EC9706 Cells Research Articles

ZNF8 promotes progression of gastrointestinal cancers via a p53-dependent mechanism

p53 is a critical tumor suppressor, and the disruption of its normal function is often a prerequisite for the development or progression of tumors. Our previous works revealed that multiple members of Krüppel-associated box (KRAB) domain zinc-finger proteins (KZFPs) family regulate p53 transcriptional activity by interacting with it. But the tumor biology functions of these members have not been fully elucidated. Here, the pan-cancer analysis related to gastrointestinal cancers (GICs) revealed that ZNF8, a p53-interacting protein, is an unfavorable prognostic factor for patients with malignancies. ZNF8 interacts with p53 and further depresses its transcriptional activity in colon cancer cells. The knockdown of ZNF8 or the overexpression of ZNF8 inhibits or facilitates the in vitro colony formation, migration, invasion, and angiogenesis of p53+/+ colon cancer HCT116 cells, HepG2 cells and EC109 cells rather than p53−/− colon cancer HCT116 cells and p53-knockout HepG2 cells, respectively. Xenograft experiments conducted in vivo also showed that the knockdown of ZNF8 in p53+/+ but not in p53−/− HCT116 cells curbs the tumor growth and metastasis to lung, leading to an extended life span for tumor-bearing mice. Clinically, two independent immunohistochemistry cohorts of colon cancer and esophageal cancer also indicated that ZNF8 is higher expression in carcinoma tissues than adjacent tissues and this is associated with worse overall survival outcomes in patients without harboring p53 mutation. Together, our results provide insight into the p53-specific tumor oncogenic function of ZNF8. ZNF8 may prove to be a potential target for GICs treatment.

XRCC2 knockdown effectively sensitizes esophageal cancer to albumin-paclitaxel in vitro and in vivo.

Esophageal cancer (EC), a prevalent malignancy, has a high incidence and mortality. X-ray repair cross complementing 2 (XRCC2) functions on DNA damage and repair that works the progression of various cancers. Nevertheless, the role and mechanism of XRCC2 remain unknown in EC. The XRCC2 expression was examined by reverse transcription quantitative polymerase chain reaction and western blot. The function of XRCC2 in EC were investigated through cell counting kit-8, colony formation, transwell, flow cytometry, chromatin immunoprecipitation, luciferase, and western blot experiments. Besides, the role of XRCC2 in EC was assessed by western blot and immunohistochemistry experiments after nude mice were injected with EC109 cells and treated with nab-paclitaxel. The XRCC2 expression was upregulated in EC. Knockdown of XRCC2 diminished cell viability, and the number of colonies, migration cells and invasion cells of KYSE150 and EC109 cells. Silencing of XRCC2 diminished the cell viability of both two cells with a lower IC50, whereas boosted the apoptosis rate of both cells with the treatment of albumin-paclitaxel. All these outcomes were reverse with the upregulation of XRCC2 in both two cells. Mechanically, XRCC2 was transcriptionally regulated by specificity protein 1 (SP1), and silencing of SP1 inhibited the cell growth of EC. In vivo, transfection of shXRCC2 with or without albumin-paclitaxel treatment both decreased the tumor size and weight, as well as the expression of XRCC2 and Ki-67 in xenografted mice. XRCC2 transcriptionally regulated by SP2 promoted proliferation, migration, invasion, and chemoresistance of EC cells.

Baitouweng decoction suppresses growth of esophageal carcinoma cells through miR-495-3p/BUB1/STAT3 axis.

Esophageal carcinoma (EC) is one of the most prevalent cancers in human populations worldwide. Baitouweng decoction is one of the most important Chinese medicine formulas, with the potential to treat cancer. To investigate the role and mechanism of Baitouweng decoction on EC cells. Differentially expressed genes (DEGs) in EC tissues and normal tissues were screened by the cDNA microarray technique and by bioinformatics methods. The target genes of microRNAs were predicted based on the TargetScan database and verified by dual luciferase gene reporter assay. We used Baitouweng decoction to intervene EC cells, and detected the activity of EC9706 and KYSE150 cells by the MTT method. Cell cycle and apoptosis were measured by flow cytometry. The expression of BUB1 mRNA and miR-495-3p was measured by qRT-PCR. The protein levels of BUB1, STAT3, p-STAT3, CCNB1, CDK1, Bax, Caspase3, and Caspase9 were measured by Western blot analysis. The migration and invasion abilities of the cells were measured by wound-healing assay and Transwell invasion assay, respectively. DEGs identified are involved in biological processes, signaling pathways, and network construction, which are mainly related to mitosis. BUB1 was the key hub gene, and it is also a target gene of miR-495-3p. Baitouweng decoction could upregulate miR-495-3p and inhibit BUB1 expression. In vitro experiments showed that Baitouweng decoction significantly inhibited the migration and invasion of EC cells and induced apoptosis and G2/M phase arrest. After treatment with Baitouweng decoction, the expression of Bax, Caspase 3, and Caspase 9 in EC cells increased significantly, while the expression of BUB1, CCNB1, and CDK1 decreased significantly. Moreover, the STAT3 signaling pathway may play an important role in this process. Baitouweng decoction has a significant inhibitory effect on EC cell growth. BUB1 is a potential therapeutic target for EC. Further analysis showed that Baitouweng decoction may inhibit the growth of EC cells by upregulating miR-495-3p targeting the BUB1-mediated STAT3 signal pathway.

Luteolin enhances drug chemosensitivity by downregulating the FAK/PI3K/AKT pathway in paclitaxel‑resistant esophageal squamous cell carcinoma.

Drug resistance is a key factor underlying the failure of tumor chemotherapy. It enhances the stem‑like cell properties of cancer cells, tumor metastasis and relapse. Luteolin is a natural flavonoid with strong anti‑tumor effects. However, the mechanism(s) by which luteolin protects against paclitaxel (PTX)‑resistant cancer cell remains to be elucidated. The inhibitory effect of luteolin on the proliferation of EC1/PTX and EC1 cells was detected by cell counting kit‑8 assay. Colony formation and flow cytometry assays were used to assess clonogenic capacity, cell cycle and apoptosis. Wound healing and Transwell invasion tests were used to investigate the effects of luteolin on the migration and invasion of EC1/PTX cells. Western blotting was used to detect the protein levels of EMT‑related proteins and stem cell markers after sphere formation. Parental cells and drug‑resistant cells were screened by high‑throughput sequencing to detect the differential expression of RNA and differential genes. ELISA and western blotting were used to verify the screened PI3K/Akt signaling pathway, key proteins of which were explored by molecular docking. Hematoxylin and eosin staining and TUNEL staining were used to observe tumor xenografts on morphology and apoptosis in nude mice. The present study found that luteolin inhibited tumor resistance (inhibited proliferation, induced cell cycle arrest and apoptosis and hindered migration invasion, EMT and stem cell spherification) in vitro in PTX‑resistant esophageal squamous cell carcinoma (ESCC) cells. In addition, luteolin enhanced drug sensitivity and promoted the apoptosis of drug‑resistant ESCC cells in combination with PTX. Mechanistically, luteolin may inhibit the PI3K/AKT signaling pathway by binding to the active sites of focal adhesion kinase (FAK), Src and AKT. Notably, luteolin lowered the tumorigenic potential of PTX‑resistant ESCC cells but did not show significant toxicity in vivo. Luteolin enhanced drug chemosensitivity by downregulating the FAK/PI3K/AKT pathway in PTX‑resistant ESCC and could be a promising agent for the treatment of PTX‑resistant ESCC cancers.

Hypoxia exposure induces lactylation of Axin1 protein to promote glycolysis of esophageal carcinoma cells

The hypoxic microenvironment in esophageal carcinoma is an important factor promoting the rapid progression of malignant tumor. This study was to investigate the lactylation of Axin1 on glycolysis in esophageal carcinoma cells under hypoxia exposure. Hypoxia treatment increases pan lysine lactylation (pan-kla) levels of both TE1 and EC109 cells. Meanwhile, ECAR, glucose consumption and lactate production were also upregulated in both TE1 and EC109 cells. The expression of embryonic stem cell transcription factors NANOG and SOX2 were enhanced in the hypoxia-treated cells. Axin1 overexpression partly reverses the induction effects of hypoxia treatment in TE1 and EC109 cells. Moreover, lactylation of Axin1 protein at K147 induced by hypoxia treatment promotes ubiquitination modification of Axin1 protein to promote glycolysis and cell stemness of TE1 and EC109 cells. Mutant Axin1 can inhibit ECAR, glucose uptake, lactate secretion, and cell stemness in TE1 and EC109 cells under normal or hypoxia conditions. Meanwhile, mutant Axin1 further enhanced the effects of 2-DG on inhibiting glycolysis and cell stemness. Overexpression of Axin1 also inhibited tumor growth in vivo, and was related to suppressing glycolysis. In conclusion, hypoxia treatment promoted the glycolysis and cell stemness of esophageal carcinoma cells, and increased the lactylation of Axin1 protein. Overexpression of Axin1 functioned as a glycolysis inhibitor, and suppressed the effects of hypoxia exposure in vitro and inhibited tumor growth in vivo. Mechanically, hypoxia induces the lactylation of Axin1 protein and promotes the ubiquitination of Axin1 to degrade the protein, thereby exercising its anti-glycolytic function.

Unraveling the independent role of METTL3 in m6A modification and tumor progression in esophageal squamous cell carcinoma

METTL3 and METTL14 are traditionally posited to assemble the m6A methyltransferase complex in a stoichiometric 1:1 ratio, modulating mRNA fate via m6A modifications. Nevertheless, recent investigations reveal inconsistent expression levels and prognostic significance of METTL3 and METTL14 across various tumor types, challenging their consistent functional engagement in neoplastic contexts. A pan-cancer analysis leveraging The Cancer Genome Atlas (TCGA) data has identified pronounced disparities in the expression patterns, functional roles, and correlations with tumor burden between METTL3 and METTL14, particularly in esophageal squamous cell carcinoma (ESCC). Knockdown experiments of METTL3 in EC109 cells markedly suppress cell proliferation both in vitro and in vivo, whereas METTL14 knockdown shows a comparatively muted effect on proliferation and does not significantly alter METTL3 protein levels. mRNA sequencing indicates that METTL3 singularly governs the expression of 1615 genes, with only 776 genes co-regulated with METTL14. Additionally, immunofluorescence co-localization studies suggest discrepancies in cellular localization between METTL3 and METTL14. High-performance liquid chromatography–mass spectrometry (HPLC–MS) analyses demonstrate that METTL3 uniquely associates with the Nop56p-linked pre-rRNA complex and mRNA splicing machinery, independent of METTL14. Preliminary bioinformatics and multi-omics investigations reveal that METTL3’s autonomous role in modulating tumor cell proliferation and its involvement in mRNA splicing are potentially pivotal molecular mechanisms. Our study lays both experimental and theoretical groundwork for a deeper understanding of the m6A methyltransferase complex and the development of targeted tumor therapies focusing on METTL3.

Abstract 4720: Effects of removing heat and nourishing yin prescription on malignant phenotype and PI3K/AKT signaling pathway of human esophageal carcinoma cells

Abstract Introduction: Esophageal carcinoma (EC) ranks eighth in the incidence of malignant tumors and the sixth mortality worldwide. This study aims to screen out the Chinese herbs with strong inhibitory effects on the proliferation of four kinds of EC cells EC9706, EC-1, TE-1 and Eca109 from 366 Chinese herbs produced in Henan Province, and study the effect of composed Removing Heat and Nourshing Yin Prescription(RHNYP) on the proliferation, migration, invasion and PI3K/AKT signaling pathway of four EC cells. Methods: MTT and orthogonal design was applied to screen out the best effective Chinese herbs and determine the optimal dosage ratio of prescription on EC9706, EC-1, TE-1, Eca109 cells from 366 Chinese herbs alcohol extracts, the effects of this prescription on the proliferation, migration, invasion, clone formation, cell cycle and apoptosis of four EC cells were investigated by MTT, RTCA, soft agarose colony forming test, flow cytometry and western blot analysis. Then exploring the effects of RHNYP on EC related signaling pathways and key genes expression by western blot and RT-PCR assay. Results: Rhizoma Fagopyri Dibotryis(Jinqiaomai), Rhizoma Anemarrhenae(Zhimu), Asparagus Cochinchinensis(Tiandong), and Carpesium Abrolanoides Linne(Heshi) were screened out and composed Removing Heat and Nourshing Yin Prescription(RHNYP) according to their efficacy and orthogonal design. RHNYP could obviously inhibit the proliferation of EC9706, TE-1, EC-1, ECa109 cells with dose-effect and time-dependent manner. IC50 values of Jinqiaomai, Zhimu, Tiandong, Heshi in RHNYP for EC9706 cell were 27.639μg/ml, 40.015μg/ml, 9.044μg/ml, 11.208μg/ml respectively; for EC-1 cell were 24.575μg/ml, 56.991μg/ml, 9.361μg/ml, 13.584μg/ml respectively; for TE-1 cell were 23.534μg/ml, 25.862μg/ml, 6.788μg/ml, 8.911μg/ml respectively; for ECa109 cell were 33.990μg/ml, 33.604μg/ml, 19.873μg/ml, 15.227μg/ml respectively. RHNYP could effectively inhibit the proliferation, migration, invasion, cloning ability, cell cycle and reduce apoptosis of four EC cells. PI3K/AKT/mTOR pathway had closely relationship with the occurrence and development of EC and RHNYP could effectively regulate the proteins expression on the pathway. CDCK3, KIF4A and RAD51AP1 genes were negatively correlated with the overall survival of EC patients and RHNYP could significantly inhibit their expression in four EC cells. Conclusion: RHNYP can effectively inhibit EC cell proliferation, migration, invasion and cell cycle, induce apoptosis, which may be related to the regulation of PI3K/AKT/mTOR pathway. Citation Format: Fuchun Si, Wenyu Zhao, Gao Si, Xuejie Song. Effects of removing heat and nourishing yin prescription on malignant phenotype and PI3K/AKT signaling pathway of human esophageal carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4720.

Bioinformatics Analysis and Verification of Metabolic Abnormalities in Esophageal Squamous Carcinoma.

Although esophageal carcinoma (EC) is one of the most common cancers in the world, details of its pathogenesis remain unclear. Metabolic reprogramming is a main feature of EC. Mitochondrial dysfunction, especially the decrease in mitochondrial complex I (MTCI), plays an important role in the occurrence and development of EC. The objective of the study was to analyze and validate the metabolic abnormalities and the role of MTCI in esophageal squamous cell carcinoma. In this work, we collected transcriptomic data from 160 esophageal squamous carcinoma samples and 11 normal tissue samples from The Cancer Genome Atlas (TCGA). The OmicsBean and GEPIA2 were used to conduct an analysis of differential gene expression and survival in clinical samples. Rotenone was used to inhibit the MTCI activity. Subsequently, we detected lactate production, glucose uptake, and ATP production. A total of 1710 genes were identified as being significantly differentially expressed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis suggested that these differentially expressed genes (DEGs) were significantly enriched in various pathways related to carcinoma tumorigenesis and progression. Moreover, we further identified abnormalities in metabolic pathways, in particular, the significantly low expression of multiple subunits of MTCI genes (ND1, ND2, ND3, ND4, ND4L, ND5, and ND6). Rotenone was used to inhibit the MTCI activity of EC109 cells, and it was found that the decrease in MTCI activity promoted HIF1A expression, glucose consumption, lactate production, ATP production, and cell migration. Our results indicated the occurrence of abnormal metabolism involving decreased mitochondrial complex I activity and increased glycolysis in esophageal squamous cell carcinoma (ESCC), which might be related to its development and degree of malignancy.

Design, synthesis and biological evaluation of 1,2,3-triazole benzothiazole derivatives as tubulin polymerization inhibitors with potent anti-esophageal cancer activities

In this work, we utilized the molecular hybridization strategy to design and synthesize novel 1,2,3-triazole benzothiazole derivatives K1-26. The antiproliferative activities against MGC-803, Kyse30 and HCT-116 cells were explored, and their structure-activity relationship were preliminarily conducted and summarized. Among them, compound K18, exhibited the strongest proliferation inhibitory activity, with esophageal cancer cells Kyse30 and EC-109 being the most sensitive to its effects (IC50 values were 0.042 and 0.038 μM, respectively). Compound K18 effectively inhibited tubulin polymerization (IC50 = 0.446 μM), thereby hindering tubulin polymerize into filamentous microtubules in Kyse30 and EC-109 cells. Additionally, compound K18 induced the degradation of oncogenic protein YAP via the UPS pathway. Based on these dual molecular-level effects, compound K18 could induce G2/M phase arrest and cell apoptosis in Kyse30 and EC-109 cells, as well as regulate the expression levels of cell cycle and apoptosis-related proteins. In summary, our findings highlight a novel 1,2,3-triazole benzothiazole derivative K18, which possesses significant potential for treating esophageal cancers.

Expression of circRNA-0036474 in esophageal cancer and its clinical significance.

Circular RNAs are considered to play important roles in the progression of different cancers such as esophageal squamous cell carcinoma. However, the functions of circular RNAs in esophageal squamous cell carcinoma are still not clear. This study aimed to investigate the role and mechanism of circRNA-0036474 in the progression of esophageal squamous cell carcinoma. The hsa_circ_0036474 expression levels were found to be elevated in both EC109 cells and esophageal squamous cell carcinoma tissue samples. Moreover, knockdown of circRNA-0036474 expression in the EC109 cells induced migration and invasion, characterized by the down-regulation of E-cadherin, and up-regulation of N-cadherin and vimentin. In addition, the over-expressed hsa_circ_0036474 significantly decreased the activity of EC109 cells, elevated E-cadherin expression but declined N-cadherin and vimentin expression. Moreover, over-expressed mir-223-3p levels and interfered RERG expression verified the role of hsa_circ_0036474 in inhibiting the invasion and migration of EC109 cells, reducing the expression of N-cadherin and vimentin, and promoting the expression of E-cadherin. In conclusion, circRNA-0036474 mitigated the progression of esophageal squamous cell carcinoma through regulating mir-223-3p/RERG axis, presenting a potential therapeutic target for the treatment.

Hypoxia-induced factor-1α promotes radioresistance of esophageal cancer cells by transcriptionally activating LINC01116 and suppressing miR-3612 under hypoxia.

Esophageal cancer (EC) is a challenging tumor to treat with radiotherapy, often exhibiting resistance to this treatment modality. To explore the factors influencing radioresistance, we focused on the role of hypoxia-induced factor-1α (HIF-1α), and its interaction with the long noncoding RNA long intergenic nonprotein coding RNA 1116 (LINC01116). We analyzed the LINC01116 expression in EC and EC cell lines/human normal esophageal epithelial cell line (Het-1A). LINC01116 was silenced/overexpressed in EC109/KYSE30 cells under hypoxia, followed by radioresistance assessment. We measured HIF-1α levels in hypoxic EC cells and further validated the binding of HIF-1α with LINC01116, analyzing their interaction in EC cells. We then performed experiments in EC109 cells by transfection them with sh-HIF-1α/oe-LINC01116 to verify the effects. Additonally, we analyzed the localization of LINC01116 and its binding with miR-3612, followed by a combined experiment performed to validate the results. Our findings indicated that LINC01116 was highly expressed in EC and further elevated in hypoxic EC cells. LINC01116 was expressed at a high level in EC, which was further elevated in EC cells under hypoxic conditions. Knockdown of LINC01116 triggered EC cell apoptosis, thus suppressing radioresistance. Further investigation revealed that HIF-1α transcriptionally activated LINC01116 expression under hypoxia, and silencing HIF-1α lowered EC cell radioresistance by downregulating LINC01116. Under hypoxic conditions, LINC01116 could function as a sponge for miR-3612 and inhibit its expression. This interaction between LINC01116 and miR-3612 played a crucial role in mediating radioresistance in EC cells. Briefly, under hypoxic conditions, HIF-1α facilitates radioresistance of EC cells by transcriptionally activating LINC01116 expression and downregulating miR-3612.

Tretinoin (2,4-difluoro-phenyl) triazole activates proapoptotic protein expression and targets NRP2 protein to inhibit esophageal carcinoma cell growth.

The present study investigated the effect of tretinoin (2,4-difluoro-phenyl) triazole (TDFPT) on the growth and proliferation of Kyse-270 and EC9706 esophageal carcinoma cells and explored the underlying mechanism. The results demonstrated that TDFPT treatment of Kyse-270 and EC9706 cells led to a dose-dependent reduction in cell proliferation. Colony formation was significantly (p < .05) reduced in Kyse-270 and EC9706 cells on treatment with various concentrations of TDFPT. In TDFPT-treated Kyse-270 and EC9706 cells, the expression of Bcl-2 protein showed a remarkable decrease, whereas the level of Bax protein was found to be higher compared with the control cells. Cell invasion showed a prominent decrease in Kyse-270 and EC9706 cells on treatment with TDFPT. Treatment with TDFPT led to a prominent suppression in the expression of MMP-9 and NRP2 in Kyse-270 and EC9706 cells. In silico studies using the AutoDock Vina and discovery studio software revealed that various confirmations of TDFPT bind to NRP2 protein with the affinity ranging from -8.6 to -6.1 kcal/mol. It was found that the TDFPT interacts with NRP2 protein by binding to alanine (ALA A:295), proline (PRO A:306), glutamine (GLN A:307), and isoleucine (ILE A:293) amino acid residues. In summary, TDFPT exposure suppresses esophageal carcinoma cell proliferation, inhibits colony formation ability, and activates apoptotic pathway. Thus, TDFPT acts as an effective antiproliferative agent for esophageal carcinoma cells and needs to be investigated further as chemotherapeutic molecule.

FOXN3: A Novel Tumor Suppressor Inhibits the Progression of Esophageal Cancer via Downregulating the SIRT1/PI3K/AKT Axis

In this study, we investigated the impact of FOXN3 on esophageal cancer progression and its underlying mechanism. Through online databases, we observed a significant decrease in FOXN3 levels in esophageal cancer tissues and EC9706 cells. Conversely, SIRT1 expression was elevated in EC109 and EC9706 cells. FOXN3 was found to interact with SIRT1, AKT1, and PIK3CA. To explore FOXN3′s effects, we treated EC9706 cells with pcDNA-FOXN3, which led to increased FOXN3 levels. Consequently, SIRT1, p-AKT/AKT, p-PI3K/PI3K ratios, cell proliferation,migration, invasion, and expression of Ki67, PCNA, MMP3, MMP9, N-cadherin, Vimentin, and Bcl-2 were reduced. In contrast, cell apoptosis, E-cadherin, and Bax levels increased. Further analysis revealed that FOXN3 inhibited cell proliferation and epithelial-mesenchymal transition (EMT) while promoting apoptosis by down-regulating the SIRT1/PI3K/AKT pathway. In conclusion, FOXN3 plays a crucial role in esophageal cancer progression by modulating the SIRT1/PI3K/AKT pathway, affecting cell proliferation, EMT, and apoptosis. This study highlights FOXN3 as a potential target for therapeutic interventions in esophageal cancer.

MicroRNA-223 Suppresses the Progression of Esophageal Cancer by Negatively Regulating SMAD Family Member 4

The purpose of this study was to elucidate the biological role of MicroRNA-223 (miRNA-223) in mediating the malignant progression of esophageal cancer and the underlying mechanism. MiRNA-223 levels were measured using qRT-PCR in 50 paired esophageal cancer tissues and adjacent paracancerous tissues. The correlation between miRNA-223 level and pathological indicators in esophageal cancer patients was analyzed. In vitro experiments assessed the impact of miRNA-223 on the proliferative, migratory, and invasive abilities of esophageal cancer cells. Additionally, rescue experiments were conducted to investigate the involvement of miRNA-223 and its downstream target, SMAD4, in the progression of esophageal cancer. Esophageal cancer tissues showed decreased levels of miRNA-223 compared to adjacent tissues. Patients with low miRNA-223 exhibited higher rates of lymphatic and distant metastasis, as well as poorer overall survival than those with high miRNA-223 levels. Increasing miRNA-223 in TE-1 and EC-109 cells reduced their proliferative, migratory, and invasive capabilities. Esophageal cancer tissues and cell lines displayed elevated SMAD4 levels, which were negatively regulated by miRNA-223. Restoring SMAD4 expression partially reversed the inhibitory effects of miRNA-223 overexpression in esophageal cancer cells. MiRNA-223 is closely correlated to lymphatic metastasis, distant metastasis and poor prognosis of esophageal cancer patients. MiRNA-223 suppresses proliferative, migratory and invasive abilities of esophageal cancer cells via negatively regulating SMAD4.

Extracellular shuttling miR-21 contributes to esophageal cancers and human umbilical vein endothelial cell communication in the tumor microenvironment and promotes tumor angiogenesis by targeting phosphatase and tensinhomolog.

Cell-cell communication by carcinoma-derived exosomes can influence the tumor microenvironment (TME) and regulate cancer progression. Based on the overexpression of microRNA-21-5p (miR-21) in plasma from patients diagnosed with esophageal squamous cell carcinoma (ESCC) and exosomes from ESCC cell lines identified earlier, this study aimed to explore the influence of exosomal miR-21 within the TME. ScRNA-Seq and Bulk RNA-Seq were integrated to elucidate the communication between cancer and endothelial cells. The functionality and mechanisms by which exo-miR-21 derived from carcinoma regulate endothelial cell-mediated angiogenesis were assessed using a cocultivation model of EC9706 cells and recipient human umbilical vein endothelial cells (HUVECs), through blood vessel formation experiments, luciferase reporter assays, RT-qPCR, and western blot analysis. A total of 3842 endothelial cells were extracted from the scRNA-seq data of ESCC samples and reclustered into five cell subtype. Cell-cell communication analysis revealed cancer cells presented a strong interaction with angiogenesis-like endothelial cells in secreted signaling. MiR-21 was unregulated in ESCC and the carcinoma-derived exo-miR-21 was significantly raised in HUVECs. The exo-miR-21 promoted the proliferation and migration of HUVECs while also enhancing, closed mesh count, and junction number in HUVECs. Mechanistically, dual-luciferase reporter assay revealed that PTEN was the target of miR-21. Meanwhile, p-Akt was significantly increased and suppressed by inhibition of miR-21 and PI3K inhibitor LY294002. Exo-miR-21-mediated communication between endothelial and cancer cells plays a pivotal role in promoting the angiogenesis of ESCC. Therefore, controlling exo-miR-21 could serve as a novel therapeutic strategy for ESCC by targeting angiogenesis.

Endogenous hydrogen sulfide inhibition suppresses tumor growth by promoting apoptosis and pyroptosis in esophageal cancer cells

BackgroundHydrogen sulfide (H2S) has been identified as the third gaseous signaling molecule. Endogenous H2S plays a key role in the progression of various types of cancer. However, the effect of endogenous H2S on the growth of esophageal cancer (EC) remains unknown. MethodsIn this study, three kinds of H2S-producing enzymes inhibitors, DL-propargylglycine (PAG, inhibitor of cystathionine-γ-lyase), aminooxyacetic acid (AOAA, inhibitor of cystathionine-β-synthase), and L-aspartic acid (L-Asp, inhibitor of 3-mercaptopyruvate sulfurtransferase) were used to determine the role of endogenous H2S in the growth of EC9706 and K450 human EC cells. ResultsThe results indicated that the combination (PAG+AOAA+L-Asp) group showed higher inhibitory effects on the viability, proliferation, migration, and invasion of EC cells than PAG, AOAA, and L-Asp group. Inhibition of endogenous H2S promoted apoptosis via activation of mitogen-activated protein kinase pathway in EC cells. Endogenous H2S suppression triggered pyroptosis of EC cells by activating reactive oxygen species-mediated nuclear factor-κB signaling pathway. In addition, the combine group showed its more powerful growth-inhibitory effect on the growth of human EC xenograft tumors in nude mice without obvious toxicity. ConclusionOur results indicate that inhibition of endogenous H2S production can significantly inhibit human EC cell growth via promotion of apoptosis and pyroptosis. Endogenous H2S may be a promising therapeutic target in EC cells. Novel inhibitors for H2S-producing enzymes can be designed and developed for EC treatment.

Different Expression Pattern of G Protein-Coupled Estrogen Receptor GPER1 in Esophageal Squamous Cell Carcinoma and Adenocarcinoma.

Esophageal carcinoma is a male-dominant malignancy worldwide, and esophageal adenocarcinoma (EAC) shows more significant sex bias than esophageal squamous cell carcinoma (ESCC) in morbidity and mortality. The G protein-coupled estrogen receptor 1 (GPER1) is involved in several sex-related cancers; however, its expression level in esophageal carcinoma has been poorly investigated and its role is not precisely defined, depending on histological types. In the present study, the mRNA levels of GPER1 in esophageal carcinoma were collected from GEPIA and Oncomine databases for meta-analyses. The protein expression levels of GPER1 were detected by immunohistochemistry in the tissue microarray of EAC and ESCC. The GPER1 selective agonist G1, antagonist G15, and siRNA were applied in vitro to investigate their impacts on esophageal cell lines. Analysis of the RNA levels from the databases showed a decreased expression of GPER1 in overall esophageal carcinoma, and low expression levels of GPER1 were found to be associated with low survival of tumor patients. However, in the subgroup of EAC and its precancerous lesion, Barrett's esophagus, overexpression of GPER1 RNA was increased when compared with the normal tissues. The average staining scores of GPER1 protein in the tissue microarray of EAC were significantly higher than normal esophageal samples, and the rate of positive staining increased with the grade of poor tumor differentiation. The scores of GPER1 protein in ESCC tissues were lower than those in the normal tissues. The results from cell line experiments in vitro showed that the GPER1 agonist G1 inhibited proliferation and promoted apoptosis of ESCC cells EC109 with positive expression of GPER1. G1 had no obvious effect on normal esophageal NE2 cells with weak expression of GPER1. In addition, GPER1 RNA knockdown and application of antagonist G15 reversed the effects of G1 on EC109. The results of this study indicate that the expression levels of GPER1 are higher in EAC than in ESCC, which might be correlated with the dimorphic estrogen signaling pathway in different types of esophageal carcinoma.

CircRNA PDE3B regulates tumorigenicity via the miR-136-5p/MAP3K2 axis of esophageal squamous cell carcinoma.

CircRNA has a covalently closed circular conformation and a stable structure. However, the exact role of circRNA in esophageal squamous cell carcinoma (ESCC) remains uncertain. The purpose of this study was to explore the role of hsa_circ_0000277 (circ_PDE3B) in ESCC. The expression levels of circ_PDE3B, miR-136-5p and mitogen-activated protein kinase kinase kinase 2 (MAP3K2) in ESCC tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. The proliferation ability of EC9706 and KYSE30 cells was detected by clonal formation, 5-ethynyl-2'-deoxyuridine (EdU) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays. Flow cytometry was used to detect the apoptosis rate of cells. Transwell assay was used to detect the invasion ability of EC9706 and KYSE3 cells. The relationship between miR-136-5p and circ_PDE3B or MAP3K2 was verified by dual-luciferase reporter assay and RNA pull-down, and the effect of circ_PDE3B on tumor growth in vivo was explored through tumor transplantation experiment. Immunohistochemistry (IHC) assay was used to detect MAP3K2 and Ki67 expression in mice tumor tissues. The results showed that circ_PDE3B was highly expressed in ESCC tissues and cells. Downregulated circ_PDE3B expression in ESCC cells significantly reduced cell proliferation, migration and invasion. Circ_PDE3B served as a sponge for miR-136-5p, and miR-136-5p inhibition reversed the roles of circ_PDE3B knockdown in ESCC cells. MAP3K2 was a direct target of miR-136-5p, and miR-136-5p targeted MAP3K2 to inhibit the malignant behaviors of ESCC cells. Furthermore, circ_PDE3B regulated MAP3K2 expression by sponging miR-136-5p. Importantly, circ_PDE3B knockdown inhibited tumor growth in vivo. In conclusion, circ_PDE3B acted as oncogenic circRNA in ESCC and accelerated ESCC progression by adsorption of miR-136-5p and activation of MAP3K2, supporting circ_PDE3B as a potential therapeutic target for ESCC.

MUC13 promotes the development of esophageal cancer by upregulating the expression of o-glycan process-related molecules

BackgroundEsophageal cancer is one of the most common malignant tumors in the world, which is characterized by poor prognosis, aggressiveness, and poor survival. Mucin 13 (MUC13) is a member of the membrane-bound mucin and located on chromosome 3q21.2 and consists of α and β subunits. It has been found that MUC13 is overexpressed in a variety of tumor cells and acts a vital role in the invasiveness and malignant progression of several types of tumors. However, the role and regulatory mechanism of MUC13 in the progression of esophageal cancer remain unclear.MethodsThe expression level of MUC13 was detected in 15 esophageal cancer tissues and 15 pairs of adjacent nontumor tissues by immunohistochemistry (IHC). In addition, the expression of MUC13 mRNA level in human esophageal cancer cell lines (EC9706 and ECA109 and TE-1) was measured by qRT-PCR. In vitro, after silencing MUC13 with lentiviral interference technology, CCK8 assay, clone formation assay, and flow cytometry were applied to investigate the proliferation activity, clone formation ability and anti-apoptosis ability of EC9706 and ECA109 cells. The tumor xenograft growth assay was used to confirm the influence of MUC13 knockdown on the growth of esophageal tumors in vivo. The qRT-PCR assay and western blot experiments were taken to study the mechanism of MUC13 regulating the proproliferation and antiapoptotic of esophageal cancer.ResultsThe results showed that MUC13 was overexpressed in esophageal cancer tissues and cell lines (EC9706 and ECA109 and TE-1), especially in EC9706 and ECA109 cells, but low expressed in human esophageal epithelial cell line (HEEC). Next, silencing MUC13 inhibits proliferation, blocks cell cycle progression, and promotes cell apoptosis in vitro, and restrains the growth of esophageal cancer tissues in vivo. Finally, MUC13 affects the proproliferation and antiapoptotic by regulating the expression of GLANT14, MUC3A, MUC1, MUC12, and MUC4 that closely related to O-glycan process.ConclusionsThis study proved that MUC13 is an important molecule that regulates the O-glycan process and then affects the progress of esophageal cancer. MUC13 may be a novel therapeutic target for patients with esophageal cancer.

HMGB1-mediated-TLR4/MyD88 signaling regulates cell proliferation, migration and invasion in esophageal squamous cell carcinoma

Purpose: To study the effect of high mobility group box protein 1 (HMGB1)-mediated toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) on esophageal squamous cell carcinoma (ESCC). &#x0D; Methods: Cell viability was determined using CCK-8 test and colony formation assay, while cell migration and invasion were assessed by Transwell assay. Western blotting was used to determine protein and mRNA expression levels. The effect of TLR4/MyD88 signaling pathway on proliferation of ESCC cells was investigated via in vitro knockdown of HMGB1. &#x0D; Results: Data from in vitro experiments indicated that HMGB1 knockdown significantly decreased the proliferation, migration, and invasion of EC9706 cells in siHMGB1 group, when compared with siNC group (number of invasive cells in siHMGB1 vs. corresponding number in siNC: 26.7 ± 4.5 vs. 68.7 ± 2.5; p &lt; 0.01), and also decreased proliferation, migration and invasion of TE-9 cells in siHMB1, relative to siNC group (number of invasive cells in siHMGB1 vs. corresponding number in siNC: 29.3 ± 3.5 vs. 55.7 ± 3.1; p &lt; 0.01). Moreover, MGB1 knockdown via siTLR4 transfection significantly down-regulated the expressions of TLR4/MyD88 and epithelial-mesenchymal transition (EMT), but over-expression of TLR4 reversed the inhibition by HMGB1 knockdown on ESCC cells (p &lt; 0.01). &#x0D; Conclusion: These findings suggest that HMGB1-mediated TLR4/MyD88 signal pathway is a potential treatment route for ESCC.