【作者】 晋亮；

【导师】 卢兹凡；

【作者基本信息】 第四军医大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 细胞凋亡是机体细胞在正常生理或病理状态下发生的一种自发的,程序性死亡过程,作为一种机体广泛存在的生物学行为,参与了发育、内环境稳态、应激和肿瘤等众多生理和病理学过程。凋亡信号通路通常分为胞外配体途径和胞内应激信号途径。胞外配体如Fas、TNFα和TRAIL等,结合胞膜上的相应受体,使得受体胞内段募集接头蛋白(FADD和TRADD等)和凋亡起始者的Caspase-8,-10,这些分子组成了死亡诱导信号复合体(DISC),进一步活化下游的凋亡效应者Caspase-3, -6, -7,继而引发细胞凋亡。胞内应激信号,如生长因子的撤除、DNA损伤、氧化应激和癌基因的激活等,可导致线粒体外膜的泄露,细胞色素C的释放并引发凋亡小体的产生,其组成包括细胞色素C、Apaf-1和Caspase-9。作为凋亡起始者Caspase-9的激活,可进一步活化下游的凋亡效应者Caspase-3, -6, -7,继而引发细胞凋亡。因其信号通路众多,且相互影响,细胞内对凋亡的调控就更加复杂。近年来,由RNA结合蛋白和miRNA介导的转录后调控越来越受到人们的重视,已有报道称细胞凋亡过程中该调节方式扮演重要作用。我们课题组一直关注的QKI就是一类RNA结合蛋白,属于STAR(signal transduction and activation of RNA)家族,在进化过程中高度保守,在神经系统发育过程中参与髓鞘发育。除了在神经系统中对于髓鞘的形成发挥重要功能以外,QKI蛋白在血管发生、细胞凋亡、细胞黏附、细胞生长及形态形成和器官发生等方面具有举足轻重的功能。由于转录产物的不同剪接,QKI有3种亚型,分别为QKI-5/6/7,差别主要集中在它们C末端和3’非翻译区的不同。其中QKI-5包含了一段核定位信号肽,因此主要定位于胞核中,但也可在胞核与胞浆中穿梭;而QKI-6和QKI-7则定位于胞浆中。这一差别决定了各自的亚细胞定位和功能有所不同。根据QKI与RNA结合的序列特异性,对整个mRNA进行筛选发现:QKI可能调控的下游靶基因中有众多如Bid、FOXO1、Sirt1等参与凋亡调节过程的重要基因,高度提示QKI可能在细胞凋亡中发挥作用。本研究通过Real time PCR、Western Blotting和启动子双荧光素酶报告系统实验观察了化疗药物诱导的肿瘤细胞凋亡模型中QKI的表达变化,并通过PARP检测、Annexin V-FITC/PI流式细胞术和ROS检测等实验研究了QKI表达变化对凋亡的影响,最后探讨了QKI表达变化对化疗药物诱导的肿瘤细胞凋亡影响的相关机制。本研究的初步结论如下:1.构建并改造了QKI的显性负突变体,这一突变体包含二聚体形成序列但没有RNA结合功能。同时我们利用QKI异构体细胞定位的不同,在显性负突变体C端融合表达了QKI-5的核定位信号,通过与QKI-5的共定位实现了针对QKI-5亚型的特异性功能抑制。2.利用ViraPower?腺病毒表达系统成功构建了可表达QKI-6的重组腺病毒表达载体,并包装出高滴度的重组腺病毒。3.观察到在3种不同的化疗药物诱导的Hela细胞凋亡模型中,随着凋亡的增加QKI的mRNA和蛋白表达水平都逐渐降低,通过QKI启动子的双荧光素酶报告系统实验、QKI mRNA稳定性实验和蛋白酶体抑制实验证明,QKI表达的下调主要发生在转录和转录后水平;4.在细胞水平过表达QKI-5蛋白后,表柔比星和顺铂诱导的Hela细胞凋亡比例明显减少;相反,在细胞水平过表达QKI-7蛋白后,表柔比星和顺铂诱导的Hela细胞凋亡比例明显增多;同时,在细胞水平过表达QKI-6蛋白后,表柔比星和顺铂诱导的Hela细胞凋亡比例无明显变化。在主要表达QKI-5的Hela细胞中降低QKI所有异构体表达,可观察到表柔比星和顺铂诱导的细胞凋亡比例明显增多。5.在正常培养状态下,过表达QKI-7后,Hela细胞ROS的产生增高约1.4倍,而在阿霉素或顺铂诱导细胞凋亡过程中,这种增高提高到了约3倍,表明QKI-7有促进ROS生成的作用,尤其是在化疗药物诱导的应激条件下,这种效应更加明显。在QKI-5抗凋亡作用机制研究中,通过生物信息学预测发现,FOXO3a 3’UTR存在QKI反应元件。Western blotting检测也观察到在顺铂刺激条件下,伴随着QKI-5表达的降低,FOXO3a的表达增高。在Hela细胞中过表达QKI-5可导致FOXO3a的表达降低,降低QKI的表达可引起FOXO3a的表达增高,这些都提示FOXO3a参与了QKI-5的凋亡调节效应。具体机制有待进一步研究。综上所述,本课题组首次发现在化疗药物诱导的肿瘤细胞凋亡过程中QKI发挥重要的调节功能。在这个过程中,QKI-5蛋白可能通过降低FOXO3a的表达而发挥抗凋亡效应,QKI-7蛋白则通过增强ROS的生成而发挥促凋亡效应,提示QKI同源异构体表达水平的变化可能参与了肿瘤细胞耐药性的形成,有望成为新的肿瘤辅助治疗靶点。更多还原

【Abstract】 Apoptosis, termed programmed cell death, occurs both in the physiological and pathological situation. As a biological behavior in vivo, apoptosis involves in many physiological and pathological processes, such as development, homeostasis, stress, tumor and etc. Apoptosis signal pathways include extracellular ligands pathway and intracellular stress signals pathway. When extracellular ligands such as Fas, TNFa or TRAIL bind to their receptors, the intracellular death domains of these receptors recruit adaptor proteins (such as FADD and TRADD) and initiator caspase-8 and -10, resulting in the formation of the death-inducing signaling complex (DISC). Caspase-8 and -10 are activated at the DISC, which cleave the executioner caspases, caspase-3, -6 and -7. The latter three caspases are the main proteases that degrade the cell. Intracellular stress signals, such as growth factor withdrawal, DNA damage, oxidative stress or oncogene activation, lead to permeabilization of the mitochondrial outer membrane and release of cytochrome c. The consequent release of cytochrome c leads to the formation of a complex– the apoptosome– which contains cytochrome c, Apaf-1 and initiator caspase-9. Caspase-9 is auto-activated by induced proximity in the apoptosome. Active caspase-9 cleaves and thereby activates the executioner caspases. Complicated and intercrossed signal pathways for apoptosis make the regulation of apoptosis a complex and diverse process.Nowadays, more attention is paid to the post-transcriptional regulation mediated by RNA binding protein and miRNA. Posttranscriptional regulation is believed to play a vital role in the apoptosis. RNA binding protein QKI belongs to the evolutionarily conserved STAR family and has a critical role in the myelination of CNS development. However, the wide expression pattern of QKI in numerous tissues besides CNS implicates a fundamental role of QKI in other biological behavior, such as vascular development, cell apoptosis, cell adhesion, cell growth and organogenesis. The QKI gene produces a diverse set of proteins by alternative splicing. The three well studied isoforms (QKI-5, -6, and -7) appear to have different roles in development. They are constructed with the same 311-amino acid body but have different carboxyl tails consisting of 30, 8, and 14 amino acids, respectively. Because QKI-5 contains a nuclear location signal peptide (NLS), it mainly locates in the cell nuclus. QKI-6 and 7 mainly locate in the cell plasma. But sometimes QKI-5 translocates between cell plasma and cell nuclus. The distinction of location decides that isoforms of QKI have different function. According to the predicted 1400 candidate target mRNAs reported in Nat Stuct Mol Bio, many putative downstream targets are related with apoptosis, such as Bid、FOXO1、Sirt1, leading to the possibility of QKI in apoptosis regulation.To this end, we observed the alteration of QKI expression level during the chemotherapy induced cancer cell apoptosis through Real time PCR, Western Blotting and Dual-luciferase reporter assay system. Then we investigated the influence of the QKI expression changes on apoptosis through PARP detection, Annexin V-FITC/PI flow cytometry and ROS detection. Finally, we elucidate a potential mechanism study on the role of QKI on the chemotherapy induced cancer cell apoptosis. The main findings of our study are as follows:1. We constructed a QKI dominant negative mutation vector.The dominant negative mutation expresses peptide with the dimer sequence and without the RNA bingding ability. In the vector, the C terminal of the DN mutation was fused with the nuclear location signal of QKI-5, which thus has the same localization of QKI-5 isoform and specifically inhibits QKI-5 function.2. The recombinant adenovirus of QKI-6 has been successfully constructed through ViraPower? adenovirus expression system, which has been expressed even at a very low MOI.3. RT-PCR and Western-blot results showed that the mRNA and protein level of QKI decreased under the stimulation of three different chemotherapy drugs. The activity of QKI promoter was downregulated under the stimulation. QKI mRNA stability assay showed that QKI mRNA stability decreased under the stimulation. In addition, proteasome inhibitor could not reverse the downregulation of QKI expression under those stimuli. These results showed that the alteration of QKI expression level occurred on the transcriptional and post-transcriptional level.4. Overexpression of QKI-5 resulted in down-regulation of epricubicin and cisplatin induced apoptosis in Hela cells, while overexpression of QKI-7 resulted in significant more obvious apoptosis. In addition, overexpression of QKI-6 had no obvious apoptosis on epricubicin and cisplatin induced apoptosis. Repression of endogenous of all the QKI isoforms resulted in up-regulation of epricubicin and cisplatin induced apoptosis in Hela cells, which maybe due to the predominant expression of QKI-5 in Hela cells. 5. Overexpression of QKI-7 resulted in 1.4 fold increase of ROS in cells even under the rest stage, and the increase was up to 3 fold during epricubicin and cisplatin induced apoptosis, suggesting QKI-7 promotes the production of ROS especially during the chemotherapy. For the role of QKI-5 in antagonizing apoptosis, we found that FOXO3a 3’UTR contained two QKI response elements by bioinformatics analysis. Western blotting showed that FOXO3a expression decreased when QKI-5 expression increased under the stimulation of ciaplatin in Hela cells. At the same time we observed that overexpression of QKI-5 resulted in the reduction of FOXO3a expression, suggesting a role of FOXO3a in QKI-5 mediated apoptosis regulation. Further study is needed to confirm the speculation.In summary, we first defined an important role of QKI in chemotherapy induced apoptosis. QKI-5 has an anti-apoptosis function possibly through reducing the FOXO3a expression. In contrast, QKI-7 promotes the apoptosis of cells through enhancing the production of ROS during the chemotherapy drugs induced apoptosis. These results indicate that the alteration of QKI isoforms expression may be involved in the apoptosis under the stimulation of chemotherapy drugs. Aberrant QKI alteration under chemotherapy may be responsible for drug resistance, which is of great value for further examination. QKI may become a new target of tumor adjuvant chemotherapy.更多还原