【作者】 张莹；

【导师】 曹流；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2018， 博士

【摘要】 目的:结肠癌是发生于结肠部位的常见的消化道恶性肿瘤,占胃肠道肿瘤的第三位^[1],其治疗手段与其它肿瘤一样有手术治疗、放射治疗、化学治疗、免疫治疗、基因治疗及中医药治疗等。由于手术治疗无法根治而放射疗法有很强的副作用,基因靶向治疗带来的副作用很小,成为治疗肿瘤的新手段。近年来研究发现,在遗传物质不变的情况下,蛋白质修饰能够影响基因组的稳定性,最终导致肿瘤的发生。其中乙酰化^[2]和磷酸化^[3-4]修饰在其中起至关重要的作用,那么研究两个修饰之间的相互作用就成为近几年研究的热点。去乙酰化酶SIRT2作为sirtuins家族的主要成员之一在有丝分裂进程的调节^[5],氧化应激反应^[6],代谢^[7],微管稳定性^[8],细胞迁移^[9],凋亡^[10],神经毒性^[11-12]以及抑制分化^[7,13]中起到重要作用。SIRT2主要定位于细胞质中,但在G₂/M转换过程中,SIRT2可以入核,先前有研究报道当抑制SIRT2,FoxO1的乙酰化水平升高,乙酰化状态的FoxO1与ATG7结合,作为E1样泛素化连接酶的ATG7,通过影响细胞自噬导致细胞死亡。这为开发SIRT2抑制剂靶向治疗结肠癌提供了理论依据^[14]。染色质结构维持蛋白1（SMC1）是我们通过质谱技术发现的一个SIRT2新的底物。在姐妹染色单体的粘附,染色质压缩,基因计量补偿,细胞周期调节以及DNA损伤修复中具有重要作用^[15-16]。而SMC1 Ser ⁹⁵⁷以及Ser ⁹⁶⁶的磷酸化是SMC1行使功能的重要表现行使,但有关SMC1的乙酰化/去乙酰化修饰方面的研究尚未被报道,乙酰化与磷酸化之间的调控更未见任何报道。本研究以蛋白质之间的互作为前提条件,在证实了SIRT2与SMC1在体内外均有相互作用基础上,探讨了氧自由基压力下,SIRT2是如何去乙酰化SMC1而影响SMC1的Ser ⁹⁵⁷以及Ser ⁹⁶⁶磷酸化水平,以及这一通路在结肠癌中起到何种作用。同时我们生产SIRT2去乙酰化SMC1位点的特异抗体,并且利用该抗体进一步验证SIRT2对SMC1的去乙酰化调控,且通过外源转染人结肠癌细胞系HCT116该位点的活性形式以及磷酸化激活型乙酰化位点激活形式后检测G₂/M周期,凋亡等生物学功能,再进一步利用体内裸鼠成瘤探究该位点对于肿瘤增殖的作用,这一研究为肿瘤的靶向治疗提供新思路。研究方法:本研究首先利用质谱技术发现了SIRT2与SMC1具有相互作用。在氧自由基压力下,免疫共沉淀进一步证实SIRT2与SMC1互作增强。那么接下来我们证实SIRT2与SMC1不仅存在互作,且SMC1作为SIRT2底物,可以被去乙酰化。首先利用体内免疫共沉淀实验证明SMC1是乙酰化蛋白,随后利用TSA,NAM处理后,SMC1的乙酰化水平升高,证实SMC1可以被HDAC家族调控。接着利用免疫共沉淀证实SIRT2能够去乙酰化SMC1,且在氧自由基压力下,SIRT2表达升高同时SMC1乙酰化水平下降。而通过外源转染SIRT2催化活性死型质粒,证实SMC1能够被去乙酰化确实依赖于SIRT2的活性区域。为了更直接证明SIRT2对SMC1的去乙酰化机制,我们利用Cell signaling technology（CST）,Phosphosite plus数据库网站提供的SMC1潜在的乙酰化位点为依据,构建了SMC1乙酰化位点死型的质粒转染到细胞中,利用免疫共沉淀找寻SIRT2去乙酰化SMC1的潜在位点。随后我们证实CBP是SMC1的乙酰基转移酶,并且二者有强烈互作。接下来为了证实SIRT2对SMC1去乙酰化修饰促进SMC1磷酸化的发生。首先,在氧自由基压力下,当SIRT2缺失以及催化活性丧失、外源转染CBP以及利用HDAC抑制剂情况下,利用WB技术检测SMC1磷酸化水平。为了探求SIRT2对SMC1的去乙酰化位点在细胞周期G₂/M以及细胞凋亡生物学功能中的作用,利用外源转染人结肠癌细胞系HCT116该位点的活性形式以及磷酸化激活型乙酰化位点激活形式后检测G₂/M周期指标,凋亡指标等生物学功能,最后将上述质粒外源过表达HCT116-shSMC1筛选并鉴定该稳转细胞系,随后皮下注射裸鼠,利用体内成瘤探究该位点对于肿瘤增殖的作用。结果:1、生理条件下,SIRT2与SMC1具有互作,且氧自由基压力下互作增强。2、SMC1是乙酰化蛋白,HDAC抑制剂能够使得SMC1乙酰化水平升高。3、SIRT2是SMC1的去乙酰化酶,且在氧自由基压力下,SIRT2表达升高,SMC1的乙酰化水平降低,并且SIRT2对SMC1的去乙酰化位点是Lys579,随后我们生产了这三个位点的特异性抗体,特异性良好,可以用于后续实验。4、CBP是SMC1的乙酰基转移酶,二者存在互作。5、氧自由基压力下,SIRT2表达量以及活性的缺失使SMC1磷酸化水平降低,两种修饰呈负相关。6、氧自由基压力下,SIRT2通过其去乙酰化酶活性提高SMC1-P水平。7、外源转染人结肠癌细胞系HCT116-shSMC1 K579的活性形式K579Q与WT以及S957DS966DK579Q相比细胞周期G₂/M周期阻滞增加,凋亡升高。8、利用体内裸鼠成瘤证实HCT116-shSMC1-K579Q肿瘤增长速度最慢,K579Q具有抑制肿瘤增殖的作用为肿瘤的靶向治疗提供了新的理论依据。结论:1、SIRT2对粘连蛋白SMC1去乙酰化修饰位点是第579位的赖氨酸。2、氧自由基压力下,SIRT2对SMC1去乙酰化修饰增强了ATM激酶与SMC1结合,最终促进了其磷酸化的发生。3、抑制SIRT2去乙酰化SMC1降低其磷酸化的发生能抑制结肠癌增殖速率。更多还原

【Abstract】 Objective:Colon cancer is a common gastrointestinal malignancy,accounting for the third most common form of gastrointestinal cancer^[1].Similar to other tumors,it is treated with surgery,radiotherapy,chemotherapy,immunotherapy,gene therapy and traditional Chinese medicine.Surgery and radical therapy are not preferred due to their inefficiency and side effect.Gene–targeted therapies emerged as a new option.Recently,studies have found that in the same genetic material,protein modification can affect the stability of the genome,eventually leading to the occurrence of tumors.Among them,acetylation^[2]and phosphorylation^[3-4]plays a crucial role in them,so the study of the interaction between the two modifications has become a hot topic in recent years.aAs a representative of the Sirtuins family,SIRT2 plays an important role in mitosis progression regulation^[5],oxidative stress reaction^[6],metabolism^[7],microtubule stability^[8],cell migration^[9],apoptosis^[10],neurotoxicity^[11]and the inhibition of differentiation^[7,13].SIRT2 is predominantly cytoplasmic.But during the G₂/M transition phase,it can shuttle into the nucleaPrevious studies have reported that when SIRT2 is inhibited,FoxO1 has an increased level of acetylation and FoxO1binds to acetylated ATG7 as an E1-like ubiquitin ligase,leads to cell death by affecting autophagy.This provides a theoretical basis for the develop ment of targeted therapy of colon cancer with SIRT2 inhibitors^[9].Chromatin structural maintenance protein 1（SMC1）is a new SIRT2 substrate we found by mass spectrometry.It plays an important role in the adhesion of sister chromatids,chromatin compress ion,gene metrological compensation,cell cycle regulation and DNA damage repair^[10-11].The phosphorylation of SMC1 Ser 957 and Ser 966 is an important manifestation of the function of SMC1.However,the studies on acetylation/deacetylation of SMC1 have not been reported,and the cross-talk between acetylation and phosphorylation of SMC1 remains largely unexplored.In this study,we confirm the interaction between SIRT2 and SMC1,both in vivo and in vitro.Under oxidative stress,we elucidated how deacetylated SMC1 affect SMC1 phosphorylation in Ser ⁹⁵⁷ and Ser ⁹⁶⁶,and how this pathway plays a role in colon cancer.At the same time,we produced a SIRT2-deacetylated SMC1-specific antibody and further verified the deacetylation regulation of SMC1 by SIRT2Exogenous transfection of human colon cancer cell line HCT116 with the activated acetylation site and activated acetylation&phosphorylation site was used to detect G₂/M cycle maker,apoptosis and other biological functions.Furthermore,nude mice were used to explore the role of the site for tumor proliferation.It can lead to a new avenue for the targeted therapy of cancer.Methods:O ur study is based on the results of mass spectrometry.Subsequently,we conform the interaction between SIRT2 and SMC1 by co-immunoprecipitation and GST-pull down,in which is strengthened under oxidative stress.First of all,it was proved that SMC1 was acetylated protein in vivo by co-immunoprecipitation,and then the acetylation level of SMC1 was increased after TSA and NAM treatment.The results showed that SMC1 could be regulated by the HDAC family.Then,we confirmed that SIRT2 deacetylates SMC1 by co-immunoprecipitation.Under the oxidative stress,the expression of SIRT2 was increased and the level of SMC1acetylation decreased dependent on the catalytic active region of SIRT2.We constructs the acetylation sites mutated plasmid of SMC1 based on the Cell signaling technology（CST）database,exploring the deacetylation of SMC1 potential sites by SIRT2 by co-immunoprecipitation.Then,we verify that the acetylase of SMC1 is CBP,by co-immunoprecipitation.Next,we confirm that SIRT2 deacetylated SMC1promoting the phosphorylation of SMC1.Firstly,underoxidative stress,the phosphorylation of SMC1 was detected through abnormal SIRT2 expression and catalytic activity,respectively.And the phosphorylation of SMC1 was detected by using C BP and HDAC inhibitor.Secondly,in order to confirm the function of deacetylatation site of SMC1 by SIRT2 in the G2/M checkpoint under oxidative stress,we transfected SMC1-WT,K579Q（acetylatation–activated type）,K579Q&S957DS966D（acetylatation–activated type&phosphorylation-activated type）plasmid to SMC1 knockdown cells,then we examine the variation of marked proteins involved in G2/M phase of the cell cycle using western blotting.Thirdly,in order to investigate SIRT2 affect the phosphorylation of SMC1 by deacetylation and participate in apoptosis,we transfected SMC1-WT,K579Q（acetylatation–activated type）,K579Q&S957DS966D（acetylatation–activated type&phosphorylation-activated type）plasmid to SMC1 knockdown cells,and detected the changes of apoptosis markered proteins,then we used flow cytometry to analyze the cells apoptosis.Finally,in order to further explain that the regulatory mechanism plays a role of tumorigenesis,we constructed shSMC1-SMC1-WT,K579Q（acetylatation–activated type）,and K579Q&S957DS966D（acetylatation–activated type&phosphorylation-activated type）HCT116 stable cell line.Subsequently,we use the cell line injected into nude mouse BABL/C,then we measued the tumor growth rate,and detected the expression of PARP1 of tumor.Results:1.Under physiological conditions,SIRT2 interacts with SMC1 and the interaction is enhanced under oxidative stress,2.SIRT2 is a sirtuin of SMC1.Under the oxygen radical pressure,SIRT2 expression is elevated and the acetylation level of SMC1 is decreased,.We found that the deacetylation site of SMC1 by SIRT2is Lys579,and therefore we produced three corresponding site-specific antibodies.The spedificity is good for the subsequent experiments.3.CBP is the acetyltransferase of SMC1.4.Under the oxidative stress,the abnormal expression and enzymic activity of SIRT2 could result in the decrease of the phosphorylation level of SMC1.6.Under the oxidative stress,,SIRT2 enhances the level of SMC1-P through its deacetylase activity.7 G₂/M was arrested with exogenous transfection of human colon cancer cell line HCT116 with the K579Q in contrasted with WT and K579Q&S⁹⁵⁷DS⁹⁶⁶D cell lines.Apoptosis was also higher than other cell lines.The tumor growth rate was lower with the K579Q in nude mouse.It is consistent with the above conclusions and it will provide a new theoretical basis for targeted therapy of colon cancer.Conclusions:1.SIRT2 deacetylates SMC1 through the lysine of K579.2.Under the oxidative stress,the deacetylatation of SMC1 by SIRT2 improves the binding between ATM and SMC1,ultimately promoting the phosphorylation of SMC1.3.Inhibition of SIRT2 deacetylation of SMC1 can reduce the phosphorylation of SMC1 and it also can inhibit the proliferation rate of colon cancer.更多还原