Effects of mTOR siRNA on mTOR/p70S6K signaling pathway in esophageal squamous cell carcinoma cells and the growth of transplanted tumor in nude mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of mTOR siRNA on mTOR-p70S6K signaling pathway in esophageal squamous cell carcinoma (ESCC) cells in vitro,and growth and apoptosis in transplanted tumor in nude mice.</p><p><b>METHODS</b>mTOR siRNA was transfected into ESCC cell line EC9706 cells. The expressions of factors of the mTOR/p70S6K signaling pathway were detected by RT-PCR and Western blot. DNA contents and cell apoptosis were determined by flow cytometry, and cell proliferation was measured by CCK-8 assay. The effects of mTOR siRNA on the transplanted tumor growth were assessed in nude mice.</p><p><b>RESULTS</b>The levels of mTOR and p-p70S6K were significantly decreased (P < 0.05) while the level of p70S6K was increased (P < 0.05) in the cells transfected with mTOR siRNA, compared with that in untransfected cells and cells transfected with control siRNA. After being interfered by mTOR siRNA, the number of apoptotic cells was increased, cell proliferation became slower and cell cycle was arrested in G(1) phase compared with that in control cells. Also, mTOR siRNA inhibited the growth of transplanted tumor in vivo.</p><p><b>CONCLUSIONS</b>mTOR siRNA can effectively interfere in mTOR-p70S6K signaling pathway, induce cell apoptosis and inhibit cell proliferation and tumor growth, suggesting that mTOR-p70S6K signaling pathway plays an important role in the carcinogenesis and development of esophageal squamous cell carcinoma.</p>

Effect of KISS-1 on invasive potential and proliferation of esophageal squamous carcinoma cell line EC-1 / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of KISS-1 expression on the potential of invasion and proliferation of esophageal squamous carcinoma cell EC-1.</p><p><b>METHODS</b>Protein and mRNA expressions of KISS-1 were evaluated by Western blot and RT-PCR in four esophageal carcinoma cell lines (EC-1, Eca109, EC9706 and TE-1). Using liposome-mediated transfection, an eukaryotic expression vector (pcDNA3.1-KISS-1) of KISS-1 gene was transfected into EC-1 cells. Boyden chamber model, MTT and clone formation assay were used to detect the potential of invasion and proliferation.</p><p><b>RESULTS</b>Western blot and RT-PCR showed a baseline low level of expression of KISS-1 protein (0.715 +/- 0.109) and mRNA (0.670 +/- 0.176) in EC-1 cells. pcDNA3.1-KISS-1 expression vector was successfully transfected into EC-1 cells. Western blot and RT-PCR showed that the expression of KISS-1 protein (1.143 +/- 0.218) and mRNA (0.877 +/- 0.162) in EC-1 cells transfected with pcDNA3.1-KISS-1 were significantly higher than those transfected with the control vector pcDNA3.1 (0.745 +/- 0.130, 0.685 +/- 0.128; t = 3.850, 2.481, P < 0.05) and the control cells (0.855 +/- 0.184, 0.677 +/- 0.138; t = 2.275, 2.306, P < 0.05). Boyden chamber analysis showed that the invasiveness of the cells transfected with KISS-1 at 24 h (91.8 +/- 11.7), 48 h (117.8 +/- 11.1) and 72 h (139.2 +/- 11.8) were significantly reduced than that of the cells transfected with the control vector pcDNA3.1 (118.1 +/- 14.7, 141.7 +/- 13.2, 162.2 +/- 22.7; t = 3.153, 4.215, 3.569, P < 0.01) and the control cells (112.2 +/- 15.6, 138.1 +/- 13.0, 162.3 +/- 14.0; t = 4.154, 3.797, 2.702, P < 0.05). MTT showed that the proliferation potential of cells after transfection with KISS-1 at 48 h (0.517 +/- 0.127) and 72 h (0.394 +/- 0.137) were significantly reduced than that of cells transfected with the control vector pcDNA3.1 (0.636 +/- 0.186, 0.513 +/- 0.150; t = 2.054, 2.709, P < 0.05) and the control cells (0.646 +/- 0.135, 0.511 +/- 0.153; t = 2.276, 2.205, P < 0.05). Clone formation assay suggested that cells transfected with KISS-1 (157.2 +/- 36.4) showed significantly decreased clone formation than cells transfected with the control vector pcDNA3.1 (236.3 +/- 78.1; t = 3.441, P < 0.01) and the control cells (242.5 +/- 48.6; t = 2.250, P < 0.05).</p><p><b>CONCLUSION</b>KISS-1 gene inhibits the potential of invasion and proliferation of EC-1 cells.</p>

Transforming growth factor beta1 regulation of epithelial-mesenchymal transition in esophagus squamous cell carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the functional role of transforming growth factor beta1(TGFbeta1) in the regulation of epithelial-mesenchymal transition (EMT) and the effect of TGFbeta1-ASODN blockage of EMT in esophagus squamous cell carcinoma.</p><p><b>METHODS</b>Esophageal squamous cell carcinoma cell line EC9706 was transfected with chemically synthesized TGFbeta1-ASODN. RT-PCR, immunohistochemistry and flow cytometry were used to detect the protein and mRNA expressions of TGF-beta1, E-cadherin and vimentin before and after the transfection. Morphological changes were documented and scarification test was used to detect the migration potential of EC9706 before and after the transfection.</p><p><b>RESULTS</b>After TGFbeta1-ASODN transfection, mRNA (0.25 +/- 0.07) and protein (35.07% +/- 1.42%) expressions of TGFbeta1 in EC9706 were significantly lower than those before transfection (mRNA: 0.43 +/- 0.09; protein: 43.57% +/- 1.77%, chi(2) = 13.847 and chi(2) = 84.120, P < 0.05). The mRNA (0.38 +/- 0.09) and protein (17.13% +/- 1.45%) expressions of E-cadherin were significantly higher than those before transfection (0.22 +/- 0.06; 12.53% +/- 1.31%, chi(2) = 0.160 and chi(2) = 40.008, P < 0.05) and the mRNA (0.73 +/- 0.07) and protein (14.15% +/- 1.46%) expressions of vimentin were significantly lower than those (0.89 +/- 0.09; 17.97% +/- 1.42%) before transfection (chi(2) = 0.160 and chi(2) = 21.103, P < 0.05). Scarification test showed that after transfection, the mobility of EC9706 was significantly inhibited and its migration length (0.45 +/- 0.05) was significantly shorter than that before the transfection (0.81 +/- 0.11, chi(2) = 16.854, P < 0.05).</p><p><b>CONCLUSIONS</b>TGFbeta1 may contribute to EMT in esophageal squamous cell carcinoma. TGFbeta1-ASODN leads to an over-expression of E-cadherin and a down-regulation of vimentin, along with the morphological alterations and migration inhibition, indicating that a blockage of TGFbeta1 suppresses EMT in esophagus squamous cell carcinoma.</p>

Small interfering RNA suppression of transducer and activator of transcription 3 (STAT3) signaling pathway: inhibitory effect on proliferation of human esophageal squamous carcinoma cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the efficiency of blockage of constitutively activated STAT3 signaling by small interfering RNA (siRNA), and to explore the inhibitory effects on the proliferation of human esophageal squamous carcinoma cells (EC9706 and Eca109).</p><p><b>METHODS</b>EC9706 and Eca109 were transfected with chemical synthesized STAT3 siRNA (100 nmol/L). RT-PCR and Western blot were used to detect STAT3 mRNA and protein expression, including phosphorylated-STAT3 (p-STAT3) before and after the transfection respectively. The changes of DNA-binding activity and cell proliferation were evaluated by electrophoretic mobility gel shift assay and MTT, respectively. Stages of cell cycle were determined by flow cytometry.</p><p><b>RESULTS</b>Expression levels of STAT3 mRNA and STAT3, p-STAT3 proteins were progressively inhibited by STAT3 siRNA at various time points after transfection. STAT3-DNA-binding activity was suppressed after transfection evidenced by electrophoretic mobility gel shift assay. The cell cycle was arrested at G(0)/G(1) phase along with a significant inhibition of cell proliferation after STAT3 siRNA treatment.</p><p><b>CONCLUSION</b>STAT3 siRNA specifically and efficiently blocks the constitutively activated STAT3 signaling pathway in human esophageal squamous carcinoma cells, resulting in cell cycle arrest and proliferation inhibition.</p>

Constitutive activation of signal transducers and activators of transcription 3 and expression of its target gene products in human ESCC cell line / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of signal transducers and activators of transcription 3 (STAT3) and its target gene products including vascular endothelial growth factor (VEGF) and Bcl-2 in two human esophageal squamous cell carcinoma (ESCC) cell lines for understanding whether STAT3 signaling transduction pathway was constitutively activated in ESCC cell lines.</p><p><b>METHODS</b>Western blotting was used to determine the protein levels of STAT3, p-STAT3 (activated STAT3), VEGF and Bcl-2 in two ESCC cell lines (EC9706 and Eca109). Nuclear proteins from the ESCC cell lines were extracted to evaluate the DNA-binding activity by electrophoretic mobility gel shift assay (EMSA). RT-PCR was used to detect the mRNA of STAT3, VEGF and Bcl-2.</p><p><b>RESULTS</b>Western blotting and RT-PCR revealed that STAT3, VEGF and Bcl-2 protein and mRNA were overexpressed in the two ESCC cell lines, which contained constitutively activated STAT3 signaling transduction pathway. The results of EMSA of the nuclear protein showed high DNA-binding activity of STAT3.</p><p><b>CONCLUSION</b>STAT3 signaling transduction pathway is constitutively activated in human ESCC cell lines, suggesting that STAT3 may play a critical role in the carcinogenesis of the esophagus.</p>