【作者】 张琳；

【导师】 张捷；

【作者基本信息】 吉林大学 ， 内科学， 2018， 博士

【摘要】 背景:近年来肺腺癌已成为世界上发病率与死亡率最高的恶性肿瘤之一,在全球每年新增的160万肺癌患者中,肺腺癌占40%,严重威胁了人类的生命健康。为了解决这一现状,分子靶向治疗药物的研究已逐渐成为热点,靶向治疗药物的应用为患者的治疗带来了福音,肺腺癌患者的生存率有所提高。然而,靶向药物的长期作用会使患者体内产生耐药性抵抗,目前肺癌患者的5年生存率依然低于17%,而关肺腺癌的分子发病机制的研究依旧不透彻,因此,研制一种新的靶向治疗药物,探索一种新的与肺腺癌发生发展相关的基因势在必行,探索其发生发展的分子机制,了解其分子生物学以及免疫学基础,对肺腺癌的治疗以及靶向治疗药物的开发具有重大的意义。MID1(Midline 1)基因最早发现于Opitz BBB/G综合征(OS)中,除OS外,MID1在其他神经系统的疾病例如亨廷顿舞蹈病、阿尔兹海默症等疾病中陆续被发现,因此该基因早期被认为是一种神经系统中线相关疾病的特有基因。随后,MID1在前列腺癌、乳腺癌以及大肠癌中被发现,同时,我们的实验研究也发现MID1在特发性肺纤维化(IPF)以及哮喘中可以通过气道高反应性促进炎症介质的释放,加重疾病的进展。MID1是一种E3泛素连接酶,通常情况下MID1通过其蛋白结构中B-Box1区域与PP2A的催化亚基相结合,降低PP2A全酶的活性。蛋白磷酸酶2A(protein phosphatase 2A,PP2A)是PPP家族成员之一,由结构亚基A、调节亚基B以及催化亚基C组成,三种亚基构成PP2A全酶,发挥着PP2A的结构与功能,PP2A是一类肿瘤生长的抑制因子,抑制肿瘤细胞生长、增殖和DNA复制及翻译,干扰细胞周期,促进细胞凋亡。综上,目前尚未有相关文献报道MID1在肺腺癌的发生发展过程中的作用与功能,而MID1在肿瘤系统中的作用是癌基因或是抑癌基因也未从得知,正是由于MID1在肿瘤、哮喘、IPF等疾病发生中起到了不同的作用,而PP2A与细胞的增殖、周期、凋亡密切相关,我们推测MID1、PP2A或许可以与肺腺癌的发生发展的机制相关。因此,研究MID1-PP2A信号转导通路在肺腺癌发生中的作用机制,明确MID1以及PP2A的表达及功能,深入探究MID1-PP2A信号转导通路对肺腺癌细胞的增殖、周期、凋亡等生物学行为的调控,这对后续临床上抗肿瘤药物的研发以及基因靶向治疗技术的推进提供了重要的细胞学与分子学依据。方法与内容:1、比较三种腺癌细胞系(A549、H1975、H1650)以及支气管上皮细胞(BEAS-2B)中MID1与PP2A的表达情况取稳定培养,处于对数生长期的细胞,提取四种细胞的mRNA及蛋白,利用实时荧光定量PCR(RT-qPCR)、Western Blot以及ELISA法检测MID1和PP2A在肺腺癌细胞以及BEAS-2B细胞中的表达情况。2、比较MID1与PP2A在患者肺腺癌组织以及癌旁组织中的表达情况收集临床手术患者的肺腺癌及对应的癌旁组织样本各30例,利用RT-qPCR以及ELISA检测MID1和PP2A在人体组织中的表达,并且分析MID1以及PP2A的蛋白含量与患者的性别、年龄、吸烟史、TNM分期的相关性。同时,利用免疫组织化学的方法对组织染色处理,明确MID1及PP2A在肺腺癌以及癌旁中的定位与表达。3、比较在A549、H1975、H1650细胞系转染MID1-siRNA介导沉默MID1,以及加入FTY-720(10nM)试剂下调PP2A后MID1与PP2A的表达情况向肺腺癌细胞中转染MID1-siRNA以抑制细胞内MID1的表达水平,利用RT-qPCR、Western Blot以及ELISA法检测MID1 mRNA的转录水平和蛋白的表达情况,计算转染效率。用同样的方法检测PP2A的表达情况,观察MID1是否对其表达水平产生影响。向肺腺癌细胞中加入FTY-720试剂,利用Western Blot以及ELISA法检测PP2A蛋白水平的表达,检测其是否可以被FTY-720激活。4、比较下调MID1,上调PP2A对肺腺癌细胞增殖、周期、凋亡的影响对A549、H1975、H1650分别转染MID1-siRNA,以及加入FTY-720试剂来下调MID1以及上调PP2A后,利用MTS检测细胞增殖及活性,同时使用Western Blot方法检测与细胞周期以及凋亡相关的基因Bax、Bcl-2、p53、p21、Cleave-Caspase9、CDK4、CDK6、CyclinD1、ERK1/2、Phopho-ERK1/2的表达。结果:1、分别在A549、H1975、H1650三组肺腺癌细胞,支气管上皮细胞BEAS-2B中检测MID1基因的表达。结果显示,与BEAS-2B比较,MID1基因的mRNA以及蛋白在肺腺癌细胞系中表达均上调,明显高于BEAS-2B(P<0.05),PP2A在三种肺腺癌细胞系中的表达均低于BEAS-2B(P<0.05),提示二者可能分别发挥着癌基因与抑癌基因的功能并参与肺腺癌的形成2、在人体组织中,MID1基因的mRNA以及蛋白在肺腺癌组织中的表达高于癌旁组织(P<0.05),更重要的是,MID1的表达量与患者的年龄呈正相关,年龄越大,MID1表达越高(P<0.001)。而PP2A蛋白在肺腺癌组织中的表达低于癌旁组织(P<0.05),患者年龄越大,PP2A的表达越低(P<0.001)。进一步说明了MID1在肺腺癌中或许发挥着癌基因的功能,PP2A的作用恰好相反。3、在体外三种肺腺癌细胞系中转染MID1-siRNA后,MID1的表达水平受到明显抑制(P<0.05),同时PP2A蛋白水平表达显著升高(P<0.05)。另外,用PP2A的激活剂FTY-720作用于肺腺癌细胞后,PP2A的表达水平显著升高(P<0.05)。这证明了MID1负调控PP2A的表达。4、在三种肺腺癌细胞系中下调MID1以及上调PP2A后,结果显示,与对照组相比,处理组细胞周期阻滞在G1期,G1期相关蛋白CDK4、CDK6以及CyclinD1表达水平显著下调(P<0.05);促进细胞凋亡,促凋亡基因Cleave-Caspase9以及Bax表达水平显著上调,凋亡抑制基因Bcl-2表达水平下调(P<0.05);细胞的活性被抑制,细胞增殖水平降低(P<0.05);与细胞周期、凋亡、增殖均相关的蛋白p21、p53表达上调,ERK1/2磷酸化水平降低(P<0.05)。进一步证明了下调MID1、上调PP2A可以抑制肺腺癌细胞增殖、促进细胞凋亡,诱导细胞发生周期阻滞。结论:1、本实验首次研究了MID1-PP2A信号转导通路在肺腺癌中的调控机制,提示MID1与PP2A可能分别发挥着癌基因与抑癌基因的功能,并参与肺腺癌的形成。MID1与PP2A与患者年龄具有相关性,这提示二者的表达可能与年龄的累积,肿瘤微环境的长期作用有关。2、本实验在体外实验中进一步探究了MID1对PP2A的调控作用,沉默MID1可以激活PP2A的表达,这证明了MID1可以通过与PP2A的催化亚基结合下调PP2A的活性。3、本实验进一步探究了MID1-PP2A信号通路的作用与功能,揭示了MID1-PP2A信号通路对肺腺癌细胞增殖、周期、凋亡具有重要的的影响。上述研究提高了我们对肿瘤发生的分子机制的认识,同时也为肿瘤治疗以及分子靶向药物的开发提供新的靶点。更多还原

【Abstract】 Background:One of the major diseases endangering human health continues to be lung adenocarcinoma,which has the highest mortality rate of all malignancies worldwide and attracted the attention of many experts and scholars.The 5-year relative survival of which is only 17% despite innovations in techniques and the application of targeted therapy drugs.However,the molecular mechanisms active in NSCLC are still poorly understood,thus investigating new active pathways in the disease to understand its occurrence and development is imperative,and may provide new insights into pharmacological mechanisms for antitumor therapies.The gene Midline 1(MID1)was found in 1997 in a patient with Opitz G/BBB syndrome(OS),which is a congenital disorder that affects the formation of ventral midline structures as a member of tripartite motif(TRIM)family proteins.Besides OS,MID1 was found in other nervous system diseases,such as Alzheimer’s disease and Huntington’s disease(HD).In follow-up,MID1 was found in prostate cancer,breast cancer and colorectal cancer.In the same time,we had demonstrated that MID1 played a key role in respiratory system diseases,such as asthma and idiopathic fibrosis,with airway hyper reactivity(AHR)and the release of inflammation factor,inducing the development of disease.MID1 is an E3 ubiquitin ligase(the RING,B-box1 and B-box2 domains have ubiquitin E3 ligase activity)that associates with microtubules.A known MID1 target is protein phosphatase 2A(PP2A),which is targeted by the B-box1 domain,reduce the activity of protein phosphatase 2A(PP2A)holoenzyme.The PP2 A complex is a member of the protein serine/threonine phosphatase(PP)type 2A family,composed of three types of subunits,scaffo lding subunit A,regulatory subunit B and catalytic subunit C.It has been reported that PP2 A levels are decreased in many cancers,including colorectal and breast cancers,PP2 A plays many roles in carcinogenesis including regulating apoptosis,proliferation,cell migration,cytoskeletal rearrangement and regulating cell cycle.As mentioned above,this is related literature on the role and function of MID1 in the pathogenesis and development of lung adenocarcinoma,and the function of MID1 in tumor system is still unknown as a tumor suppressor or oncogene.Thus,to study on mechanism of MID1-PP2 A signal pathway in lung adenocarcinoma,to clarify the expression and function of MID1 and PP2 A,and to explore the regulation of MID1-PP2 A signal pathway in the proliferation,cycle and apoptosis of lung adenocarcinoma cells could provide an important cytological and molecular basis for the further development of antitumor drugs and the advancement of gene targeting therapy technology.Methods:1.Comparing the expression of MID1 and PP2 A in lung adenocarcinoma cells(A549、H1975、H1650)and bronchial epithelial cell(BEAS-2B)Taking the cells which were stable in vitro,total mRNA and protein were extracted from A549、H1975、H1650 and BEAS-2B.Detecting the expressio n of MID1 and PP2 A using quantitative real-time PCR(RT-qPCR),Western Blot and ELISA methods.2.Comparing the expression of MID1 and PP2 A in lung adenocarcinoma and adjacent tissuesThirty-paired human lung adenocarcinoma and adjacent tissues were ob tained from the Department of Thoracic Surgery of the Second Hospital of Jilin University(Changchun,People’s Republic of China).Detecting the expression of MID1 and PP2 A using quantitative real-time PCR(RT-qPCR),Western Blot and ELISA methods.And we analyzed the correlation between MID1,PP2 A protein and clinical characteristics of lung adenocarcinoma patients,such as age,sex,TNM stage,smoking history and so on.Immunohistochemistry(IHC)was done to analyze the expression and localization of MID1 and PP2 A in human lung adenocarcinoma and adjacent tissues.3.Comparing the expression of MID1 and PP2 A after MID1-siRN A transfection and activation by FTY-720(10nM)in A549、H1975、H1650 cel sAfter transfection in MID1-siRNA and negative control siRNA(NC-siRNA)in A549,H1975 and H1650 cells.RT-qPCR、Western Blot and ELISA methods was done to detect the MID1 expression and calculate transfection efficiency,besides PP2 A expression.After FTY720 was diluted with culture medium to a final concentration of 10 nM for 24 h as a PP2 A activator,and detect PP2 A expression.4.Comparing the regulation of MID1-PP2 A signaling pathway in cell cycle progression,proliferation and apoptosis in vitroAfter the treatment of MID1-siRNA transfection and activation by FTY-720,using MTS methods to detect cell proliferation and activity.And also detecting the expression of cell cycle and apoptosis related gene such as Bax、Bcl-2、p53、p21、Cleave-caspase9、CDK4、CDK6、CyclinD1、ERK1/2、Phopho-ERK1/2 by using Western Blot methods.Result:1.lung adenocarcinoma cells showed significantly higher MID1 expression compared with the nonmalignant BEAS-2B cell line.These results showed that adenocarcinoma cell lines had higher MID1 levels than BEAS-2B cells(P<0.05).And the expression of PP2 A had an opposite result(P<0.05).2.lung adenocarcinoma tissues showed significantly higher MID1 expression compared with the adjcent tissues.We then studied the correlation between MID1 expression and clinical characteristics of lung adenocarcinoma patients.The older patients had more MID1 expression than young patients(P<0.001).However,no significant differences were observed with respect to sex,TNM stage or smoking history in lung adenocarcinoma.These results showed that MID1 was significantly upregulated in lung adenocarcinoma compared with normal tissues and cell lines,and that MID1 expression may have a relationship with patient age.The 30 lung adenocarcinoma tissues had lower PP2 A expression than adjacent normal tissues(P<0.05).Consistent with MID1 expression,we also studied correlations between PP2 A expression and clinical characteristics of lung adenocarcinoma patients.Again,only age had a significant correlation,older patients had lower PP2 A expression than younger ones(P<0.001).No significant differences were observed with respect to sex,TNM stage or smoking history in lung adenocarcinoma.According to these results,we suggest that PP2 A is a tumor suppressor that is downre gulated in lung adenocarcinoma,patient age may influence PP2 A expression.3.After transfection with MID1-siRNA,MID1 expression was significantly reduced at the mRNA and protein levels compared with NC-siRNA(P<0.05),PP2 A protein levels were found to have increased compared with cells transfected with NC-siRNA(P<0.05).The protein expression of PP2 A was higher upregulated by the treatment of FTY-720(P<0.05).4.After the three lung adenocarcinoma cell lines(A549,H1975,H1650)were treated with MID1-siRN A and NC-siRNA,MID1-siRNA significantly increased levels of C leave-Caspase9 and Bax,but decreased levels of Bcl-2,ERK1/2 and phospho-ERK1/2,the phospho-ERK/ERK ratio also decreased.We also analyzed the relative levels of cell cycle regulator expression,which indicated that treatment of MID1-siRNA reduced the relative levels of C DK4,CDK6,Cyclin D1,but increased the relative levels of p53 and p21.After using FTY-720 to active PP2 A,the expression of PP2 A was significantly increased.Furthermore,the expression of cleave-Caspase9,Bax,Bcl-2,ERK1/2,phopho-ERK1/2,p53,p21,C DK4,CDK6,and cyclin D1 were the same as treatment with MID-siRNA in A549,H1975 and H1650 cells.We also did cell proliferation assays,after treated with transfection and activation of FTY-720,the viability of cells was downregulated(P<0.05).Conclusion:1.We demonstrated the function of MID1 in lung adenocarcinoma for the first time.We examined MID1 and PP2 A expression in lung adenocarcinoma tissues and cell lines compared with nonmalignant samples ones,finding that MID1 was highly expressed in lung cancer.This suggested that MID1 could act as an oncogene in lung adenocarcinoma,opposite of PP2 A.Moreover,our studies also showed that both MID1 upregulation and PP2 A downregulation were significantly associated with patients age;the older the patient,the higher their MID1 expression and the lower their PP2 A expression.2.We silenced MID1 in vitro,we found that PP2 A expression was higher than in control cells.Thus,we conclude that increased MID1 e xpression leads to a reduction in PP2 A,making PP2 A a tumor suppressor in this signaling pathway.3.We also demonstrated that the MID1-PP2 A signaling pathway could induce apoptosis,proliferation and cell cycle arrest.O verall,our results revealed that the MID1-PP2 A signaling pathway plays a role in lung adenocarcinoma.O ur findings are novel,improve our understanding of the molecular mechanisms underlying lung tumorigenesis,and may provide new insights into pharmacological mechanisms for antitumor therapies.更多还原