CUL4A调控NF-κB信号通路在促进胃癌侵袭中的分子机制

发布时间：2017-12-27 12:39

本文关键词：CUL4A调控NF-κB信号通路在促进胃癌侵袭中的分子机制　出处：《南昌大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:探讨CUL4A在炎症促进胃癌侵袭性中影响,以及阐明CUL4A通过调控NF-κB信号通路参与这一过程的分子机制。方法:1、选择HGC27胃癌细胞株,转染CUL4A siRNA以及转染无义序列NC。在炎症刺激物LPS的刺激下,采用细胞划痕、Transwell实验检测四组胃癌细胞(NC、LPS、CUL4A si RNA、LPS+CUL4A siRNA)的迁移及侵袭能力。2、采用western blot技术检测LPS对CUL4A和NF-κB蛋白刺激作用以及刺激的最佳时间和最适浓度。3、采用western blot和RT-PCR技术检测CUL4A siRNA对CUL4A蛋白及mRNA水平表达的抑制情况。在最佳时间和最适浓度LPS刺激下,采用western blot技术检测分别转染CUL4A siRNA和无义序列NC胃癌细胞的NF-κB的蛋白表达情况和用RT-PCR技术检测NF-κB信号通路下游炎症因子的mRNA水平。4、采用免疫组织化学(Immunohistochemistry,IHC)和western blot技术同时检测CUL4A和NF-κB蛋白在胃癌及癌旁组织标本中的表达的情况,并分析它们在胃癌组织中表达之间的相关性。结果:1、细胞划痕实验及Transwell实验结果表明,LPS能够促进胃癌细胞的侵袭和迁移能力,但下调CUL4A则这一促进作用减弱。2、LPS能够增强CUL4A和NF-κB的蛋白表达,且最佳时间和最是浓度都分别是2h和100ng/mL。3、Western blot和RT-PCR结果表明,CUL4A siRNA能够有效的在蛋白水平及mRNA水平上抑制CUL4A的表达。与此同时,转染了CUL4A siRNA的胃癌细胞组与NC组相比NF-κB的表达明显下降,且下游的一些炎症因子mRNA表达也下降。4、免疫组织化学实验中,CUL4A蛋白主要染色在胃癌组织的细胞膜和细胞核中,而NF-κB蛋白染色主要在细胞核中。CUL4A和NF-κB在胃癌组织中的表达是呈正相关的。胃癌及癌旁组织的western blot结果显示,CUL4A和NF-κB蛋白在胃癌组织中的过度表达。结论:CUL4A能够通过调控NF-κB信号通路促进胃癌的侵袭性。
[Abstract]:Objective: To investigate the effect of CUL4A on the invasion of gastric cancer by inflammation, and to elucidate the molecular mechanism of CUL4A involved in the regulation of NF- kappa B signaling pathway. Methods: 1, HGC27 gastric cancer cell line was selected, CUL4A siRNA was transfected and NC was transfected with non sense sequence. Under the stimulation of LPS, the migration and invasion ability of four groups of gastric cancer cells (NC, LPS, CUL4A Si RNA, LPS+CUL4A siRNA) were detected by cell scratch test and Transwell test. 2. The best time and optimum concentration of LPS on CUL4A and NF- kappa B were detected by Western blot. 3. Western blot and RT-PCR were used to detect the inhibition of CUL4A siRNA on the expression of CUL4A protein and mRNA. Under the optimal time and the best concentration of LPS stimulation, Western blot technique was used to detect the protein expression of NF- NF- B in gastric cancer cells transfected with CUL4A siRNA and nonsense sequence NC respectively, and RT-PCR level was used to detect the level of NF- in the downstream of NF- kappa signaling pathway. 4, the expression of CUL4A and NF- kappa B protein in gastric cancer and paracancerous tissue specimens were detected by immunohistochemistry (Immunohistochemistry, IHC) and Western blot technology, and the correlation between their expression in gastric cancer tissues was analyzed. Results: 1. The results of cell scratch test and Transwell test showed that LPS could promote the invasion and migration of gastric cancer cells, but down regulation of CUL4A promoted this effect. 2, LPS can enhance the protein expression of CUL4A and NF- kappa B, and the best time and the most concentration are 2H and 100ng/mL respectively. 3, Western blot and RT-PCR results showed that CUL4A siRNA could effectively inhibit the expression of CUL4A at protein level and mRNA level. At the same time, the expression of NF- kappa B in the gastric cancer cells transfected with CUL4A siRNA was significantly lower than that in the NC group, and the expression of some of the downstream inflammatory factors mRNA was also decreased. 4. In immunohistochemical experiments, CUL4A protein is mainly stained in the cell membrane and nucleus of gastric cancer tissue, while the NF- kappa B protein staining is mainly in the nucleus. The expression of CUL4A and NF- kappa B in gastric carcinoma is positively correlated. Western blot results of gastric cancer and paracancerous tissue showed that the overexpression of CUL4A and NF- kappa B protein in gastric cancer tissues. Conclusion: CUL4A can promote the invasiveness of gastric cancer by regulating the NF- kappa B signaling pathway.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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