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NANOG Promotes Cell Proliferation, Invasion, and Stemness via IL-6/STAT3 Signaling in Esophageal Squamous Carcinoma.

ABSTRACT: Background: Cancer cells have properties similar to those of stem cells, including high proliferation and self-renewal ability. NANOG is the key regulatory gene that maintains the self-renewal and pluripotency characteristics of embryonic stem cells. We previously reported that knockdown of the pluripotent stem cell factor NANOG obviously reduced the proliferation and drug-resistance capabilities of esophageal squamous cell carcinoma (ESCC). In this study, we gained insights into the potential regulatory mechanism of NANOG, particularly in ESCC. Methods: NANOG was ectopically expressed in the Eca-109 cell line via pcDNA3.1 vector transfection. The mRNA expression of different genes was detected using quantitative real-time polymerase chain reaction, and protein quantification was performed by western blotting. The enzyme-linked immunosorbent assay was used to detect the expression of interleukin 6 (IL-6). The capabilities of proliferation, migration, and invasion were investigated using cell count and Transwell assays. The tumor sphere-forming assay was used to investigate the sphere formation capacity of cancer stem cells. Results: The expression of NANOG promoted the cell proliferation and sphere formation capacity of cancer stem cells in a dose-dependent manner. IL-6-mediated activation of signal transducer and activator of transcription 3 (STAT3) was closely related to the expression of NANOG in ESCC. Consistently, the target genes of STAT3, including CCL5, VEGFA, CCND1, and Bcl-xL, were upregulated upon the overexpression of NANOG. Conclusion: These results revealed that the expression of NANOG promotes cell proliferation, invasion, and stemness via IL-6/STAT3 signaling in ESCC.

SUBMITTER: Deng L

PROVIDER: S-EPMC8723181 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Despite investigations into microRNA (miRNA) expression in esophageal cancer (EC) tissue, miRNAs that participate in EC pathogenesis and their subsequent mechanisms of action remain to be determined. The present study aimed to identify important miRNAs that contribute to EC development, and to assess miRNA biomarkers that could be used in EC diagnosis, prognosis and therapy. Bioinformatics analysis was performed to reanalyze EC tissue miRNA expression microarray dataset GSE113776, which was followed by in vitro verification of miRNA functions using reverse transcription?quantitative PCR, western blot analysis and a dual?luciferase reporter assay. Out of 93 miRNAs extracted, only miR?200a was significantly increased in EC tissues. Transfection of KYSE150 esophageal squamous cell carcinoma (ESCC) cells with miR?200a mimics significantly increased their proliferative, migratory and invasive ability, whereas the opposite cell behaviors were observed in ESCC cells transfected with a miR?200a inhibitor. A total of six miR?200a target genes [catenin ?1 (CTNNB1), cadherin?1 (CDH1), PTEN, adenomatous polyposis coli (APC), catenin ?1 (CTNNA1) and superoxide dismutase 2 (SOD2)] were selected for further analysis based on Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathway analysis, protein?protein interaction network map data and protein expression in esophageal tissue. These target genes were downregulated under miR?200a expression and upregulated in the presence of the miR?200a inhibitor. The association between miR?200a and the 3'?untranslated region of target genes in ESCC cells was confirmed using a dual?luciferase reporter assay. In conclusion, the present study demonstrated that miR?200a may participate in the promotion of ESCC cell proliferation, migration and invasion, and provided novel evidence for the direct interaction between miR?200a and CTNNB1, CDH1, PTEN, APC, CTNNA1 and SOD2, which may contribute to the observed altered cell behavior.

Project description:Oral squamous cell carcinoma (OSCC) is an extremely aggressive disease associated with a poor prognosis. Previous studies have established that cancer stem cells (CSCs) actively participate in OSCC development, progression and resistance to conventional treatments. Furthermore, CSCs frequently exhibit a deregulated expression of normal stem cell signalling pathways, thereby acquiring their distinctive abilities, of which self-renewal is an example. In this study, we examined the effects of GLI3 knockdown in OSCC, as well as the differentially expressed genes in CSC-like cells (CSCLCs) expressing high (CD44high) or low (CD44low) levels of CD44. The prognostic value of GLI3 in OSCC was also evaluated. The OSCC cell lines were sorted based on CD44 expression; gene expression was evaluated using a PCR array. Following this, we examined the effects of GLI3 knockdown on CD44 and ESA expression, colony and sphere formation capability, stem-related gene expression, proliferation and invasion. The overexpression of genes related to the Notch, transforming growth factor (TGF)?, FGF, Hedgehog, Wnt and pluripotency maintenance pathways was observed in the CD44high cells. GLI3 knockdown was associated with a significant decrease in different CSCLC fractions, spheres and colonies in addition to the downregulation of the CD44, Octamer-binding transcription factor 4 (OCT4; also known as POU5F1) and BMI1 genes. This downregulation was accompanied by an increase in the expression of the Involucrin (IVL) and S100A9 genes. Cellular proliferation and invasion were inhibited following GLI3 knockdown. In OSCC samples, a high GLI3 expression was associated with tumour size but not with prognosis. On the whole, the findings of this study demonstrate for the first time, at least to the best of our knowledge, that GLI3 contributes to OSCC stemness and malignant behaviour. These findings suggest the potential for the development of novel therapies, either in isolation or in combination with other drugs, based on CSCs in OSCC.

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NANOG Promotes Cell Proliferation, Invasion, and Stemness via IL-6/STAT3 Signaling in Esophageal Squamous Carcinoma.

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