本文選題：食管鱗癌 + LSD1　； 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：目的探明食管鱗癌中LSD1與Notch信號通路的相互調(diào)節(jié)作用及調(diào)節(jié)機(jī)制。方法1.LSD1與Notch通路相關(guān)蛋白在不同食管鱗癌細(xì)胞株中的表達(dá)情況。蛋白免疫印跡法(Western blot)檢測食管鱗癌細(xì)胞株KYSE450、KYSE790、ECa109、EC9706和TE1中LSD1及Notch通路相關(guān)蛋白Notch1、DTX1和Hes1的表達(dá)情況。2.LSD1對Notch信號通路的調(diào)控作用。用LSD1抑制劑TCP與LSD1 si RNA處理食管鱗癌細(xì)胞,Western blot檢測處理前后細(xì)胞中LSD1、組蛋白H3K4me2和Notch信號通路相關(guān)蛋白DTX1和Hes1的表達(dá)情況;3.染色質(zhì)免疫沉淀反應(yīng)(Chromatin Immunoprecipitation,Ch IP)驗證ECa109細(xì)胞中LSD1與Notch信號通路靶基因Hes1、Notch3、DTX1和CR2啟動子不同區(qū)域的結(jié)合情況。4.新型Notch信號通路抑制劑FLI-06對食管鱗癌ECa109和EC9706細(xì)胞的增殖、凋亡及周期的影響。CCK-8法檢測FLI-06對食管鱗癌細(xì)胞的增殖作用;然后采用流式細(xì)胞術(shù)檢測FLI-06對食管鱗癌細(xì)胞凋亡和周期的影響。5.Notch信號通路對LSD1的調(diào)控。食管鱗癌細(xì)胞中,用不同濃度FLI-06與γ-分泌酶抑制劑GSI-DAPT分別處理食管鱗癌ECa109和EC9706細(xì)胞48 h后,Western blot檢測Notch通路相關(guān)蛋白Notch3、DTX1和Hes1及LSD1與組蛋白H3K4me2的表達(dá)情況。結(jié)果1.五株食管鱗癌細(xì)胞中均存在LSD1及Notch信號通路相關(guān)蛋白的表達(dá),且表達(dá)水平不同。2.LSD1抑制劑TCP處理48 h后,KYSE450、KYSE790、ECa109、EC9706和TE1細(xì)胞中LSD1的表達(dá)均降低,其組蛋白H3K4me2的表達(dá)升高,Notch信號通路相關(guān)蛋白的表達(dá)也均降低;LSD1 si RNA同樣使ECa109細(xì)胞中LSD1與Notch信號通路相關(guān)蛋白表達(dá)受到抑制。3.染色質(zhì)免疫沉淀反應(yīng)證實LSD1能與Notch靶基因的啟動子區(qū)域結(jié)合。4.FLI-06抑制食管鱗癌ECa109和EC9706細(xì)胞的增殖,并且具有濃度依賴性,其中FLI-06對ECa109和EC9706細(xì)胞48 h的IC50分別為(5.814±0.053)μM和(10.741±0.049)μM;流式細(xì)胞術(shù)結(jié)果顯示,FLI-06以不同濃度處理ECa109和EC9706細(xì)胞48 h后,隨著藥物濃度增加,細(xì)胞的凋亡率顯著增加,G1期細(xì)胞數(shù)增加,表明FLI-06能誘導(dǎo)細(xì)胞的凋亡,且能將細(xì)胞阻滯于G1期。5.FLI-06和GSI-DAPT處理食管鱗癌ECa109和EC9706細(xì)胞48 h后,Notch3、DTX1和Hes1等Notch通路相關(guān)蛋白表達(dá)降低,Notch信號通路受到抑制;LSD1表達(dá)降低,組蛋白H3K4me2表達(dá)水平?jīng)]有變化。結(jié)論1.食管鱗癌細(xì)胞中LSD1對Notch信號通路有調(diào)節(jié)作用,且LSD1可能是通過與Notch靶基因的啟動子區(qū)域結(jié)合從而對Notch信號通路起到調(diào)節(jié)作用。2.在食管鱗癌細(xì)胞中,Notch信號通路對LSD1也具有調(diào)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 鹵0.053) 渭 M and (10.741 鹵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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.1

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