本文選題：IKBKE + Hippo通路��； 參考：《天津醫(yī)科大學》2017年博士論文

【摘要】：最近的研究表明,IKBKE(inhibitor of nuclear factor kappa-B kinase subunit epsilon)已經證實在多種惡性腫瘤中高表達,且沉默IKBKE蛋白表達可以抑制腫瘤進展。在本篇文章中,我們首先使用CCK-8檢測方法計算CYT387對2種代表性的膠質母細胞瘤細胞系U87-MG及LN229的半抑制濃度(half maximal inhibitory concentration,IC50)確定膠質母細胞瘤對此種小分子敏感;并且驗證CYT387,作為一種潛在的IKBKE抑制劑,能夠顯著地抑制膠質瘤細胞系細胞的增殖、遷移以及侵襲能力。此外,CYT387還可誘導細胞凋亡和并使細胞周期阻滯在G2期。與此同時我們發(fā)現(xiàn),沉默IKBKE蛋白含量可以增強Hippo通路,降低Hippo通路下游因子的轉錄水平。另外,我們證實了CYT387不僅僅抑制IKBKE激酶活性同時也降低其在蛋白水平的表達,而這種改變不涉及IKBKE mRNA水平。接下來,通過免疫印跡的辦法證實了CYT387從劑量及時間依賴性上促進Hippo信號通路來控制腫瘤的惡性進展。為探查更深一步的機制,我們使用免疫共沉淀(Co-immunoprecipitation,co-IP)的方法及特異性磷酸化抗體證實了IKBKE與YAP1及TEAD2相互作用,促進YAP1及TEAD2入核,調控腫瘤增殖生長。在接下來的體內中,我們發(fā)現(xiàn)CYT387能明顯抑制裸鼠皮下腫瘤的生長但無法遏制顱內腫瘤的生長,這可能是由于CYT387穿透血腦屏障(blood brain barrier,BBB)差造成的。這些結果表明,CYT387有可能成為新的抗膠質瘤靶向藥物但需要尋求通過血腦屏障的方法。方法 我們使用CCK-8檢測方法設立不同藥物濃度,通過檢測一定藥物濃度梯度下腫瘤細胞成活率,確定膠質母細胞瘤細胞系U87-MG及LN229在不同時間下的半抑制濃度(half maximal inhibitory concentration,IC50)。我們使用CCK-8的檢測方法及克隆形成實驗測定小分子抑制劑CYT387對腫瘤細胞增殖能力;利用劃痕試驗及transwell方法檢測藥物對膠質瘤細胞系遷移及侵襲能力的影響。接下來應用流式細胞儀檢測小分子抑制劑CYT387對膠質瘤細胞凋亡及周期的影響;并通過免疫印跡的方法,檢測相應周期蛋白及凋亡蛋白的表達;那下面,機制的研究中,我們先使用IKBKE-shRNA沉默IKBKE的蛋白表達,檢測Hippo通路核心蛋白LATS2,YAP1,TEAD2,p-YAP1磷酸化127位點及下游效應蛋白Axl,c-myc的表達含量的變化,并通過使用重組質粒過表達IKBKE的蛋白含量,重新檢測Hippo通路核心蛋白及下游效應蛋白表達變化,并使用小分子抑制劑CYT387從劑量及時間依賴性上處理膠質瘤細胞系檢測這些指標的變化;在接下來對進一步機制的研究中,我們使用免疫共沉淀(Co-immunoprecipitation,co-IP)的方法,從內源性及外源性兩個方面說明IKBKE與YAP1,TEAD2存在相互結合的作用;分別提取胞漿及胞核蛋白,分別使用免疫印記的方法說明沉默IKBKE可以抑制YAP1及tead2入核啟動轉錄過程,并應用免疫熒光的辦法說明沉默IKBKE可以抑制YAP1的入核;使用real-time RT-PCR證實IKBKE改變YAP1,TEAD2的含量是否牽扯到轉錄或者翻譯水平。使用U87-MG細胞注射4-5周大小裸鼠皮下建立裸鼠皮下腫瘤模型,模型建立一周后使用灌胃針經口灌胃CYT387(100mg/kg/day),飼養(yǎng)30天監(jiān)測皮下腫瘤大小,隨后處死全部裸鼠,獲取對照組與實驗組全部腫瘤稱重,免疫組化的方法檢測IKBKE,凋亡,周期,Hippo通路及下游因子指標變化;隨后使用感染了luciferase病毒的U87-MG細胞在立體定向儀的輔助下建立裸鼠顱內腫瘤模型,實驗組每天灌胃藥物CYT387(100mg/kg/day),每7天使用活體熒光成像儀監(jiān)測顱內腫瘤大小,監(jiān)測裸鼠體重及裸鼠存活情況,取鼠腦切片做免疫組化染色確定指標變化。結果 1,膠質瘤細胞系U87-MG及LN229對CYT387十分敏感,經CYT387處理后膠質瘤細胞增殖及集落形成能力大大降低。2,CYT387可以明顯抑制膠質瘤細胞系U87-MG及LN229遷移及侵襲能力。3,CYT387可以促進膠質瘤細胞系U87-MG及LN229凋亡。4,CYT387促進膠質瘤細胞系U87-MG及LN229周期阻滯,使腫瘤阻滯于G2期。5,CYT387不僅可以抑制IKBKE激酶活性還可以降低IKBKE蛋白表達含量,這種改變主要是翻譯后的修飾,在轉錄水平無明顯影響。6,CYT387通過抑制IKBKE表達及活性增強Hippo通路抑制惡性膠質瘤細胞生長。7,IKBKE可以與YAP1及TEAD2直接作用影響Hippo通路,沉默IKBKE可以抑制YAP1及TEAD2向細胞核內轉座。8,CYT387可以抑制裸鼠皮下腫瘤生長但對裸鼠顱內原位腫瘤的抑制效果不明顯。結論 1,CYT387在體外實驗中可以明顯抑制膠質母細胞瘤細胞系U87-MG及LN229的生長;促進其凋亡;2,CYT387可以活化Hippo通路抑制腫瘤細胞的生長,而這一作用主要是通過減低IKBKE激酶的活性及表達量實現(xiàn)的。3,IKBKE可與YAP1及TEAD2相互作用,促進YAP1及TEAD2向細胞內轉座從而影響Hippo通路從而控制膠質瘤進展;4,CYT387可以抑制皮下腫瘤的生長但對顱內腫瘤影響效果不明顯,可能與其透過血腦屏障差有關。
[Abstract]:Recent studies have shown that IKBKE (inhibitor of nuclear factor kappa-B kinase subunit epsilon) has been shown to be highly expressed in a variety of malignant tumors, and the expression of silent IKBKE protein can inhibit the progression of tumors. In this article, we first used CCK-8 detection to calculate CYT387 pairs of 2 representative glioblastoma cell lines. -MG and LN229's semi inhibitory concentration (half maximal inhibitory concentration, IC50) determines the sensitivity of glioblastoma to this small molecule, and verifies that CYT387, as a potential IKBKE inhibitor, can significantly inhibit the proliferation, migration and invasiveness of glioma cell lines. In addition, CYT387 can also induce apoptosis and the apoptosis of glioblastoma cells. And the cell cycle was blocked at G2 phase. At the same time, we found that the silencing of IKBKE protein content could enhance the Hippo pathway and reduce the transcriptional level of the downstream factors of the Hippo pathway. In addition, we have confirmed that CYT387 not only inhibits the activity of IKBKE kinase, but also reduces its protein level, but this change does not involve the level of IKBKE mRNA. By immunoblotting, it was demonstrated that CYT387 promotes the malignant progression of the tumor by promoting the dose and time dependence of the Hippo signaling pathway. In order to explore a further mechanism, we use the Co-immunoprecipitation (co-IP) method and specific phosphorylated antibodies to confirm the interaction of IKBKE with YAP1 and TEAD2. In the next body, we found that CYT387 can significantly inhibit the growth of subcutaneous tumors in nude mice but can not inhibit the growth of intracranial tumors in the next body, which may be caused by the poor CYT387 penetration of the blood brain barrier (blood brain barrier, BBB). These results suggest that CYT387 may become a new one. These results suggest that CYT387 may become a new one. Anti glioma targeting drugs need to be sought through the blood brain barrier method. Methods we used CCK-8 detection methods to establish different drug concentrations. By detecting the survival rate of tumor cells under a certain concentration gradient, the U87-MG and LN229 concentration of glioblastoma cell lines at different time (half maximal inhibitory CO) were determined. Ncentration, IC50). We used CCK-8 detection method and clone formation test to determine the proliferation ability of small molecule inhibitor CYT387 to tumor cells; use scratch test and Transwell method to detect the effect of drug on the migration and invasion ability of glioma cell lines. Then, flow cytometry was used to detect small molecule inhibitor CYT387 to glia. The effect of apoptosis and cycle of tumor cells, and the expression of corresponding cyclin and apoptotic protein by immunoblotting, and in the study of mechanism, we first use IKBKE-shRNA silencing IKBKE protein expression to detect the core protein of Hippo pathway LATS2, YAP1, TEAD2, p-YAP1 phosphorylation site 127 and downstream effector protein Axl, c-myc The changes in the expression of the content of IKBKE were redetected by using recombinant plasmids to overexpress the protein content of the Hippo pathway, and the changes in the expression of the core protein and downstream effector protein were redetected, and the changes of the glioma cell lines were treated with the dose and time dependence of the small molecular inhibitor CYT387. In the study, we use the method of Co-immunoprecipitation (co-IP) to explain the interaction between IKBKE and YAP1 and TEAD2 from two aspects of endogenous and exogenous. The cytoplasm and nucleoprotein were extracted respectively. The method of immuno imprinting was used respectively to show that IKBKE could inhibit the transcription of YAP1 and tead2, and the transcription process of YAP1 and tead2 was inhibited. The immunofluorescence method was used to show that silent IKBKE could inhibit the nucleation of YAP1; real-time RT-PCR was used to confirm that IKBKE changed YAP1, whether the content of TEAD2 was involved in the transcription or translation level. The subcutaneous tumor model of nude mice was established under the subcutaneous injection of U87-MG cells for 4-5 weeks in nude mice, and the model was established by intragastric intragastric perfusion a week later. 87 (100mg/kg/day), the size of the subcutaneous tumor was monitored for 30 days, then all nude mice were killed and all the tumor weighed in the control group and the experimental group. The immunohistochemical method was used to detect the changes of IKBKE, apoptosis, cycle, Hippo pathway and downstream factor, and then the U87-MG cells infected with luciferase virus were established with the aid of stereotactic instrument. The model of nude mice intracranial tumor, the experimental group was intragastric CYT387 (100mg/kg/day) every day, the size of intracranial tumor was monitored every 7 days by living fluorescent imager, the weight of nude mice and the survival of nude mice were monitored. The rat brain slices were determined by immunohistochemical staining. Results 1, the U87-MG and LN229 of glioma cell line were very sensitive to CYT387, and CYT387 After treatment, the proliferation and colony formation ability of glioma cells greatly reduced.2. CYT387 could obviously inhibit the migration and invasion of glioma cell lines U87-MG and LN229.3. CYT387 can promote the apoptosis of glioma cell lines U87-MG and LN229, and CYT387 promotes the U87-MG and LN229 cycle arrest of glioma cell lines. Only inhibition of IKBKE kinase activity can also reduce the expression of IKBKE protein, which is mainly translated after translation, and there is no obvious effect on.6 at transcriptional level. CYT387 inhibits the growth of malignant glioma cells by inhibiting the expression of IKBKE and the activity of Hippo to inhibit the growth of malignant glioma cells. IKBKE can affect the Hippo pathway directly with YAP1 and TEAD2, silent IK. BKE can inhibit the transposition of YAP1 and TEAD2 into the nucleus.8, CYT387 can inhibit the growth of nude mice, but the inhibitory effect on nude mouse intracranial tumor in situ is not obvious. Conclusion 1, CYT387 can obviously inhibit the growth of U87-MG and LN229 in the cell line of glioblastoma in vitro; promote its apoptosis; 2, CYT387 can activate the Hippo pathway. Inhibit the growth of tumor cells, and this effect is mainly by reducing the activity and expression of IKBKE kinase.3. IKBKE can interact with YAP1 and TEAD2 to promote YAP1 and TEAD2 transposing to the Hippo pathway to control the progression of glioma; 4, CYT387 can inhibit the growth of subcutaneous tumors but affect the effect of intracranial tumors. The fruit is not obvious. It may be related to poor blood brain barrier.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R739.41

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