食管鳞癌中LSD1与Notch信号通路相互调节作用及机理研究

发布时间：2018-07-03 12:17

  本文选题：食管鳞癌 + LSD1　； 参考：《郑州大学》2017年硕士论文

【摘要】：目的探明食管鳞癌中LSD1与Notch信号通路的相互调节作用及调节机制。方法1.LSD1与Notch通路相关蛋白在不同食管鳞癌细胞株中的表达情况。蛋白免疫印迹法(Western blot)检测食管鳞癌细胞株KYSE450、KYSE790、ECa109、EC9706和TE1中LSD1及Notch通路相关蛋白Notch1、DTX1和Hes1的表达情况。2.LSD1对Notch信号通路的调控作用。用LSD1抑制剂TCP与LSD1 si RNA处理食管鳞癌细胞,Western blot检测处理前后细胞中LSD1、组蛋白H3K4me2和Notch信号通路相关蛋白DTX1和Hes1的表达情况;3.染色质免疫沉淀反应(Chromatin Immunoprecipitation,Ch IP)验证ECa109细胞中LSD1与Notch信号通路靶基因Hes1、Notch3、DTX1和CR2启动子不同区域的结合情况。4.新型Notch信号通路抑制剂FLI-06对食管鳞癌ECa109和EC9706细胞的增殖、凋亡及周期的影响。CCK-8法检测FLI-06对食管鳞癌细胞的增殖作用;然后采用流式细胞术检测FLI-06对食管鳞癌细胞凋亡和周期的影响。5.Notch信号通路对LSD1的调控。食管鳞癌细胞中,用不同浓度FLI-06与γ-分泌酶抑制剂GSI-DAPT分别处理食管鳞癌ECa109和EC9706细胞48 h后,Western blot检测Notch通路相关蛋白Notch3、DTX1和Hes1及LSD1与组蛋白H3K4me2的表达情况。结果1.五株食管鳞癌细胞中均存在LSD1及Notch信号通路相关蛋白的表达,且表达水平不同。2.LSD1抑制剂TCP处理48 h后,KYSE450、KYSE790、ECa109、EC9706和TE1细胞中LSD1的表达均降低,其组蛋白H3K4me2的表达升高,Notch信号通路相关蛋白的表达也均降低;LSD1 si RNA同样使ECa109细胞中LSD1与Notch信号通路相关蛋白表达受到抑制。3.染色质免疫沉淀反应证实LSD1能与Notch靶基因的启动子区域结合。4.FLI-06抑制食管鳞癌ECa109和EC9706细胞的增殖,并且具有浓度依赖性,其中FLI-06对ECa109和EC9706细胞48 h的IC50分别为(5.814±0.053)μM和(10.741±0.049)μM;流式细胞术结果显示,FLI-06以不同浓度处理ECa109和EC9706细胞48 h后,随着药物浓度增加,细胞的凋亡率显著增加,G1期细胞数增加,表明FLI-06能诱导细胞的凋亡,且能将细胞阻滞于G1期。5.FLI-06和GSI-DAPT处理食管鳞癌ECa109和EC9706细胞48 h后,Notch3、DTX1和Hes1等Notch通路相关蛋白表达降低,Notch信号通路受到抑制;LSD1表达降低,组蛋白H3K4me2表达水平没有变化。结论1.食管鳞癌细胞中LSD1对Notch信号通路有调节作用,且LSD1可能是通过与Notch靶基因的启动子区域结合从而对Notch信号通路起到调节作用。2.在食管鳞癌细胞中,Notch信号通路对LSD1也具有调控作用。
[Abstract]:Objective to investigate the interaction and mechanism of LSD1 and Notch signaling pathway in esophageal squamous cell carcinoma. Methods 1. Expression of LSD1 and Notch pathway related protein in different esophageal squamous cell carcinoma cell lines. Expression of LSD1 and Notch Pathway-associated proteins Notch1DTX1 and Hes1 in esophageal squamous Cell carcinoma Cell Line KYSE450KYSE790 EC9706 and TE1 were detected by Western blot. 2. The regulation of Notch signaling pathway by LSD1. The expression of histone H3K4me2 and Notch signaling pathway related proteins DTX1 and Hes1 in esophageal squamous cell carcinoma cells treated with LSD1 inhibitor TCP and LSD1 si RNA was detected by Western blot. Chromatin immunoprecipitation (Ch IP) was used to identify the binding of LSD1 with Notch signal pathway target gene Hes1 Notch3tX1 and CR2 promoter in ECa109 cells. Effects of novel Notch signaling pathway inhibitor FLI-06 on proliferation, apoptosis and cell cycle of esophageal squamous cell carcinoma ECa109 and EC9706. Then the effect of FLI-06 on apoptosis and cell cycle of esophageal squamous cell carcinoma cells was detected by flow cytometry. 5. Notch signaling pathway regulated LSD1. Esophageal squamous cell carcinoma cells were treated with different concentrations of FLI-06 and GSI-DAPT, respectively. After 48 hours of treatment, the expression of Notch pathway related proteins Notch3pDTX1, Hes1, LSD1 and histone H3K4me2 were detected by Western blot. Result 1. LSD1 and Notch signaling pathway related proteins were expressed in all five esophageal squamous cell carcinoma cells, and the expression level of LSD1 was decreased in KYSE450KYSE790, ECa109 EC9706 and TE1 cells after 48 h treatment with different levels of LSD1 inhibitor TCP. The expression of histone H3K4me2 increased and the expression of Notch signaling pathway related proteins decreased. The expression of LSD1 si RNA also inhibited the expression of LSD1 and Notch signaling pathway related proteins in ECa109 cells. Chromatin immunoprecipitation assay demonstrated that LSD1 could bind to Notch target promoter region. 4. FLI-06 inhibited the proliferation of esophageal squamous cell carcinoma ECa109 and EC9706 cells in a concentration-dependent manner. The IC50 of ECa109 and EC9706 cells treated with FLI-06 for 48 h was (5.814 卤0.053) 渭 M and (10.741 卤0.049) 渭 M. flow cytometry showed that the apoptosis rate of ECa109 and EC9706 cells increased significantly with the increase of drug concentration. The results showed that FLI-06 could induce apoptosis of esophageal squamous cell carcinoma ECa109 and EC9706 cells, and could block the cells in G1 phase. 5. FLI-06 and GSI-DAPT treated ECa109 and EC9706 cells for 48 h. After 48 hours, the expression of Notch pathway related proteins such as Notch3, DTX1 and Hes1 were decreased. There was no change in the expression of histone H 3 K 4 m 2. Conclusion 1. LSD1 regulates Notch signaling pathway in esophageal squamous cell carcinoma cells, and LSD1 may regulate Notch signaling pathway by binding to Notch target gene promoter region. The Notch signaling pathway also regulates LSD1 in esophageal squamous cell carcinoma cells.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

【相似文献】

相关期刊论文 前10条

1 张燕;郑志z，

本文编号：2093611