Abstract
BackgroundSignal transducer and activator of transcription 3 (STAT3) is persistently activated in a wide variety of epithelial cancers. Aberrant activity of STAT3 correlates with tumor growth, invasion and metastasis, which makes it a potential therapeutic target of cancer. To explore the biological role of STAT3 in esophageal cancer, we used small hairpin RNA to knockdown the expression of the STAT3 gene in the esophageal carcinoma ECA109 cell line and the cell apoptosis, cell cycle and cell migration were investigated.MethodsThe cell apoptosis was tested using DNA ladder, mitochondrial membrane potential assay, TUNEL assay, annexin V-PI staining. Cell cycle phases were estimated using flow cytometry analysis. The mRNA and proteins related to apoptosis and cell cycle were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. And cell migration was investigated by in vitro Transwell assay. The data were analyzed with two-sample Student’s t test and ANOVA followed by the LSD post hoc test.ResultsOur results showed that knockdown of STAT3 in ECA109 cells induced noticeable apoptotic morphological changes like cell shrinkage, apoptotic vacuoles, membrane blebbing time-dependently. In addition, DNA ladder, TUNEL assay, Annexin V-PI staining and declined level of cleaved Caspase-3 indicated that down-regulation of STAT3 could induce apoptosis in ECA109 cells. Flow cytometry analysis displayed the induction of G1-phase cell cycle arrest of ECA109 cells by STAT3 decreasing, consistent with the descend of c-Myc and cyclin D1 in protein levels. Furthermore, STAT3 knockdown suppressed the expression of matrix metalloproteinases-9, sushi domain containing 2 and urokinase plasminogen activator in ECA109 cells and inhibited cell migration ability.ConclusionsKnockdown of STAT3 could induce the apoptosis and G1 cell cycle arrest in esophageal carcinoma ECA109 cells, and inhibit the migration ability of cells as well.
Highlights
Signal transducer and activator of transcription 3 (STAT3) is persistently activated in a wide variety of epithelial cancers
Several cytokines and growth factors like IL-6 or epidermal growth factor (EGF) family members, as well as hepatocyte growth factor (HGF)contribute to the phosphorylation of STAT3, the active STAT3 interacts with its target gene to prevent apoptosis [16] and stimulates transcription of key cancer genes linked with cell cycle [17]
Inhibition of STAT3 expression in ECA109 cells through plasmid‐based small hairpin RNA (shRNA) To investigate the biological functions of STAT3 downregulation, we utilized recombinant plasmid of shRNA to inhibit endogenous STAT3 in ECA109 cells
Summary
Signal transducer and activator of transcription 3 (STAT3) is persistently activated in a wide variety of epithelial cancers. Aberrant activity of STAT3 correlates with tumor growth, invasion and metastasis, which makes it a potential therapeutic target of cancer. The potential oncogenic role of STAT3 was established by the expression of constitutively activated STAT3 in various tumor cell lines including breast, melanoma, pancreas, prostate, colon and esophagus and stomach [12,13,14,15]. Several cytokines and growth factors like IL-6 or epidermal growth factor (EGF) family members, as well as hepatocyte growth factor (HGF)contribute to the phosphorylation of STAT3, the active STAT3 interacts with its target gene to prevent apoptosis [16] and stimulates transcription of key cancer genes linked with cell cycle [17]. We targetedly down-regulated the expression of STAT3 in esophageal cancer ECA109 cell line using STAT3-specific shRNA vector pSi-STAT3 plasmid and investigated the effects of STAT3 down-regulation on ECA109 cells
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