本文關(guān)鍵詞：CUL4A調(diào)控NF-κB信號通路在促進胃癌侵襲中的分子機制　出處：《南昌大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

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【摘要】：目的:探討CUL4A在炎癥促進胃癌侵襲性中影響,以及闡明CUL4A通過調(diào)控NF-κB信號通路參與這一過程的分子機制。方法:1、選擇HGC27胃癌細(xì)胞株,轉(zhuǎn)染CUL4A siRNA以及轉(zhuǎn)染無義序列NC。在炎癥刺激物L(fēng)PS的刺激下,采用細(xì)胞劃痕、Transwell實驗檢測四組胃癌細(xì)胞(NC、LPS、CUL4A si RNA、LPS+CUL4A siRNA)的遷移及侵襲能力。2、采用western blot技術(shù)檢測LPS對CUL4A和NF-κB蛋白刺激作用以及刺激的最佳時間和最適濃度。3、采用western blot和RT-PCR技術(shù)檢測CUL4A siRNA對CUL4A蛋白及mRNA水平表達的抑制情況。在最佳時間和最適濃度LPS刺激下,采用western blot技術(shù)檢測分別轉(zhuǎn)染CUL4A siRNA和無義序列NC胃癌細(xì)胞的NF-κB的蛋白表達情況和用RT-PCR技術(shù)檢測NF-κB信號通路下游炎癥因子的mRNA水平。4、采用免疫組織化學(xué)(Immunohistochemistry,IHC)和western blot技術(shù)同時檢測CUL4A和NF-κB蛋白在胃癌及癌旁組織標(biāo)本中的表達的情況,并分析它們在胃癌組織中表達之間的相關(guān)性。結(jié)果:1、細(xì)胞劃痕實驗及Transwell實驗結(jié)果表明,LPS能夠促進胃癌細(xì)胞的侵襲和遷移能力,但下調(diào)CUL4A則這一促進作用減弱。2、LPS能夠增強CUL4A和NF-κB的蛋白表達,且最佳時間和最是濃度都分別是2h和100ng/mL。3、Western blot和RT-PCR結(jié)果表明,CUL4A siRNA能夠有效的在蛋白水平及mRNA水平上抑制CUL4A的表達。與此同時,轉(zhuǎn)染了CUL4A siRNA的胃癌細(xì)胞組與NC組相比NF-κB的表達明顯下降,且下游的一些炎癥因子mRNA表達也下降。4、免疫組織化學(xué)實驗中,CUL4A蛋白主要染色在胃癌組織的細(xì)胞膜和細(xì)胞核中,而NF-κB蛋白染色主要在細(xì)胞核中。CUL4A和NF-κB在胃癌組織中的表達是呈正相關(guān)的。胃癌及癌旁組織的western blot結(jié)果顯示,CUL4A和NF-κB蛋白在胃癌組織中的過度表達。結(jié)論:CUL4A能夠通過調(diào)控NF-κB信號通路促進胃癌的侵襲性。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.2

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相關(guān)期刊論文 前2條

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本文編號：1341761