Normal Human Esophageal Epithelial Cells Research Articles

NEK2 Promotes ESCC Malignant Progression by Inhibiting Cellular Senescence via the FOXM1/c-Myc/p27 Signaling Pathway.

Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) is a crucial serine-threonine kinase involved in the process of cell mitosis. However, the precise relationship between NEK2 and esophageal squamous cell carcinoma (ESCC) remains inadequately understood. NEK2 expression in ESCC tissues was assessed through bioinformatics analysis, reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry, revealing a correlation with ESCC patient prognosis. Cultured ESCC cells and human normal esophageal epithelial cells (HEEC) were used to investigate the effects of NEK2 knockdown on the development and progression of ESCC by integrated confluence algorithm, colony formation, wound-healing, transwell, and ESCC xenograft tumor model, in vitro and in vivo. In ESCC tissues, NEK2 was found to be significantly upregulated, and its expression correlated with poor prognosis in ESCC patients. NEK2 may facilitate ESCC development by regulating cell proliferation, migration, and invasion. Additionally, results from in vivo experiments suggested that NEK2 knockdown can inhibit tumor growth. Moreover, forkhead box M1 (FOXM1) was identified as a potential downstream target of NEK2 in the regulation of ESCC, with its overexpression reversing the effects of NEK2 knockdown on ESCC. Mechanistic studies also indicated that NEK2 may promote the malignant progression of ESCC by inhibiting cellular senescence through the activation of the FOXM1/c-Myc/p27 signaling pathways, which may provide a novel perspective for the management of ESCC.

Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids.

Background: Esophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbecco's Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. Methods: We have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize the ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-β receptor-mediated signaling, both key regulators of the proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. Results: The formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGFβ-receptor-mediated signaling. Optimized HOME0 improved normal human esophageal organoid formation. In the HOME0-grown organoids, IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media.

The role of LncRNA TUG1 in DNA damage and malignant transformation induced by Helicobacter pylori and N-methyl-N′-nitro-N-nitrosoguanidine on human esophageal epithelial cells

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) and Helicobacter pylori might synergistically promote the malignant transformation of human esophageal epithelial cells (HEECs) through inducing DNA double-strand breaks (DSBs) and inhibition of PAXX protein expression. The long noncoding RNA (lncRNA) TUG1 is associated with multiple cancers, and its overexpression can promote cancer by interfering with the functions of oncogenes. LncRNA TUG1 is also associated with DNA methyltransferase 1 (DNMT1) and the epithelial signaling pathway of H. pylori infection. To explore the role of LncRNA TUG1 in the malignant transformation of HEECs induced by H.pylori + MNNG, a stable strain of HEECs with LncRNA TUG1 knockdown (LncRNA TUG1-KD) was constructed using RNA interference technology with lentivirus as a vector. Set up negative controls LncRNA TUG1-NC (null carrier lentivirus was selected to transfect HEECs) and block controls (normal HEECs without exposure). H. pylori + MNNG were added to the LncRNA TUG1-KD and LncRNA TUG1-NC groups for 6 h and then passaged until their malignant transformation. From each group, cells in the early, intermediate, and late stages of malignant transformation were used for the alkaline comet assay and determination of protein expression, including γ-H2AX and PAXX, by western blotting assay to assess DNA damage and repair processes. Clone formation assay in soft agar and nude mouse xenograft model was used to assess malignancy. This study suggests that H. pylori + MNNG promotes the malignant transformation of HEECs by inducing DNA DSBs and inhibiting PAXX expression, and this effect may be alleviated by LncRNA TUG1 knockdown. It elucidates the pathogenesis of EC from the perspective of the combined effect of epigenetic and environmental carcinogens, offering new insights for the comprehensive prevention and treatment of EC.

Circ_0003855 involvement of esophageal cancer progression through miR-622/FLOT1.

To confirm the relationship between Circ_0003855 and EC, we purchased the Human esophageal carcinoma cell line Eca109 and normal human esophageal epithelial cells HEEC, and the expression levels of Circ_0003855, miR-622, and FLOT1 were detected. The results show that Circ_0003855 and FLOT1 were highly expressed in Eca109 cells, while miR-622 was lowly expressed (p < 0.05). Subsequently, Circ_0003855 small interfering RNA (si-Circ_0003855) and its negative control (si-NC) were used to detect changes in cellular biological behaviors. We found that the activity of Eca109 cells was reduced after interfering with the expression of Circ_0003855, and miR-622 expression was elevated, while FLOT1 was decreased (p < 0.05). Additionally, si-Circ_0003855 and miR-622 inhibitor sequence (miR-622-inhibition) were co-transfected into cells with miR-622-inhibition alone, and untreated Eca109 cells were used as a control to detect the expression of FLOT1. Co-transfection of si-Circ_0003855 and miR-622-inhibition showed no significant difference in FLOT1 expression compared to the control cells (p > 0.05). Synthesizing the results of these experiments above, we believe that interfering with the expression of Circ_0003855 can inhibit the activity of EC cells, and its mechanism is related to miR-622 and FLOT1.

Linc01614 Regulates the Proliferation, Apoptosis, and Chemotherapy Resistance in Esophageal Squamous Cell Carcinoma by Targeting Mir-4775.

Esophageal squamous cell carcinoma (ESCC), a widely known esophageal disease, severely affects people's health. Numerous investigations demonstrated that long non-coding RNAs (lncRNAs) performed key jobs inside a wide scope of organic cycles and stand out in malignant growth. Our study planned to investigate the roles and mechanisms of linc01614 in ESCC. A Total of 60 ESCC tissue samples including 30 patients with cisplatin sensitivity and 30 patients with cisplatin resistance, who received DDP-based treatment, were obtained from Zhuhai People's Hospital, Zhuhai City during 2021. These tissues were frozen and saved in a -80 °C ultra-low temperature freezer. We performed CCK-8, clone formation, flow cytometry assays to determine the effect of linc01614 on ESCC progression, and explored the specific mechanism of linc01614 in ESCC cell proliferation, apoptosis, and chemotherapy resistance. linc01614 expression was upregulated in ESCC tissues and cells compared with non-tumor tissues and human normal esophageal epithelial cells (Het-1A). Knockdown of linc01614 repressed cell expansion, chemotherapy opposition, and advanced cell apoptosis in ESCC. Besides, linc01614 regulated the expression of miR-4775 as a competitive endogenous RNA (ceRNA). The linc01614/miR-4775 axis played an important role in ESCC progression and drug resistance, revealing that linc01614 is a promising target in ESCC treatment.

Research on essential performance of oxidized chitosan-crosslinked acellular porcine aorta modified with bioactive SCPP/DOPA for esophageal scaffold with enhanced mechanical strength, biocompatibility and anti-inflammatory

Acellular porcine aorta (APA) is an excellent candidate for an implanted scaffold but needs to be modified with appropriate cross-linking agent to increase its mechanical property and storage time in vitro as well as to give itself some bioactivities and eliminate its antigenicity for acting as a novel esophageal prosthesis. In this paper, a polysaccharide crosslinker (oxidized chitosan, OCS) was prepared by oxidizing chitosan using NaIO4 and further used to fix APA to prepare a novel esophageal prosthesis (scaffold). And then the surface modification with dopamine (DOPA) and strontium-doped calcium polyphosphate (SCPP) were performed one after another to prepare DOPA/OCS-APA and SCPP-DOPA/OCS-APA to improve the biocompatibility and inhibit inflammation of the scaffolds. The results showed that the OCS with a feeding ratio of 1.5:1.0 and a reaction time of 24 h had a suitable molecular weight and oxidation degree, almost no cytotoxicity and good cross-linking effect. Compared with glutaraldehyde (GA) and genipin (GP), OCS-fixed APA could provide a more suitable microenvironment for cell proliferation. The vital cross-linking characteristics and cytocompatibility of SCPP-DOPA/OCS-APA were evaluated. Results suggested that SCPP-DOPA/OCS-APA exhibited suitable mechanical properties, excellent resistance to enzymatic degradation/acid degradation, suitable hydrophilicity, and the ability to promote the proliferation of Human normal esophageal epithelial cells (HEECs) and inhibit inflammation in vitro. In vivo tests also confirmed that SCPP-DOPA/OCS-APA could diminish the immunological response to samples and had a positive impact on bioactivity and anti-inflammatory. In conclusion, SCPP-DOPA/OCS-APA could act as an effective, bioactive artificial esophageal scaffold and be expected to be used for clinical in the future.

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.

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.

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.

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.

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.

MicroRNA‑454‑3p inhibits cell proliferation and invasion in esophageal cancer by targeting insulin‑like growth factor 2 mRNA‑binding protein 1

Esophageal cancer (ESCA) is the eighth most common cause of cancer-associated mortality in humans. An increasing number of studies have demonstrated that microRNAs (miRs) serve important roles in mediating tumor initiation and progression. miR-454-3p has been found to be involved in the development of various human malignancies; however, little is known about the role of miR-454-3p in esophageal cancer. In the present study, the protein and gene expression levels of miR-454-3p in ESCA tissues and cells were downregulated compared with adjacent normal tissues and normal human esophageal epithelial cells. Additionally, miR-454-3p downregulation resulted in improved survival rates in patients with ESCA, and miR-454-3p overexpression significantly suppressed cell proliferation, migration and invasion and promoted apoptosis in four ESCA cell lines (EC9706, ECA109, TE-1 and TE-8). It was found that miR-454-3p overexpression inhibited the expression of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) at the protein and mRNA expression levels. Furthermore, it was demonstrated that miR-454-3p inhibited ESCA cell proliferation, migration and apoptosis by targeting IGF2BP1 via the ERK and AKT signaling pathways in a subcutaneous xenograft tumor mouse model. These results showed that miR-454-3p functioned as an important tumor suppressor in ESCA by targeting IGFBP1. Therefore, miR-454-3p may be a novel prognostic biomarker and therapeutic target for patients with ESCA.

Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes.

During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.

Effect of hsa_circ_RNA0023397 regulation of miR-106b expression on proliferation and apoptosis of esophageal cancer cells.

To explore the potential function of a candidate circular ribonucleic acid (circRNA) [human serum albumin (hsa)_circ_RNA0023397] in esophageal cancer cells. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the expression level of hsa_circ_RNA0023397 in three esophageal cancer cell lines (KYSE-150, ECA109, and TE-1), which was compared with that in normal human esophageal epithelial cell line (HET-1A). The expression plasmid of hsa_circ_RNA0023397 was constructed, and the effect of overexpression of hsa_circ_RNA0023397 on cell proliferation was determined by cell counting kit-8 (CCK-8) and colony formation assay. The effect of overexpression of hsa_circ_RNA0023397 on cell apoptosis was detected by flow cytometry. Further bioinformatics analysis and Luciferase reporter gene analysis were carried out to explore the role of hsa_circ_RNA0023397 as a sponge of micro RNAs (miRNAs). Compared with that in normal human esophageal epithelial cell line HET-1A, the expression of hsa_circ_RNA0023397 was down-regulated in three esophageal cancer cell lines in vitro. Overexpression of hsa_circ_RNA0023397 overtly inhibited KYSE-150 cell proliferation and promoted its apoptosis. Bioinformatics prediction and Luciferase reporter gene assay confirmed that hsa_circ_RNA0023397 could bind to miR-160b. MiR-106b participated in hsa_circ_RNA0023397-mediated inhibition of proliferation of esophageal cancer KYSE-150 cells. Hsa_circ_RNA0023397 is down-regulated in esophageal cancer cells and can act as miR-106b to affect the biological function of esophageal cancer cells.

The Depletion of ABI3BP by MicroRNA-183 Promotes the Development of Esophageal Carcinoma.

Background Esophageal cancer (EC), as a serious threat to human life and health, is one of the most common cancers around the world. Many studies have suggested that many microRNAs are involved in tumorigenesis and progression. Methods To search for a novel and promising predictive therapeutic target or biomarker to achieve the goal of the early diagnosis and treatment of EC, we used the EC cell lines Eca-109 and KYSE-150 and normal human esophageal epithelial cells (HEECs) to investigate the effect of ABI3BP on EC. Results We found that ABI family member 3 binding protein (ABI3BP) was downregulated in EC and suppressed the proliferation, activity, migration, and invasion of EC cells. ABI3BP was downregulated by miR-183, which plays the role of an oncogene. Conclusion ABI3BP and miR-183 can be considered potential biomarkers for the diagnosis of patients with EC and can be effective targets for antitumor therapy.

Cucurbitacin E inhibits esophageal carcinoma cell proliferation, migration, and invasion by suppressing Rac1 expression through PI3K/AKT/mTOR pathway.

As an oxygenated tetracyclic triterpenoid, Cucurbitacin E (CuE) possesses potential antitumor properties in sorts of malignancies. However, its effect on human esophageal carcinoma cells has not been previously unearthed, and the mechanism underlying its anticarcinoma activity remains vague. Hence, this study was arranged to probe the function of CuE on esophageal carcinoma cells and its specific mechanism. Human esophageal carcinoma cells (ECA109 and EC9706) and human normal esophageal epithelial cells (Het-1A) were selected for subsequent experiments. The expression levels of Rac1 in esophageal carcinoma cells were measured. After transfection of sh-Rac1 or pCDNA3.1-Rac1, esophageal carcinoma cells were exposed to CuE. Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-ethynyl-2'-deoxyuridine staining were utilized for measurement of cell proliferation ability, cell scratch assay for inspection of cell migration rate, and Transwell for detection of cell invasion ability. The phosphorylation levels of protein kinase B and mTOR were analyzed by Western blot. Rac1 was highly expressed in esophageal carcinoma cells. Transfection of sh-Rac1 in esophageal carcinoma cells resulted in suppression on cell proliferation, migration, and invasion, as well as downregulated phosphorylation levels of AKT and mammalian target of rapamycin (mTOR) in esophageal carcinoma cells, while transfection of pCDNA3.1-Rac1 had an opposite effect, implicating that Rac1 can promote the viability of esophageal carcinoma cells. Esophageal carcinoma cells subjected to CuE treatment had decreased expression of Rac1, suppressed cell viability, and decreased phosphorylation levels of AKT and mTOR. Transfection of pCDNA3.1-Rac1 and CuE treatment in esophageal carcinoma cells enhanced viability of esophageal carcinoma cells and promoted the phosphorylation levels of AKT and mTOR in comparison with cells treated with CuE alone. CuE inhibits proliferation, invasion, and migration of esophageal carcinoma cells via downregulating Rac1 to block the phosphoinositide 3-kinase/AKT/mTOR pathway.

Ubiquitination of NOTCH2 by DTX3 suppresses the proliferation and migration of human esophageal carcinoma.

The NOTCH2 gene plays a role in the development of many tumors. Deltex E3 ubiquitin ligase 3 (DTX3) was identified as a novel E3 ligase for NOTCH2 and as a potential therapeutic target for esophageal cancer. However, whether DTX3 could regulate NOTCH2 to suppress the progression of esophageal carcinoma remains unknown. In our study, NOTCH2 had higher expression in human esophageal carcinoma cell lines compared to normal human esophageal epithelial cell line, and ablation of NOTCH2 suppressed the proliferation and migration of esophageal carcinoma cells. A novel E3 ligase for NOTCH2 was identified by yeast two‐hybrid (Y2H) screening, and DTX3 promoted the ubiquitination and degradation of NOTCH2. Further study showed that DTX3 overexpression suppressed the proliferation and tumorigenicity of human oesophageal carcinoma cells. The analysis of tissue samples from patients revealed that the expression of NOTCH2 was high while the expression of DTX3 was low in esophageal cancer. Furthermore, the expression of DTX3 and NOTCH2 showed a significant negative correlation in human oesophageal cancer samples. Our study suggested that the DTX3‐NOTCH2 axis plays an important role in the progression of esophageal cancer, and DTX3 acts as an anti–oncogene in esophageal carcinoma, potentially offering a therapeutic target for esophageal cancer.

EphA5 knockdown enhances the invasion and migration ability of esophageal squamous cell carcinoma via epithelial-mesenchymal transition through activating Wnt/\u03b2-catenin pathway

BackgroundThe erythropoietin-producing hepatocellular (Eph) receptor A5 (EphA5) has been found to be overexpressed in some malignant tumors and is associated with disease prognosis. However, the role of EphA5 in esophageal squamous cell carcinoma (ESCC) is not clear.MethodsIn the present study, we measured the expression of EphA5 in ESCC tissues and cell lines including KYSE150 and KYSE450 cells. siRNA transfection was used to interfere with EphA5 expression in ESCC cell lines. Cell viability, colony formation, scratch and invasion assays were performed to explore the roles of EphA5 in ESCC cell lines. Flow cytometry analysis was performed to investigate whether EphA5 could affect the cell apoptosis and cycle. The biomarkers related to epithelial-mesenchymal transition (EMT) and molecules associated with Wnt/β‑catenin signaling were also measured by western blot and immunofluorescence.ResultsThe protein and mRNA expression of EphA5 were significantly higher in fresh ESCC tissues and cell lines compared with normal control groups and human normal esophageal epithelial cells (HEEC). The cell viability assay and colony formation assay revealed that EphA5 knockdown enhanced the proliferation of KYSE150 and KYSE450 cells in vitro. The invasion and migration of ESCC cells were accelerated after EphA5 knockdown. The expression of EMT biomarkers was altered in ESCC cells transfected with siRNA targeting EphA5. Moreover, EphA5 downregulation enhanced the protein levels of β‑catenin and p-GSK-3βSer9, which play a key role in the Wnt/β‑catenin pathway.ConclusionsEphA5 knockdown promotes the proliferation of esophageal squamous cell carcinoma,enhances invasion and migration ability via epithelial-mesenchymal transition through activating Wnt/β‑catenin pathway.

LncRNA CCAT2 promotes radiotherapy resistance for human esophageal carcinoma cells via the miR‑145/p70S6K1 and p53 pathway.

The long non‑coding RNA colon cancer‑associated transcript2 (CCAT2) is abnormally expressed in various types of malignant tumor tissues and considered to be an oncogene, including for esophageal cancer (EC). Radiotherapy is an important and widely used cancer treatment. However, some patients with EC do not respond to radiotherapy. This study was designed to investigate effects of CCAT2 expression on radiotherapy dynamics for ECcells and to explore underlying molecular mechanisms. Reverse transcription‑quantitative PCR was used to measure CCAT2 expression in EC tissues, normal esophageal mucosa, ECcells and normal human esophageal epithelial cells. TUNEL assays were used to assess the effect of CCAT2 on X‑ray‑induced apoptosis of ECcells. Protein expression was detected by western blot. CCAT2 was highly expressed in EC tissues and ECcells, and was negatively associated with radiotherapy efficacy in patients with EC. Invitro, knockdown of CCAT2 enhanced radiosensitivity of ECcells and promoted apoptosis by increasing Bax/Bcl2 and active‑caspase3/caspase3 following X‑ray treatment. In addition, CCAT2 negatively regulated miR‑145 and P70 ribosomal protein S6 kinase1 (p70S6K1) expression, and inhibited phosphorylation of Akt, ERK and p70S6K1 in ECcells. After X‑ray treatment, CCAT2 negatively regulated protein levels of p53, P21 and c‑Myc. These results showed that CCAT2 promoted the radiotherapy resistance of ECcells via negative regulation of the miR‑145/p70S6K1 and the p53 signaling pathways and associated elements may be potential targets for improving the sensitivity of EC radiotherapy.