Survivin shRNA对食管癌细胞c-myc、核因子-κB基因表达的影响
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摘要
目的探讨Survivin对c-myc、核因子(NF)-κB基因的调控作用。方法 shRNA干扰沉默食管癌ECA109细胞系中Survivin基因表达,观察c-myc、NF-κB基因的表达变化,流式细胞术检测细胞周期的改变。结果1.食管癌ECA109细胞系中,Survivin shRNA干扰下调Survivin表达后,c-myc mRNA及蛋白的表达水平明显降低;2.Survivin表达后,NF-κB mRNA及NF-κB总蛋白无明显变化,但其蛋白活化表达水平明显降低,NF-κB上游调控核因子κB抑制物激酶(IKK)α、IKKβmRNA表达下调; 3.Survivin表达下调导致G2期延长,G2期细胞比例增加,细胞增殖阻滞。结论 Survivin在转录及蛋白水平对c-myc具有正向调控作用,Survivin表达下调抑制了IKKα、IKKβmRNA的表达及NF-κB的磷酸化。
Objective To study the effect of Survivin shRNA interference on c-myc and NF-κB expression levels in esophageal cells ECA109.Methods Survivin shRNA interference technology was applied to liposomes transfection esophageal ECA109 cells.Half quantitative RT-PCR and Western blotting method were applied to detect genes expression of c-myc and NF-κB gene.Flow cytometry was used to detect the proliferation and apoptosis of cell lines index changes.Results 1.After Survivin shRNA transfected esophageal ECA109 cells,Survivin mRNA and protein expression obviously down-regulated,and c-myc mRNA and protein expression also down-regulated;2.After Survivin down-regulated,NF-κB mRNA expression had no obvious change,however the phosphorylated proteins level of NF-κB was reduced,whose upstream factor nuclear factor kappa B inhibitor kinase predominate(IKK) alpha,IKK beta mRNA express was cut;3.Esophageal cell ECA109 apoptosis increased significantly.Cells ratio increased in G2 phase but decreased in S phase.The cell cycle blocked.Conclusion Survivin is effective on adjusting c-myc gene expression on mRNA and protein level.Survivin regulates NF-κB activation by down-regulation of IKK alpha,IKK beta mRNA expression.
Objective To study the effect of Survivin shRNA interference on c-myc and NF-κB expression levels in esophageal cells ECA109.Methods Survivin shRNA interference technology was applied to liposomes transfection esophageal ECA109 cells.Half quantitative RT-PCR and Western blotting method were applied to detect genes expression of c-myc and NF-κB gene.Flow cytometry was used to detect the proliferation and apoptosis of cell lines index changes.Results 1.After Survivin shRNA transfected esophageal ECA109 cells,Survivin mRNA and protein expression obviously down-regulated,and c-myc mRNA and protein expression also down-regulated;2.After Survivin down-regulated,NF-κB mRNA expression had no obvious change,however the phosphorylated proteins level of NF-κB was reduced,whose upstream factor nuclear factor kappa B inhibitor kinase predominate(IKK) alpha,IKK beta mRNA express was cut;3.Esophageal cell ECA109 apoptosis increased significantly.Cells ratio increased in G2 phase but decreased in S phase.The cell cycle blocked.Conclusion Survivin is effective on adjusting c-myc gene expression on mRNA and protein level.Survivin regulates NF-κB activation by down-regulation of IKK alpha,IKK beta mRNA expression.
引文
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[7]Kar R,Palanichamy JK,Banerjee A,et al.Survivin siRNAincreases sensitivity of primary cultures of ovarian cancer cells to paclitaxel[J].Clin Transl Oncol,2015,17(9):737-742.
[8]Wang H,Ye YF.Effect of survivin siRNA on biological behaviour of breast cancer MCF7 cells[J].Asian Pac J Trop Med,2015,8(3):225-228.
[9]Li J,Han Y,Zhou D,et al.Down regulation of Survivin gene expression affects ionizing radiation resistance of human T98 glioma cells[J].Cell Mol Neurobiol,2018,38(4):861-868.
[10]Tracey L,Pérez-rosado A,Artiga MJ,et al.Exp ression of the NF-kappa B targets Bcl-2 and BIRC5/Survivin characterizes small B-cell and aggressive B-cell lymphomas,respectively[J].JPathol,2005,206(2):123-134.
[2]Peery RC,Liu JY,Zhang JT.Targeting survivin for therapeutic discovery:past,present,and future promises[J].Drug Discov Today,2017,22(10):1466-1477.
[3]Mathieu R,Klatte T,MargulisⅤ,et al.Survivin is not an independent prognostic factor for patients with upper tract urothelial carcinoma:a multi-institutional study[J].Urol Oncol,2015,33(11):495.e15-22.
[4]Santarelli A,Mascitti M,Lo Russo L,et al.Survivin-based treatment strategies for squamous cell carcinoma[J].Int J Mol Sci,2018,19(4):971-995.
[5]Yin H,Que R,Liu C,et al.Survivin targeted drug screening platform identifies a matrine derivative WM-127 as a potential therapeutics against hepatocellular carcinoma[J].Cancer Lett,2018,425:54-64.
[6]Sun XF,Li XB,Yang GT,et al.Radix Actinidiae extractive inhibits the growth of transplanted tumor of esophagus cancer cell EC109 in nude mice[J].Acta Anatomica Sinica,2009,40(1):32-36.(in Chinese)孙雪飞,李希波,杨国涛,等.藤梨根提取物对食管癌细胞系EC109细胞裸鼠移植瘤的抑制作用[J].解剖学报,2009,40(1):32-36.
[7]Kar R,Palanichamy JK,Banerjee A,et al.Survivin siRNAincreases sensitivity of primary cultures of ovarian cancer cells to paclitaxel[J].Clin Transl Oncol,2015,17(9):737-742.
[8]Wang H,Ye YF.Effect of survivin siRNA on biological behaviour of breast cancer MCF7 cells[J].Asian Pac J Trop Med,2015,8(3):225-228.
[9]Li J,Han Y,Zhou D,et al.Down regulation of Survivin gene expression affects ionizing radiation resistance of human T98 glioma cells[J].Cell Mol Neurobiol,2018,38(4):861-868.
[10]Tracey L,Pérez-rosado A,Artiga MJ,et al.Exp ression of the NF-kappa B targets Bcl-2 and BIRC5/Survivin characterizes small B-cell and aggressive B-cell lymphomas,respectively[J].JPathol,2005,206(2):123-134.