than (0.845±0.327) in normal esophageal epithelial tissues (P<0.001). The miR-182-5p level exhibited negative correlation with CADM2 level in ESCC tissues (r=-0.5004, P<0.001). In addition, CADM2 expression was closely correlated with TNM staging and lymph node metastasis (P<0.05). The survival ratio of ESCC patients with high CADM2 level was evidently higher than that of ESCC patients with low CADM2 level (P<0.05). MiR-182-5p inhibitor significantly upregulated the expression of CADM2 protein, but suppressed the protein expressions of FAK, p-Akt and Akt, whereas miR-182-5p mimic markedly downregulated the expression of CADM2 protein, but promoted the protein expressions of FAK, p-Akt and Akt. Conclusion: MiR-182-5p is implicated in the carcinogenesis and development of ESCC, and thus may be a potential molecular target for ESCC patients.

LINC00673 Represses CDKN2C and Promotes the Proliferation of Esophageal Squamous Cell Carcinoma Cells by EZH2-Mediated H3K27 Trimethylation.

Long non-coding RNAs (lncRNAs), some of the most abundant epigenetic regulators, play an important role in esophageal squamous cell carcinoma (ESCC). In the current study, the functions and mechanisms of the lncRNA LINC00673 were investigated. The expression levels of LINC00673 and its potential target genes were assessed by quantitative real-time polymerase chain reaction (qPCR) in ESCC surgical specimens and ESCC cell lines. RNA fluorescence in situ hybridization (RNA FISH) was employed to detect the subcellular location and the levels of LINC00673 in ESCC samples from patients with different survival times. LINC00673 function in ESCC carcinogenesis was also evaluated in vivo and in vitro. Cell cycle synchronization was performed using serum withdrawal; the cell cycle was monitored by fluorescence analysis and cellular DNA was detected by flow cytometry. The molecular mechanisms underlying LINC00673 were explored via Western blotting, chromatin immunoprecipitation (ChIP), and ChIP-PCR. Up-regulated LINC00673 was associated with poor prognosis in ESCC patients and promoted the proliferation of ESCC cells both in vitro and in vivo. Compared to the control group, depletion of LINC00673 in ESCC cells arrested the cell cycle, at least, at the G1/S checkpoint. Knockdown of LINC00673 significantly enhanced posttranscriptional expression of CDKN2C, and histone 3 lysine 27 trimethylation (H3K27me3) was enriched at the promoter region of CDKN2C. After inhibiting EZH2, the CDKN2C expression levels were increased. The present findings are the first to reveal that LINC00673 represses CDKN2C expression and promotes ESCC cell proliferation by elevating EZH2-mediated H3K27me3 levels. These data suggest that LINC00673 regulates the cell cycle in ESCC and that it is a promising target for clinical therapy.

Hsa_circRNA6448-14 promotes carcinogenesis in esophageal squamous cell carcinoma.

Circular RNAs (circRNAs) play important roles in cancer progression. hsa_circRNA6448-14 originates from exon 5 to exon 11 of the TGFBI gene. We investigated the roles of hsa_circRNA6448-14 in esophageal squamous cell carcinoma (ESCC) with microarrays and quantitative real-time polymerase chain reaction (qRT-PCR), Kaplan-Meier analysis, loss-of-function and gain-of-function assays, and pull-down assays for miRNA binding. The hsa_circRNA6448-14-miRNA-mRNA network was drawn using Circos. hsa_circRNA6448-14 was significantly upregulated in ESCC tissues and cell lines. As a diagnostic biomarker, hsa_circRNA6448-14 had an area under the curve (AUC), sensitivity, and specificity of 0.906, 82.9%, and 85.5%, respectively. hsa_circRNA6448-14 upregulation was correlated with poor differentiation, advanced pTNM stage, poor disease-free survival (DFS), and poor overall survival (OS). Elevated hsa_circRNA6448-14 promoted cell proliferation, migration, invasion, and inhibited apoptosis in vitro. hsa_circRNA6448-14 functioned as a miRNA sponge to competitively bind miR-455-3p, and hsa_circRNA6448-14 expression negatively correlated with that of miR-455-3p. hsa_circRNA6448-14 promoted carcinogenesis in ESCC, suggesting that hsa_circRNA6448-14 could serve as a diagnostic and prognostic biomarker for ESCC.