DNA-PKcs在放射诱发HeLa细胞自噬中的作用及抑制自噬对细胞周期检查点的影响研究

发布时间：2018-05-19 21:18

本文选题：DNA + 依赖的蛋白激酶催化亚基　；参考：《中国人民解放军军事医学科学院》2016年硕士论文

【摘要】：DNA依赖的蛋白激酶催化亚基(DNA-dependent kinase catalytic subunit,DNA-PKcs)是一个分子量大约为460k D的蛋白激酶,其属于磷脂酰肌醇-3-激酶相关蛋白激酶家族成员,是一种被DNA激活的丝氨酸/苏氨酸激酶,其与KU70/KU80组成的异源二聚体调节亚基一起组成了异源三聚体DNA依赖的蛋白激酶(DNA-dependent kinase,DNA-PK)。DNA-PKcs的丝氨酸/苏氨酸蛋白激酶活性不仅可以激活调节损伤修复的相关蛋白和参与细胞周期调控的蛋白,而且还可以通过自身活化的方法调节其激酶活性。研究证实DNA-PKcs是一个具有多功能性的酶,其最主要的也是人们研究最透彻的一个功能就是通过非同源末端修复途径参与DNA双链断裂损伤修复。除此之外,DNA-PKcs的沉默或缺失还会引起电离辐射或化学药物引起的细胞不正常的分裂,细胞非正常的G1期或G2/M期阻滞和自噬。近年来随着对DNA-PKcs研究的不断研究,发现其在肿瘤组织中的表达要超出正常范围。很多研究已经表明DNA-PKcs的抑制是一个增加肿瘤细胞对抗电离辐射,肿瘤试剂拓扑异构酶II毒性物质和顺铂等敏感性的非常可行的方法。有研究表明,DNA-PKcs在胶质瘤细胞中参与电离辐射诱发自噬的调节,但是关于DNA-PKcs如何调节自噬的研究很少。细胞自噬是真核细胞中非常关键且保守的物质的分解与循环利用的过程,有助于降解细胞内长寿命的蛋白质,细胞溶质的成分,损伤老化的细胞器以及某些病原体使得它们得以循环重复利用,从而有助于保持细胞的内稳态,这是自噬最基本的作用。在面对错乱复杂的环境比如必需营养物质的缺乏,电离辐射以及内源性的活性氧,复制压力等,机体的细胞需要作出各种反应来应对不利环境,自噬会通过消耗自身的物质来使细胞获得临时的供应从而促进细胞存活。但是,细胞自噬在肿瘤的治疗过程中依然是一个十分有争议的话题,因为自噬可以刺激细胞存活,也可以诱导细胞死亡。研究表明当细胞遭遇某一种生理变化或者某一种特殊的处理比如致死性的药物剂量,细胞自噬就可以促进细胞的死亡。研究证实自噬的功能失调与许多疾病的起始和发展有很大关系。3-甲基腺嘌呤(3-methyladenine,3MA)是一个被研究学者广泛使用的细胞自噬抑制剂,主要作用能够通过抑制细胞自噬的正性调节子Ⅲ型PI3K来抑制自噬。目的1.以DNA-PKcs敲低的He La细胞系为对象,观察照射后细胞的放射敏感性以及自噬蛋白的表达情况,探讨DNA-PKcs调节自噬作用及其可能的分子机制;2.电离辐射可以诱发宫颈癌He La细胞G2/M期的周期阻滞,探讨自噬抑制剂3-甲基腺嘌呤在电离辐射诱发He La周期阻滞中的调节作用。方法利用实验室前期构建的DNA-PKcs沉默的表达载体,采用慢病毒介导的小干扰RNA技术,通过Lipofectamin 2000作为介质,包装慢病毒颗粒并感染He La细胞,使用嘌呤霉素抗性筛选获得稳定敲低DNA-PKcs的宫颈癌He La细胞系,并且经过免疫印迹鉴定;Hela-sh NC和Hela-sh DNA-PKcs经过单独照射或者与自噬的抑制剂3-甲基腺嘌呤联合作用处理细胞,在照射后不同时间点(0、12、24、48和72h)采用细胞增殖与毒性试验检测细胞的增殖与细胞的放射敏感性变化;He La细胞经过单独照射或者与自噬的抑制剂3-甲基腺嘌呤联合作用处理,通过细胞平板克隆形成实验检测照射后He La-sh NC和He La-sh DNA-PKcs在不同剂量射线下存活率变化;通过流式细胞术检测6Gy照射后0、2、4、6、8和12h后的细胞周期变化及有丝分裂期磷酸化组蛋白H3的变化;DNA-PKcs的激酶抑制剂NU7026与自噬的激动剂雷帕霉素共同处理细胞并通过免疫印迹实验检测多功能的泛素结合蛋白P62、微管轻链蛋白LC3、哺乳动物雷帕霉素的靶蛋白m TOR并且He La经过单独照射或者与自噬的抑制剂3-甲基腺嘌呤联合作用处理,通过免疫印迹检测周期检验点激酶2(Chk2),Cdk1及磷酸化酶Cdc25C的变化;利用分子克隆技术构建GFP-LC3表达载体并通过激光共聚焦免疫荧光技术检测绿色荧光GFP-LC3焦点的形成。结果1.免疫印迹验证He La-sh NC和He La-sh DNA-PKcs,发现DNA-PKcs在敲低细胞中表达很少,这说明DNA-PKcs稳定敲低的细胞系构建成功;2.细胞增殖与毒性检测结果显示,DNA-PKcs敲低的细胞放射敏感性增强,细胞的增殖速度减慢;在照射处理的基础上在细胞中加入自噬的抑制剂3-甲基腺嘌呤,无论是对照细胞还是DNA-PKcs敲低的细胞,其细胞的放射敏感性进一步增强;3.免疫印迹结果得到:与Hela-sh NC而言,DNA-PKcs敲低的细胞中,微观相关蛋白LC3蛋白表达增多而p62蛋白表达减少,m TOR在2481位点的磷酸化水平下调;4.使用自噬的激动剂雷帕霉素和DNA-PKcs的激酶抑制剂NU7026处理Hela细胞,通过免疫印迹实验得到DNA-PKcs的激酶活性被抑制后,细胞自噬蛋白LC3表达增加且加入雷帕霉素后自噬水平进一步增加;5.激光共聚焦免疫荧光法得到DNA-PKcs蛋白敲低后,平均每个细胞的GFP-LC3荧光斑点数目增多,加入自噬的抑制剂3MA细胞自噬被抑制,加入自噬促进物RAPA,细胞自噬增加;6.细胞经过6Gy照射处理并通过流式细胞术检测在照射后不同时间细胞周期的变化,发现细胞周期被阻滞在G2/M期,这与实验室前期的实验结果一致;在照射的基础上辅以自噬的抑制剂3-甲基腺嘌呤发现细胞的阻滞现象被破坏,这说明3MA参与了电离辐射诱导的周期阻滞的调节;7.细胞经过照射和自噬的抑制剂3-甲基腺嘌呤共同处理,通过流式细胞术检测3-甲基腺嘌呤处理后磷酸化的组蛋白H3表达增多,这说明3MA可以促进细胞从G2期进入到M期;8.免疫印迹检测在不同的时间点细胞周期相关蛋白,发现在加入自噬的抑制剂3-甲基腺嘌呤后,磷酸化的ATM(S1981)、Chk2(T68),Cdc2(T15)和Cdc25C(S216)蛋白的水平均有所下调。结论1.DNA-PKcs蛋白敲低后细胞的放射敏感性增加且电离辐射诱发的细胞自噬增加;2.DNA-PKcs可能是通过m TOR信号通路来调节自噬;3.3-甲基腺嘌呤通过ATM/Chk2/Cdc25c/Cdk1的信号通路来调节电离辐射诱发的细胞周期G2/M检查点功能。
[Abstract]:DNA dependent protein kinase catalytic subunit (DNA-dependent kinase catalytic subunit, DNA-PKcs) is a protein kinase with a molecular weight of approximately 460k D, belonging to a member of the phosphatidyl inositol -3- kinase related protein kinase family, a serine / threonine kinase activated by DNA, and a heterogenous two polymer modulating subunit consisting of KU70/KU80. The serine / threonine protein kinase activity of the DNA-dependent kinase (DNA-PK).DNA-PKcs dependent protein kinase (kinase, DNA-PK).DNA-PKcs not only activates the related proteins that regulate the damage repair and the proteins involved in the cell cycle regulation, but also can regulate the activity of the kinase through the method of self activation. The study confirms that DNA -PKcs is a multifunctional enzyme, and its most important function is to participate in the repair of DNA double strand breaks through a non homologous terminal repair pathway. In addition, the silence or deletion of DNA-PKcs may cause abnormal division of cells induced by ionizing radiation or chemical drugs, and the cells are abnormal. G1 phase or G2/M phase block and autophagy. In recent years, with the continuous study of DNA-PKcs research, it has been found that its expression in the tumor tissue is beyond the normal range. Many studies have shown that the inhibition of DNA-PKcs is an increase in the sensitivity of tumor cells to ionizing radiation, the tumor reagent topoisomerase II toxic substances and cisplatin. Method. Studies have shown that DNA-PKcs is involved in the regulation of autophagy induced by ionizing radiation in glioma cells, but there is little research on how DNA-PKcs regulates autophagy. Cell autophagy is the process of decomposition and recycling of very critical and conservative substances in eukaryotic cells, which can help to degrade proteins and cells with long life in cells. The components of the solute, the aging of the organelles and some pathogens make them circulate and reuse them, thus helping to maintain the homeostasis of the cells, which is the most fundamental role of autophagy. In the face of the complex and complex environment such as the lack of essential nutrients, ionizing radiation, endogenous reactive oxygen species, and the reproduction of pressure, Cells need to respond to adverse circumstances, and autophagy can help cells to survive by consuming their own substances to facilitate cells to survive. However, autophagy remains a very controversial topic in the treatment of tumors, because autophagy can stimulate cell survival and induce cell death. Studies have shown that cell autophagy can promote cell death when a cell is subjected to a certain physiological change or a particular treatment, such as a lethal dose of drug. The study confirms that the dysfunction of autophagy has a great relationship with the initiation and development of many diseases..3- methyl adenine (3-methyladenine, 3MA) is a widely studied scholar. A ubiquitous cellular autophagy inhibitor, which can inhibit autophagy by inhibiting the positive regulator of autophagy PI3K to inhibit autophagy. Objective 1. to observe the radiosensitivity of the cells and the expression of autophagy after exposure to the DNA-PKcs knockout He La cell lines, and to explore the role of DNA-PKcs to regulate autophagy and its possible molecules. Mechanism: 2. ionizing radiation can induce the periodic block of the G2/M phase of He La cells in cervical cancer, and explore the regulating role of the autophagy inhibitor 3- methyl adenine in the He La cycle arrest induced by ionizing radiation. Methods using the DNA-PKcs silent expression vector constructed in the early laboratory, the small interference RNA technology mediated by the slow disease virus, through Lipofectamin, through Lipofectamin, through Lipofectamin. 2000 as a medium, the lentivirus particles were packed and infected with He La cells. The He La cell line of the cervical cancer that had a stable knock down DNA-PKcs was screened using the resistance of purinomycin resistance, and was identified by immunoblotting; Hela-sh NC and Hela-sh DNA-PKcs were irradiated individually or combined with the autophagy inhibitor 3- methyl adenine. After different time points (0,12,24,48 and 72h), cell proliferation and cytotoxicity test were used to detect cell proliferation and cell radiosensitivity. He La cells were irradiated individually or combined with autophagic inhibitor 3- methyl adenine, and cell plate cloning was used to test the He La-sh NC and He La-sh DNA-PKcs after irradiation. The change of survival rate under different doses of radiation; cell cycle changes of 0,2,4,6,8 and 12h after 6Gy irradiation by flow cytometry and changes in phosphorylated histone H3 during mitosis; DNA-PKcs kinase inhibitor NU7026 and autophagy agonist rapamycin were treated together and multifunctional flooding was detected by immunoblotting. The peptide binding protein P62, microtubule light chain protein LC3, mammalian target protein M TOR of rapamycin and He La were irradiated individually or combined with autophagic inhibitor 3- methyl adenine, and the changes in periodic test point kinase 2 (Chk2), Cdk1 and phosphorylase Cdc25C were detected by immunoblotting, and GFP-LC3 was constructed by molecular cloning technology. The expression vector was expressed by the laser confocal immunofluorescence technique to detect the formation of the focus of green fluorescent GFP-LC3. Results 1. Western blot verified He La-sh NC and He La-sh DNA-PKcs, and found that DNA-PKcs was rarely expressed in the knockout cells, which indicated that the cell lines with a stable and low level of low DNA-PKcs were constructed successfully; 2. cell proliferation and toxicity detection showed, DNA-P, DNA-P. The radiosensitivity of the Kcs knockout cells increased and the cell proliferation slowed down; 3- methyl adenine, an inhibitor of autophagy, was added to the cells on the basis of irradiation treatment, whether the control cells or the DNA-PKcs knockout cells, the radiosensitivity of the cells was further enhanced; 3. the results of immunoblotting were obtained with Hela-sh NC, DNA-PKcs In the lower knockout cells, the expression of microprotein LC3 protein was increased and the expression of p62 protein decreased, and the phosphorylation level of M TOR was downregulated at 2481 site. 4. the Hela cells were treated with the autophagic agonist, rapamycin and DNA-PKcs kinase inhibitor NU7026, and the kinase activity of DNA-PKcs was inhibited by immunoblotting, and the autophagic eggs of the cells were autophagy. The expression of white LC3 was increased and the autophagy level was further increased after the addition of rapamycin. 5. laser confocal immunofluorescence showed that the number of GFP-LC3 fluorescence spots in each cell increased, the autophagy of 3MA cells added to autophagy was inhibited, autophagy promoted RAPA, autophagy increased, and 6. cells were illuminated by 6Gy. The cell cycle of the cell cycle was detected by flow cytometry and the cell cycle was detected at different time after irradiation. It was found that the cell cycle was blocked in the G2/M phase, which was in accordance with the experimental results in the early laboratory. On the basis of the irradiation, the inhibitor of autophagy, 3- methyl adenine, found that the cell block was destroyed, which indicates that 3MA is involved in the ionizing radiation. The regulation of induced periodic block; 7. cells were treated with 3- methyl adenine, an inhibitor of irradiation and autophagy, and the expression of histone H3 in the phosphorylation of 3- methyl adenine was increased by flow cytometry, indicating that 3MA could promote cells to enter M phase from G2 phase; and 8. blot detection at different time points in cell cycle phase The levels of phosphorylated ATM (S1981), Chk2 (T68), Cdc2 (T15) and Cdc25C (S216) protein were all downregulated after the autophagy inhibitor 3- methyl adenine was added. Conclusion the radiosensitivity of the cells increased and the cell autophagy induced by ionizing radiation increased after the 1.DNA-PKcs protein knockdown; 2.DNA-PKcs may be through the m signaling pathway. To regulate autophagy, 3.3- methyladenine regulates the G2/M checkpoint function of ionizing radiation induced cell cycle through ATM/Chk2/Cdc25c/Cdk1 signaling pathway.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R818

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