【作者】 李仲杰；

【导师】 余应年；

【作者基本信息】 浙江大学 ， 生理学， 2011， 博士

【摘要】 苯并(a)芘-7,8-二氢二醇-9,10-环氧化物(benzo(a)pyrene-7,8-diol-9,10-epoxide, BPDE)是多环芳烃类(polycyclic aromatic hydrocarbons, PAHs)环境化学污染物苯并(a)芘(BaP)在体内经微粒体酶代谢活化的产物,被认为是BaP的终致癌物。BPDE具有亲电性碳原子活性基团,能与组成核酸的碱基和组成蛋白质的氨基酸亲核基团共价结合形成加合物,损伤生物大分子的结构和功能。BPDE损伤不是一个细胞被动接受的过程,DNA损伤会激活细胞内核苷酸切除修复、细胞周期阻滞、激活低保真度的跨损伤复制等保护性机制,以及p53, PI-3K/Akt/JNKs, MAPKs/AP-1, IKKβ/NF-κB等信号通路被激活。为了全面了解细胞对BPDE的早期应答反应,揭示其致癌机制。本实验室采用基因组学和蛋白质组学的方法,在全基因组和蛋白质水平对FL细胞在BPDE暴露后引起的早期细胞应答反应做了系统的研究。采用AfFymetrix HG-U133 Set(～33000个基因)全基因组芯片来筛选FL人羊膜上皮细胞BPDE处理后4h的应答基因发现,在基因表达水平引起1764个差异表达基因。这些BPDE的应答基因功能涉及广泛,包括细胞周期调节,转录调节,RNA剪接、蛋白质/脂类代谢、细胞骨架、胞内运输、细胞生长、凋亡、DNA修复和DNA损伤反应等；涉及的信号通路包括：p53/ Ras/MAPK/ Akt/PKB/ PKC/ Wnt和TGFbeta通路等。应用蛋白质组学方法,对应答效应蛋白进行的研究中发现量效关系中有65个蛋白斑点在BPDE处理后12小时发生了显著的变化。时效研究中一共有128个蛋白斑点的表达发生了改变。MALDI-TOF鉴定的结果表明,这些得到成功鉴定的蛋白质功能涉及面非常广,包括转录调控,细胞周期,细胞增殖,信号转导,细胞骨架,发育,代谢以及其他功能未明的等。全基因组芯片表达谱和蛋白组学的研究表明：BPDE作用后细胞内发生了广泛的变化,揭示了细胞对BPDE应答机制的复杂性。但是表达谱芯片结果发现有1764个基因表达发生改变,而蛋白质组学仅发现194个蛋白的改变,除去由于技术限制的原因,可能细胞内还存在翻译水平抑制众多基因表达的机制。对此问题的研究,引起我们浓厚的兴趣。近年来的研究表明,MicroRNA (miRNA)是一类广泛存在于真核生物中,由内源基因编码的长度为～21-24核苷酸的非蛋白编码单链小分子RNA,在基因转录后调节中起着的重要的作用。miRNA通过碱基配对介导序列特异性的基因沉默作用,包括降解靶mRNA和抑制靶mRNA的翻译。我们推测miRNA也参与了BPDE的早期细胞应答反应,为了证实这一想法,我们采用microRNA芯片和qRT-PCR研究了miRNA在BPDE早期暴露细胞应答中的作用。microRNA芯片分析采用联川公司miRNA 14.0版μParaflo(?)双色标记微阵列芯片,分析了在0.5μM浓度BPDE处理后2小时组和DMSO对照组中miRNAs表达谱的变化。结果：与对照组相比,在处理组表达改变的miRNA和miRNA*(?)有41个(p<0.1),其中18个表达下调,23个表达上调。经统计学分析筛选出有显著性差异表达的miRNAs有10个(p<0.05),其中上调的有5个,下调的有5个；：miRNAs*有4个,其中上调2个,下调2个qRT-PCR验证芯片结果用Taqman miRNA qRT-PCR试剂盒,在芯片样品和新处理样品,验证了显著性表达差异的miRNAs中选出的7个miRNA。结果：发现has-miR-509-5p是主要发生改变的miRNA基因(p<0.01),比对照表达上调1.8倍,与芯片结果相一致。Inhibtor抑制miR-509-5p基因在BPDE诱导的FL细胞中表达将合成的miR-509-5p inhibitor寡核苷酸片段和对照寡核苷酸片段,转染细胞培养72h后,分别用含BPDE或DMSO的无血清培养基处理2h,换新鲜全培养基培养液培养2h。收集细胞,TRIzol试剂提取总RNA, qRT-PCR检测miR-509-5p在各组中的表达。结果：转染对照序列组不影响BPDE诱发的miR-509-5p基因表达上调,而转染inhibtor组,DMSO处理细胞中miR-509-5p基因的表达被下调,与Control组DMSO处理细胞相比,抑制效率达60%(p<0.01); BPDE处理不再诱导其表达的上调。miR-509-5p靶基因预测用靶基因预测软件Target scan 5.1预测miR-509-5p靶基因。结果：得到受miR-509-5p调控的靶基因265个。将预测结果与本实验室全基因组筛出的有显著性差异表达的基因进行配对分析,结果发现有65个共同的基因。从中挑选出CDC14、DOCK4、EIF5B、IGF1R、MEIS1. NF1、PRKCA、RREB1、RAD23B、TDG、TET1等11个重要的靶基因,进行后续靶基因分析。qRT-PCR验证筛出的miR-509-5p靶基因用Takara SYBR Green qRT-PCR试剂盒,在芯片样品、新处理样品、转染miR-509-5p基因inhibitor样品,检测分析挑选的11个靶基因的表达变化,找出受miR-509-5p调控的靶基因。结果：在芯片样品和新处理样品中CDC14、DOCK4、IGF1R、MEIS1、NF1、PRKCA、RREB1、RAD23B等8个基因在BPDE暴露后2h表达均显著性下调(p<0.01)。在miR-509-5p基因表达抑制样品中,CDC14B, DOCK4, IGF1R, MEIS1, NF1, PRKCA, RREB1等7个基因表达显著上调(p<0.01),而RAD23B改变不明显。结论：a.首次证实miRNAs参与了BPDE引起的FL细胞早期应答反应,确认miR-509-5p是FL细胞早期应答反应的miRNA。b.首次证实了mir-509-5p通过转录后调解节抑制靶基因CDC14B, DOCK4, IGF1R, MEIS1 NFl, PRKCA, RREB1等的表达。这些基因功能涵盖细胞骨架稳定、信号转导、细胞周期调控、DNA损伤修复、转录调控等。说明mir-509-5p在BPDE引起的FL细胞早期应答反应中起着重要作用,是细胞对BPDE早期应答的分子标志物。c.这些新的发现对了解BPDE致癌效应早期的分子机制提供了新的理论基础。基因组持续受到各种内源性和外源性因子的损伤,为了保证正常的生长控制与遗传信息DNA复制的准确性,修复这些损伤是保持基因组稳定性的关键。修复失败则导致基因突变,进而增加细胞癌变的风险。其中DNA碱基修改,如脱氨,烷基化和氧化,或由于自发脱嘌呤形成的无嘌呤/嘧啶(AP)位点等损伤出现最频繁。这些小的DNA损伤主要通过碱基切除修复(base excision repair, BER)途径来完成。DNA聚合酶β(DNA polymeraseβ, po1β)是BER修复途径中不可替代的酶,因此,po1β被认为在碱基损伤修复和基因组完整性的维持中其关键作用,实验证明po1β下调或po1β基因突变都会导致由于DNA修复缺陷的基因突变。然而,近年来的研究表明,po1β可能参与了广泛的DNA代谢反应,包括DNA复制,重组,减数分裂和DNA跨损伤合成等,但在合成的DNA链上表现为高碱基错配率。这种错误配对可能是由于po1β干扰了其他精确DNA聚合酶的正常功能,而其本身又缺乏3′→5′缺乏校对活性,使得其复制保真度下降,导致基因组不稳定。因此,建立诱导型po1β稳定高表达表达和表达下调细胞系,并利用建立的细胞系,研究po1β生物学功能的变化,对了解DNA po1β表达改变引起的基因突变和基因组不稳定机制具有重要的理论意义。在本研究中,我们构建了诱导型反义阻断和表达DNAPo1β的真核表达载体,然后将构建的地塞米松(dexamethasone, DEX)诱导的真核表达载体pMAMneo-amp-pol-β-, pMAMneo-amp-pol-β+,和对照pMAMneo-amp-,分别转染FL细胞。转染细胞用含400μg/LGeneticin (G418)的MEM培养基筛选,直至细胞集落生成,挑取单集落扩大培养,建立了FL-pMAMneo-amp-, FL-pol-β和FL-po1-β+细胞系。建立的细胞系用DEX诱导72小时后的qRT-PCR和免疫印迹分析表明：FL-po1-β细胞中Po1β的表达比对照显著下调,FL-po1-β+细胞中Po1β的表达比对照显著上调。此外,MNNG的敏感性试验表明：与对照相比,Po1β表达下调的FL-po1-β细胞对烷化剂MNNG的敏感性增加,相反,Po1β过表达的FL-po1-p+细胞对烷化剂MNNG的敏感性下降。细胞周期分析表明,聚合酶p表达的变化并不显著影响细胞周期分布。结论：成功建立了诱导Po1β表达和下调的人FL细胞系,为后续深入研究Po1β在环境致癌物诱导表达升高后的致突变,致癌机制提供了良好的细胞模型。更多还原

【Abstract】 (±)-anti-benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) is one of the metabolic products of benzo(a)pyrene (BaP), which belongs to the polycyclic aromatic hydrocarbons (PAHs) environmental chemical pollutants. BPDE is considered as the ultimate carcinogen of BaP. BPDE is the most carcinogenetic form and the electrophilic species of it is able to interact with nucleophilic sites on cellular DNA, RNA and protein resulting in bulky-adduct damage. BPDE causing cellular reponse is not a passively accepted process by the cell. BPDE induces bulky-adduct DNA damage, which would activate intracellular nucleotide excision repair (NER) and cell cycle arrest, etc. protective mechanisms, also trigger the low-fidelity translesion replication mechanism that leads to DNA mutations, and stimulate some tumor-promotive stress signaling pathways such as p53, PI-3K/Akt/JNKs, MAPKs/ AP-1, IKKbeta/NF-kappaB was activated after BPDE exposure.In order to a comprehensive understand the early cellular responses of BPDE, our laboratory used genomics and proteomics methods, whole genome and protein levels of FL cells after exposure to BPDE of early cellular responses induced to do a systematic study. Using Affymetrix HG-U133 Set (～33000 genes) whole-genome microarrays, we found 1764 genes expression were significantly altered at 4 h after exposure to BPDE compared with the vehicle exposure. These responsive genes are involved in multiple functions including cell cycle control, signaling molecules, transcription factors, and metabolic enzymes, DNA repair and DNA damage response etc.; and extensive signaling pathways including MAPK, cell cycle, Wnt signaling pathway and TGFbeta pathway. Proteomics results indicated, there were 194 protein spots were significantly altered at 12 h after exposure to BPDE compared with the vehicle exposure. These identified proteins are involved in a variety of cellular process including transcription regulation, cell-cycle control, cell proliferation, signal transduction, cell skeleton, development, metabolism and some proteins with unknown functions.These results reveal the complexity mechanism of cellular response to BPDE. However, the results of cDNA microarray gene expression found in 1764 changed, and only found 194 proteins proteomics change, except for reasons due to technical limitations, there may be exites the mechanism of inhibition many gene expression in the translation levels in cells. Research on this issue, cause us interest.microRNAs are a class of non-coding RNA gene whose products ar～22 nt sequences that play important roles in the regulation of translation and degradation of mRNAs through base pairing to partially complementary sites in the untranslated regions (UTRs) of the message. The relationship between miRNAs and carcinogen exposure were reported. Therefore, we hypothesized that miRNAs also involved in early cell responses of BPDE. In order to confirm this idea, we have examined the BPDE-induced miRNA expression profile by miRNA microarray and quantitative real-time PCR (qRT-PCR) in FL cells. In addition, we have predicted identificated miRNAs target gene and verified the regulation of these specific genes upon BPDE exposure.Analysis of miRNA microarrayWe used miRNA 14.0 version of the LC Sciences company associatedμParaflo (?) microarray to study expression profiles of miRNA of FL cells treated by BPDE for 2h. The results showed that 41 miRNAs and miRNAs* expression changed in BPDE treated compared to control (DMSO treatment) (p<0.1).23 of them were up-regulated and 18 were down-regulated. Moreover, we have focused on 7 miRNAs for further study, includingmiR-509-5p, miR-628-3p, miR-183, miR-100, miR-602, miR-484, miR-99a.Validation of changed miRNAs by qRT-PCRTo verify the 7 miRNAs expression utilized the microarray sample and prepared the fresh sample.The results showed that hsa-miR-509-5p expression significantly up-regulated compared to control (p<0.01), while, other miRNAs expression alteration were not obsevered.Inhibition of miR-509-5p gene up-regulation induced by BPDE in FL cellsTransfected miR-509-5p-specific miRNA inhibitor (100 nM) and its negative control (100 nM) in FL cells, respectively. After 72 h, we treated transfected cells with 0.5μM BPDE, DMSO as control. The results showed miR-509-5p-specific miRNA inhibitor abrogated the BPDE-induced up-regulation of miR-509-5p, suggesting the miR-509-5p is specific regulated by BPDE exposure.miR-509-5p target gene predictionWe predicted the miR-509-5p target gene by Target scan 5.1. The obtained result compared with the previous date of our lab, we found 65 intrested genes. And then, we have selected 11 target genes that are involed in cell basic activity control of miR-509-5p, such as CDC14、DOCK4、EIF5B、IGF1R、MEIS1、NF1、PRKCA、RREB1、RAD23B、TDG、TET1.Validation of miR-509-5p target gene by qRT-PCRTo verify the 11 target gene expression wtih microarray samples, prepared fresh samples, or miR-509-5p know-down samples. The results showed that CDC14B, DOCK4, IGF1R, MEIS1, NF1, PRKCA, RREB1 genes expression were significan down-regulated by BPDE (p<0.01). On the contrary, CDC14B, DOCK4, IGF1R, MEIS1, NF1, PRKCA, RREB1 gene expression were significanly up-regulated in the presence of miR-509-5p-specific miRNA inhibitor (p<0.01).Conclusion:a. miRNAs are involved in the BPDE-induced FL cell early responses.b. CDC14B, DOCK4, IGF1R, MEIS1, NF1, PRKCA and RREB1 gene were regulted by the mir-509-5p in FL cell. These identified target genes are involved in a variety of cellular process including cytoskeletal stability, signal transduction, cell cycle regulation, DNA damage repair, transcriptional regulation and so on. Our results indicated the mir-509-5p plays an important role in the BPDE induced FL cell early responses.This study provides important findings to understand the miRNA-mediated cellular responses induced by BPDE, indicating a new molecular mechanism of BPDE-induced mutagenesis and carcinogenesis. The genome is continuously damaged by a variety of endogenous and exogenous agents. Repair of such damage is a crucial mechanism for maintaining genomic integrity, which is essential for normal developmental and physiological consequences. Among the damage, DNA base modifications, such as deamination, alkylation and oxidation, or apurinic/apyrimidinic (AP) sites due to spontaneous depurination arise most frequently. These small lesions are mainly repaired through base excision repair (BER). Pol P plays a crucial role specifically in in repair of DNA damage and maintaining stability and integrality of genome. However, the recent researches indicate that polymerase P might took part in a wide spectrum of DNA metabolism reactions, including DNA replication, recombination, meiosis and transleisional DNA synthesis and behaves high error rate due to its lack of 3’→5’proofreading activity and low replication fidelity. Down-regulation or mutation of Polβis mutagenic due to deficient in DNA repair, while overexpression of this error-prone Po1βmight perturb the normal function of other accurate polymerases and cause genomic instability as well. Hence, we established the Po1βover-expressed and down-regulated stable cell line with dexamethasone. And we used these constructed cell lines to study the biological function of Pol P and the involvement of Polβin genes mutation and genomic instability.In our present study, we constructed the recombinant dexamethasone (DEX) inducible eukaryotic expression vector, pMAMneo-amp- po1-β-and pMAMneo-amp-pol-(3+. Together with the empty control pMAMneo-amp- we transfected the inducible eukaryotic expression vectors in FL cells. And then we selected cell cluster with MEM medium containing 400μg/mL Geneticin (G418). The expression of Po1βin transgenic cell lines was determined after induced by DEX for 72 hours by qRT-PCR and Western blot. The results showed Po1βexpression was down-regulated in FL-Po1β- cells, and was overecpressed in FL-Po1β+cells. Furthermore, MTT assay demonstrated that the FL-Polβ- cell was sensitive to alkylaing agent MNNG exposure, and FL-Po1β+cells was tolorent to MNNG treatment. Cell cycle analysis showed that pol P expression change does not significantly affect cell cycle distribution.更多还原