【作者】 杨波；

【导师】 邵国光；

【作者基本信息】 吉林大学 ， 外科学， 2018， 博士

【摘要】 研究背景NSCLC是一种恶性程度较高的肺部肿瘤,它的侵袭性较强,预后较差,而在所有的肺癌中,NSCLC的比例约为80%。对于NSCLC,最主要的治疗为根治性的手术切除,然而由于缺乏早期特异性的诊断方法,在确诊之后有很多的患者已经失去了手术时机。化疗在NSCLC的治疗策略中仍占据着不可撼动的地位,约60%的患者化疗是有效的[2,3],然而化疗的疗效不是特别显著[4,5]。因此,找到方法从根本上提高化疗的疗效从而提升NSCLC患者的无瘤生存期有着重要的意义。以CDDP为基础的系统性及辅助性化疗是NSCLC药物治疗的主要手段[6],甚至有时可以达到根治程度。但是,在使用CDDP化疗时,肿瘤细胞本身固有的和/或化疗后期出现的对CDDP药物的抵抗极大程度的降低了治疗效果,而CDDP耐药的分子机制引发了我们的思考,因此化疗增敏让我们产生了极大的兴趣。CK2是一种在哺乳动物细胞内大量存在的第二信使非依赖性丝氨酸/苏氨酸蛋白激酶[7],为一四聚体异构而成的全酶,它包含两个催化亚基:α和/或α,;两个调节亚基:β。其中CK2α在其中起到主要作用,特别在NSCLC中CK2α较其他亚基有着明显的过表达,且CK2α具有癌基因的致癌效应,并与细胞的存活、凋亡、侵袭、迁移能力存在密切关系,这强调了CK2α与NSCLC肿瘤发生、发展及预后的相关性。同时还有报道表明CK2α在肺癌组织、细胞中扮演着重要的角色,甚至可能成为NSCLC化学药物治疗的潜在靶点,但是其具体机制尚未明确[12]。那么对于CK2α在NSCLC的化疗中所起到的作用及其机制的研究就变得尤为重要了。目的本研究旨在探讨CK2α的表达与NSCLC病人的临床指标及预后生存关系,分析CK2α在CDDP介导的NSCLC细胞增殖、侵袭、迁移和凋亡中的作用及其可能的分子机制,由此来探索对NSCLC的联合用药治疗,提高CDDP的化疗疗效,从而达到化疗增敏。方法:1.在国际生物信息中心(www.ncbi.nlm.nih.gov)搜索确认CK2α基因名称:CSNK2A1,从癌症基因组图谱(The Cancer Genome Atlas,TCGA,http://ualcan.path.uab.edu/index.html)数据库中搜索CSNK2A1所对应的数据,从中查找CSNK2A1在NSCLC与正常组织中的表达差异情况。2.本研究选取了经术后病理证实为NSCLC的病人60例,非恶性肺部肿瘤病人40例,应用免疫组织化学检测60例NSCLC癌组织、癌旁组织和40例非恶性肺部肿瘤组织中CK2α的表达,分析CK2α的表达情况与病人临床一般资料的关系。3.从基因表达综合(Gene Expression Omnibus,GEO,http://www.ncbi.nlm.nih.gov/geo/)数据库中下载GSE42127芯片数据,样本总共176例原发性NSCLC肿瘤组织样本,从中找到CSNK2A1对应探针的表达数据,根据CK2α表达水平,结合病人生存时间数据,绘制生存曲线。4.通过MTT法检测CDDP时间和浓度梯度对NSCLC细胞增殖率的影响;流式细胞术(Annexin V-FITC和PI双染)检测CDDP浓度梯度对NSCLC细胞凋亡的影响;通过Western blot法检测CDDP对NSCLC细胞内Cleaved caspase-3、Cytochrome C凋亡相关蛋白表达水平的影响及CDDP浓度梯度对NSCLC细胞内CK2α蛋白表达水平的影响。5.通过MTT法、平板克隆实验、Transwell侵袭实验、流式细胞术Rhodamine123染色法、Western blot法检测通过抑制CK2α对CDDP作用下的NSCLC细胞的增殖、侵袭、迁移能力、细胞线粒体膜电位及细胞内凋亡相关蛋白的影响。6.通过Western blot法检测p38 MAPK特异性抑制剂对CDDP诱导NSCLC细胞凋亡的p38 MAPK信号通路关键串联蛋白表达水平的影响,通过Western blot法检测CK2α抑制剂和si RNA基因沉默CK2α基因对CDDP诱导NSCLC细胞中PML蛋白表达水平的影响,通过Hochest染色法及免疫荧光观察CK2α抑制剂对CDDP诱导NSCLC细胞内Honest和早幼粒细胞白血病基因(promyelocytic leukemia gene,PML)蛋白表达水平的影响。以此探讨抑制CK2α增强CDDP诱导NSCLC细胞凋亡的作用机制。7.通过裸鼠皮下移植NSCLC瘤模型检测CK2α抑制剂对CDDP诱导体内NSCLC肿瘤生长抑制作用的影响。8.实验所得数据均经过三次以上实验重复验证,数据以均数±标准差((?)±SD)形式表示,所得相关数据使用SPASS Statistics 19.0统计软件进行数据录入及分析,相关影像学统计使用仪器配套软件进行分析,计数数据采用X2检验,采用Kaplan-Meier法绘制生存曲线,并对曲线进行logrank检验,不同实验组之间的数据对比,使用ANOVA进行分析,p<0.05表示存在统计学差异,P<0.01表示存在显著的统计学差异。结果:1.TCGA数据库中,CSNK2A1所对应的数据显示CK2α在肺腺癌(P<0.05)、鳞癌(P<0.05)组织中的阳性表达率显著高于正常组织。2.免疫组化结果显示CK2α在NSCLC组织、癌旁组织和非恶性肺部肿瘤组织中的阳性率分别为61.7%、11.7%和22.5%,在NSCLC组织中的表达率显著高于癌旁组织(61.7%vs11.7%;P<0.05),也高于非恶性肺部肿瘤组织(61.7%vs22.5%;P<0.05)。3.统计分析结果显示CK2α的阳性表达率及表达强度与患者的年龄、性别、病理类型、淋巴结转移与否及肿瘤的TNM分期无关(P>0.05),但与NSCLC组织的病理分化程度具有显著相关性(P<0.05)。4.Kaplan-Meier曲线生存分析结果显示,CK2α低表达者生存期较长,高表达者生存期较短(P<0.05)。5.MTT法检测NSCLC细胞增殖结果显示:CDDP能够抑制H157、A549、H226细胞的增殖活性,并且在一定的范围内,CDDP对NSCLC细胞增殖的抑制呈现出浓度和时间的依赖性。6.流式细胞术结果显示:随着CDDP浓度的增加,H157、A549、H226细胞的早期凋亡率随之增加。Western blot法检测结果显示:随着CDDP作用浓度和时间的增加,NSCLC细胞中Cleaved caspase-3、Cytochrome C蛋白表达水平逐渐增高。7.Western blot法检测结果显示:随着CDDP浓度的增加,NSCLC细胞中CK2α蛋白表达水平逐渐增高。8.MTT法检测结果显示:在CK2α抑制剂作用下,CDDP对NSCLC细胞的增殖抑制作用增强;平板克隆实验结果显示:CK2α抑制剂可进一步显著增强CDDP对NSCLC细胞集落形成的抑制。9.Transwell侵袭实验结果显示:CK2α抑制剂可增强CDDP对NSCLC细胞侵袭、迁移能力的抑制。10.流式细胞术Rhodamine 123染色法检测结果显示:CK2α抑制剂可进一步增强CDDP对NSCLC细胞线粒体膜电位水平的下调作用。CK2α抑制剂和si RNA基因沉默CK2α基因增强了CDDP诱导的NSCLC细胞中凋亡相关蛋白的表达水平。11.抑制CK2α增强CDDP诱导NSCLC细胞凋亡的作用机制:CDDP激活了p38 MAPK信号通路,使得p38 MAPK磷酸化,p38 MAPK磷酸化后进一步激活CK2α,CK2α使得PML磷酸化增多,这种磷酸化的发生导致了蛋白酶介导的多聚泛素化的PML降解,从而导致了PML下调。而抑制CK2α,PML的磷酸化减少,蓄积增多,从而增强了CDDP对NSCLC细胞的凋亡效应。12.裸鼠皮下移植瘤实验结果显示:与生理盐水对照组相比,TBB给药组的生长抑制不明显,CDDP给药组和CDDP联合TBB给药组的肿瘤体积和重量抑制率增高,而且CDDP联合TBB给药较CDDP单独给药,对肿瘤的生长抑制更加明显。Western blot法检测肿瘤组织胞浆内相关蛋白结果显示:CDDP联合TBB组的Cleaved caspase-3和Cytochrome C的表达水平较TBB组、CDDP组升高,CDDP联合TBB组的CK2α的表达水平较CDDP组降低,而PML的表达水平较CDDP组升高,结果与体外实验相一致。结论:1.在临床相关研究中证实了CK2α在NSCLC组织中过表达,并在研究过程中发现了CK2α表达水平与NSCLC组织病理分化程度相关,推测CK2α可能与NSCLC的发生、发展过程相关,CK2α的表达强度检测可作为判定NSCLC恶性程度的潜在指标之一。2.蛋白激酶CK2α的过表达可能与NSCLC患者的预后相关,可作为NSCLC患者的一项不良愈后指标。3.联合使用CDDP和CK2αsi RNA或者CDDP和CK2α抑制剂在抑制NSCLC细胞增殖、侵袭、迁移和促进NSCLC细胞凋亡方面比单独使用CDDP更加有效。4.抑制蛋白激酶CK2α使CDDP在NSCLC化疗中增敏的作用机制:CDDP激活了p38MAPK信号通路,使得p38MAPK磷酸化,p38MAPK磷酸化后进一步激活CK2α,CK2α使得PML磷酸化增多,这种磷酸化的发生导致了蛋白酶介导的多聚泛素化的PML降解,从而导致了PML下调。而抑制CK2α,PML的磷酸化减少,蓄积增多,从而增强了CDDP对NSCLC细胞的凋亡效应,达到化疗增敏。5.CK2α可能成为NSCLC分子治疗的潜在靶点,对CK2α抑制剂的研究和应用对NSCLC的治疗具有深远的临床意义。更多还原

【Abstract】 Background Approximately 80% of lung cancer is NSCLC.The main treatment among choices for NSCLC is radical resection.Unfortunately,due to lack of specific early diagnosis,many lung cancer patients miss the opportunity for surgery at the initial.Approximately 60% of NSCLC patients are suitable for chemotherapy,whereas chemotherapy usually gives rise to poor response rates.Therefore,it is imperative to find new therapeutics to improve the holistic disease-free survival rates.CDDP-based systemic chemotherapy is the main pharmaceutical approach for treating NSCLC.Indeed,it often leads to an initial therapeutic success.However,intrinsic and/or acquired resistance against CDDP mainly reduces the curative effect of chemotherapy.Thus,it arouses tremendous clinical interest for us to explore strategies for chemosensitization.Casein kinase 2(CK2)is a serine-threonine kinase that is ubiquitously expressed in mammalian cells.It consists of two catalytic subnits : α and/or α,,and two regulatory subunits : β.Especially in NSCLC,the overexpression of CK2α is more obvious than other subunits and it has the carcinogenic effect of oncogenes.The close relationship between CK2α and survival,apoptosis,invasion and migration ability of cells emphasizes that CK2α is correlated with the occurrence,development and prognosis of NSCLC.Besides,it was reported that CK2α played an important role in lung cancer,which may even become a potential target for NSCLC chemotherapy,but the exact mechanism was unknown.Study of the role and exact mechanism of CK2αin the chemotherapy of NSCLC becomes more and more important.Objectives In this study,the expression level of CK2α in NSCLC were detected so as to explore the relation between CK2α and clinical index,prognosis of NSCLC patients.The role and mechanism of CK2α in viability,invasion,and apoptosis of NSCLC cells caused by CDDP were explored.The goal was to achieve chemotherapeutic sensitization.Methods1.Firstly,we made sure that the gene name of CK2α is CSNK2A1 via searching from National Center of Biotechnology Information(www.ncbi.nlm.nih.gov).Then we search the gene data which was encoded by CSNK2A1 from The Cancer Genome Atlas(TCGA,http://ualcan.path.uab.edu/index.html)database to research the expression level of CSNK2A1 in NSCLC.2.In this study,we collected tissue surgically from 60 cases of NSCLC and 40 cases of non-malignant pulmonary tumor.The expression of CK2α in 60 NSCLC tissues and adjacent tissues and in 40 non-malignant lung tumor tissues was analyzed by IHC.The relationship between the expression of CK2α and clinical index of patients was analyzed.3.We downloaded GSE42127 microarray data gene from Gene Expression Omnibus(GEO,http://www.ncbi.nlm.nih.gov/geo/)database,including 176 primary NSCLC tumor samples.Then we researched the expression data of the corresponding probe of CSNK2A1.The survival curve was generated through the combination of expression level of CK2α and survival of NSCLC patients.4.MTT assay was used to examine the toxic effect of CDDP on NSCLC cells.Flow cytometry with Annexin V-FITC was used to examine the effect of CDDP on apoptosis of NSCLC cells.Western blot analysis was used to document the protein levels of Cleaved caspase-3 、 Cytochrome C of CDDP-treated NSCLC cells and effects of CDDP on CK2α expression in NSCLC cells.5.We assessed the viability of NSCLC cells treated with CDDP under the effect of CK2α inhibitor by MTT and colony formation assay.Transwell assay was used to detect the effect of CK2α inhibitor on the migration and invasion ability of CDDP-treated NSCLC cells.In order to clarify the role of CK2α inhibitor in the progress of CDDP inducing NSCLC cells apoptosis,the change of mitochondrial membrane potential was determined by the retention of the dye Rhodamine 123.After knocking down CK2α gene through small interfering RNA,the protein levels of Cleaved caspase-3 and Cytochrome C of CDDP-treated NSCLC cells were detected by Western blot.6.Western blot was used to detect the effect of specific inhibitor of p38 MAPK on the expression of critical protein in the p38 MAPK signaling pathway induced by CDDP.The effect of CK2α inhibitor and CK2α si RNA on PML protein expression in NSCLC cells induced by CDDP was examined by Western blot and representative images via fluorescence microscopy to explore the mechanism of inhibition of protein kinase CK2α sensitizing NSCLC to CDDP.7.The effect of CK2α inhibitors on CDDP-inducing tumor growth inhibition of NSCLC in vivo was examined by using nude mice with xenografts.8.Experimental procedures were repeated more than three times.Data were presented as mean ± SD((?)± SD),and SPASS 19.0 was used for data input and statistics.The relevant imaging statistics then were analyzed with related software.Proper test methods were used according to different statistical data.The relationship between the expression of CK2α and the patients’ clinical index and parameters was analyzed through X2 test.The Kaplan Meier method and logrank test were used for survival analysis of the relationship between expression of CK2α and prognosis of NSCLC patients.ANOVA was used for data analysis of different groups.And p<0.05 was considered to represent a statistical difference and p<0.01 was considered to represent a remarkable statistical difference.Results1.In the TCGA database,the corresponding data of CSNK2A1 showed that the positive rate of CK2α in AD(p<0.05)and SCC(p<0.05)were significantly higher than that in normal tissue.2.IHC revealed that the positive rate of CK2α in NSCLC tissues,adjacent tissues and non-malignant lung tumor tissues were respectively 61.7%,11.7% and22.5%.The positive expression rate in NSCLC tissues was significantly higher than that of adjacent tissues(61.7%vs11.7%;p<0.05),also higher than non-malignant lung tumor tissues(61.7% vs 22.5%;p < 0.05).3.Statistical analysis showed that neither the positive expression rate nor intensity of CK2α had any correlation to NSCLC patients’ age,gender,pathological type,lymph node metastasis,or TNM staging of the tumor(p>0.05),but was correlated significantly to the degree of differentiation of carcinoma tissue(p< 0.05).4.The results of survival analysis of Kaplan-Meier curve showed that the survival time of the low expression of CK2α was longer than those of high expression(p<0.05).5.The viability of H157、A549、H226 cells decreased significantly with the increased concentration and extended incubation time of CDDP.6.The early apoptosis rate of NSCLC cells treated with CDDP increased markedly with the increased concentration of CDDP in H157、A549、H226 cells.Western blot analysis showed that the levels of Cleaved caspase-3、Cytochrome C of NSCLC cells increased with the extended concentration and incubation time of CDDP.7.Western blot analysis showed that the expression levels of CK2α in NSCLC cells increased gradually with the extended concentration of CDDP.8.MTT assay showed that the process that CDDP inhibited the proliferation of NSCLC cells was enhanced by inhibitor of CK2α.The colony formation assay showed that inhibitor of CK2α can significantly enhance the inhibitory effects of CDDP.9.The results of transwell : CK2α inhibitor enhanced the inhibition of migration and invasion ability of CDDP-treated NSCLC cells.10.The results of the retention of the dye Rhodamine 123: CK2α inhibitor can enhance down-regulation of mitochondrial membrane potential in CDDP-treated NSCLC cells.Western blot showed that CK2α inhibitor and CK2α si RNA enhanced the expression of apoptotic protein in NSCLC cells treated with CDDP.11.The mechanism of inhibition of CK2α sensitizing NSCLC cells to CDDP:CDDP activated the p38 MAPK signaling pathway,then p38 MAPK was phosphorylated,which enhanced the expression of CK2α.CK2α promoted the phosphorylation,ubiquitination and degradation of PML.Inhibition of CK2α can up-regulate the activity of PML and enhance the apoptotic effect of CDDP on NSCLC cells.12.NSCLC xenografts studies in nude mice indicated that the growth inhibition was not significant in TBB group compared with the control group.The inhibition rate of tumor volume and weight increased in CDDP group and CDDP+TBB group.CDDP+TBB was more effective in suppressing tumor growth than CDDP group.Western blot analysis showed that the expression levels of Cleaved caspase-3 、Cytochrome C in CDDP+TBB group is higher than TBB and CDDP groups.The expression levels of CK2α of CDDP+TBB group is lower than CDDP group,and the PML in CDDP+TBB group is higher than CDDP group.The results were consistent with in vitro experimentsConclusion1.Clinical specimens of NSCLC tissues revealed that overexpression of CK2αwas related to pathological differentiation.Therefore,the overexpression of CK2αmay be associated with the occurrence and development of NSCLC.Testing the expression and expression intensity of CK2α may be used as a potential index to determine the malignant degree of NSCLC.2.The overexpression of CK2α may be associated with prognosis of NSCLC.Therefore,CK2α may be a poor prognosis indicator.3.The combination of CDDP with CK2α si RNA or CK2α inhibitor is more efficient in suppressing the viability,proliferation,invasion of NSCLC cells,and promoting apoptosis than CDDP alone.4.The mechanism of inhibition of protein kinase CK2α sensitizing NSCLC cells to CDDP: CDDP activated the p38 MAPK signaling pathway,then p38 MAPK was phosphorylated,which enhanced the expression of CK2α.CK2α promoted the phosphorylation,ubiquitination and degradation of PML.Inhibition of CK2α can up-regulate the activity of PML and enhance the apoptotic effect of CDDP on NSCLC cells.5.CK2α may become a potential target for molecular therapy of NSCLC.The research and application of CK2α inhibitor have far-reaching clinical significance for the treatment of NSCLC.更多还原