【作者】 刘涛；

【导师】 李玉民；

【作者基本信息】 兰州大学 ， 细胞生物学， 2014， 博士

【摘要】 目的NOTCH信号通路在保持细胞增殖、分化和凋亡的平衡等方面都发挥非常重要的作用,目前有大量的研究和临床证据表明NOTCH在许多肿瘤中起原癌基因或者抑癌基因的作用,因此NOTCH通路是肿瘤治疗的一个潜在靶点。我们前期研究发现NOTCH信号通路在胃癌中存在异常表达,本研究探讨靶向抑制NOTCH信号通路应用于胃癌治疗的可能性,同时阐释抑制该信号通路诱导胃癌细胞凋亡的机制,为胃癌的治疗提供新思路和科学依据。方法将胃癌重要致癌因子(幽门螺杆菌)H. pylori和正常胃粘膜细胞株共培养24h后,通过RT2Profiler PCR Array-Human Signal Transduction Pathway Finder基因芯片筛选H. pylori感染后出现异常表达的信号通路。将不同H. pylori菌株与胃粘膜细胞共培养后,利用荧光定量PCR和Western Blot验证上一步筛选出的通路。使用NOTCH信号通路抑制剂GSI和受体配体siRNA处理三种胃癌细胞株MKN-45、SGC-7901、BGC-823,利用流式细胞仪Annexin-V-FITC/PI双染法检测抑制Notch信号通路对胃癌细胞周期及凋亡的影响,通过RT2Profiler PCR Array-Human Signal Transduction Pathway Finder基因芯片分析胃癌细胞中NOTCH信号通路与其它信号通路的cross-talking,通过Human Apoptosis Antibody Array抗体芯片阐释阻断NOTCH信号通路诱导胃癌细胞凋亡的机制。结果正常胃粘膜细胞株与H. pylori11637共培养后,通过RT2Profiler PCR Array-Human Signal Transduction Pathway Finder基因芯片检测有22个基因表达水平具有差异,这些基因主要与细胞增殖、细胞周期和转录调控相关,DAVID Pathway分析显示H. pylori感染后主要影响了Cyclins and Cell Cycle Regulation和NOTCH Signaling Pathway这两条通路。PCR及Western Blot结果验证了H. pylori不同菌株11637和SS1感染胃粘膜细胞后,NOTCH配体DLL4表达水平显著上升。流式细胞仪检测证实两种菌株通过调控细胞周期相关蛋白,抑制了细胞增殖。使用20μmol/L GSI (S2188)作用于胃癌细胞株BGC-823、MKN-45、 SGC-790124h后受体NOTCH1、NOTCH2和靶基因HES1的mRNA和相应蛋白表达水平都显著受到了抑制；抑制剂使得胃癌细胞停滞于G1期,阻止细胞DNA合成进而影响其有丝分裂,抑制了细胞的增殖,并且诱导胃癌细胞发生凋亡。NOTCH2siRNA和DLL4siRNA均能显著抑制相应蛋白表达水平,通路靶基因HES1表达水平显著下降,siRNA组胃癌细胞周期无明显变化,但与GSI组相同显著诱导了胃癌细胞的凋亡。采用Array-Human Signal Transduction PathwayFinder分别对GSI、NOTCH2siRNA和DLL4siRNA处理后的胃癌细胞MKN-45进行基因表达分析发现,三组无显著差异,NOTCH信号通路与WNT信号通路、P53信号通路和缺氧信号通路密切相关。Human Apoptosis Antibody Array抗体芯片分别对GSI、NOTCH2siRNA和DLL4siRNA处理后的MKN-45进行蛋白差异表达分析发现,GSI组差异蛋白既含有死亡受体途径的蛋白,同时线粒体凋亡途径中的多个蛋白也上调；siRNA组只有肿瘤坏死因子受体蛋白上调,说明两种抑制方法通过不同机制调控胃癌细胞的凋亡。结论NOTCH信号通路可做为胃癌治疗的潜在靶点,靶向抑制NOTCH受体或配体与抑制剂效果相似,其通过调控WNT信号通路、P53信号通路和缺氧信号通路相互作用,使胃癌细胞增殖降低并诱导发生凋亡,其凋亡机制有所差异：配体或受体siRNA通过死亡受体途径,抑制剂GSI通过死亡受体途径和线粒体途径两种方式诱导凋亡。更多还原

【Abstract】 AimsNOTCH signaling plays an important role in cell proliferation, differentiation and apoptosis. A growing body of research and clinical evidence are in support of NOTCH’s oncogenic or tumor suppressive role in a wide variety of cancers. It, therefore, places Notch signaling as a potential target for cancer therapeutics. Our previous study found that abnormal expression of the NOTCH signaling pathway in gastric cancer. The aim of this study was to investigate the possibility of NOTCH signaling pathway as a potential therapeutic target in gastric cancer, and explain the molecular mechanism which inhibition of Notch pathway induces apoptosis in gastric cancer cells. It would provide a theoretical basis for the treatment of gastric cancer.MethodsGastric epithelial cell line GES-1was co-cultured with H. pylori for24h. The signaling pathways activated by H. pylori were identified by RT2Profiler PCR Array-Human Signal Transduction Pathway Finder microarray. Total RNA and protein were isolated from GES-1incubation with different strains of H. pylori and were cocultured for24h. The activated signaling pathway verified by qPCR and Western Blot. Gastric cancer lines (MKN-45, SGC-7901, BGC-823) will be treated with or without GSI, Notch receptor siRNA and ligands siRNA. Cell cycle and apoptosis were detected by Annexin-V-FITC/PI and flow cytometry. Cross-talking between NOTCH signaling pathway and other signaling pathways analyzed by RT2Profiler PCR Array-Human Signal Transduction Pathway Finder microarray. The molecular mechanism which inhibition of NOTCH pathway induces apoptosis in gastric cancer cells analyzed by Human Apoptosis Antibody Array.ResultsThere are22different expression genes between GSI with and without H. pylori11637by RT2Profiler PCR Array-Human Signal Transduction Pathway Finder microarray. These genes were associated with cell proliferation, cell cycle and transcriptional regulation. DAVID Pathway analysis showed that the H. pylori infection mainly affects Cyclins and Cell Cycle Regulation and NOTCH Signaling Pathway. The expression levels of NOTCH ligand DLL4were significantly increased by H. pylori11637and SSI. H. pylori inhibited cell proliferation through the regulation of cell cycle-related proteins.Gastric cancer cell lines (BGC-823, MKN-45, SGC-7901) were treated with or without20μmol/L GSI for24h. The expression levels of mRNA and protein of NOTCH1, NOTCH2and HES1were inhibited significantly. Inhibitor induced cell-cycle arrest in G1phase, inhibited cell proliferation and induced apoptosis. NOTCH2siRNA and DLL4siRNA significantly inhibited the expression of the corresponding protein levels and decreased significantly HES1. siRNA group did not affect gastric cancer cell cycle. Both GSI group and siRNA group induced significantly gastric cancer cells apoptosis. NOTCH signaling pathway closely related to WNT signaling pathway, P53signaling pathway and hypoxia signaling pathways by analysis of Array-Human Signal Transduction Pathway Finder in group GSI, NOTCH2siRNA and DLL4siRNA treatment gastric cancer cell MKN-45. Human Apoptosis Antibody Array was used to identify differentially expressed protein related apoptosis in the control and GSI, NOTCH2siRNA and DLL4siRNA treatment. Our results showed that a number of apoptotic signaling proteins were modulated following treatment. Some proteins related mitochondrial pathway were elevated in GSI group. Tumor necrosis factor-a and its receptors were significantly higher in all treatment group as compared with control.ConclusionNOTCH signaling can be used as a potential target for the treatment of gastric cancer. There are similar effect in inhibitors, NOTCH receptor siRNA and ligand siRNA. Inhibition of NOTCH signaling pathway could reduce gastric cancer cell proliferation and induce apoptosis, through regulating WNT signaling pathway, P53signaling pathway, and hypoxia signaling pathways. siRNA induced cell apoptosis via death receptor-mediated pathway. GSI induced cell apoptosis via both the mitochondrial and death receptor-mediated pathway.更多还原