【作者】 曹子鹏；

【导师】 陈景元；

【作者基本信息】 第四军医大学 ， 劳动卫生与环境卫生学， 2013， 博士

【摘要】 【背景】众所周知，XIAP通过抑制caspase活性参与调节细胞凋亡[1]。在多种恶性肿瘤组织中，XIAP表达上调，包括前列腺癌[2]，急性和慢性白血病[3,4]，及其他恶性肿瘤[5-7]。XIAP的表达程度与肿瘤的侵袭和转移能力有关[8,9]。已发现XIAP与一些肿瘤的发生如吸烟诱导的肺癌等的相关性[10]。除了参与细胞凋亡调节外，它也有其他非细胞凋亡的生物学功能，如参与信号转导，参与铜代谢，参与泛素化等[11]。在凋亡抑制蛋白（IAP）家族成员中，XIAP是最有潜力的肿瘤治疗靶标。研究证实，使用化学抑制剂或小干涉RNA（siRNA）抑制XIAP表达能够抑制肿瘤细胞增殖[12,13]，但已有研究并没有阐明XIAP是直接抑制肿瘤细胞增殖，或是通过其他间接效应抑制肿瘤细胞增殖。XIAP蛋白由位于N端的三个BIR（Baculovirus IAP Repeat）结构域和C端的RING（Really Interesting New Gene）结构域构成[14]。XIAP通过与caspase3/7/9结合发挥调节细胞凋亡的功能[14]。XIAP的RING结构域有E3泛素连接酶活性，通过标记靶蛋白与泛素分子连接，促进蛋白的泛素化降解。XIAP RING结构域通过调节不同靶蛋白的降解途径发挥不同的生物学功能，包括细胞死亡[15]、炎症[16]和迁移[17]。我们最近的研究证实XIAP敲除或低表达能够降低HCT116细胞迁移及侵袭，而恢复XIAP表达能够逆转这种改变[18]，首次证实XIAP具有调节恶性肿瘤细胞迁移和侵袭的能力。我们进一步的研究证实XIAP能够与RhoGDIα结合，抑制RhoGDIα在第138位组氨酸的SUMO化影响蛋白的稳定，进而影响小Rho GTP酶活性，肿瘤细胞β-actin多聚化、细胞骨架重排和细胞迁移[18,19]。因此，发现和鉴定XIAP非细胞凋亡的生物学功能具有十分重要的生物学意义。【目的】本课题拟在实验室前期研究的基础上，旨在对XIAP调节肿瘤细胞增殖的分子机制进行研究。【方法】（1）利用脂质体转染法将HA-XIAP、HA-XIAP RING、HA-XIAP BIR、HA-XIAP H467A及pcDNA3.0空质粒转染入HCT116XIAP^-/-细胞，经过抗生素筛选构建稳定转染细胞株；（2）利用软琼脂克隆形成实验检测不同稳定转染细胞株的克隆形成能力；（3）利用ATP酶活性检测实验检测不同稳定转染细胞株的细胞增殖速度；（4）利用PI染色的流式细胞检测测定细胞周期分布；（5）利用western blot实验检测细胞周期调节蛋白表达；（6）利用RT-PCR实验检测NFAT基因的转录；（7）通过NFAT报告基因转染后检测荧光素酶活性；（8）通过脂质体转染Akt激酶的显负性突变体DN-Akt质粒并建立稳定转染细胞株，观察抑制Akt活性对细胞克隆形成能力的影响；（9）通过脂质体转染cyclin E特异的shRNA质粒减低cyclin E表达；（10）通过软琼脂克隆形成实验检测抑制cyclin E表达对细胞克隆形成能力的影响；（11）利用向细胞内转染E2F1突变的cyclin E启动子报告基因质粒并检测荧光素酶活性检测E2F1转录因子在XIAP调控cyclin E转录中的作用；（12）利用免疫共沉淀实验检测XIAP BIR结构域与E2F1蛋白的结合能力；（13）通过分离提取细胞核蛋白/胞浆蛋白检测转录因子转位；（14）通过脂质体转染c-Jun显负性突变体TAM67检测其对XIAP介导的cyclin D1转录的影响；（15）利用免疫共沉淀检测c-Jun和PP2A蛋白在细胞内的结合。【结果】1．XIAP不同结构域和E3连接酶活性分别调节肿瘤细胞增殖（1）软琼脂克隆形成实验结果显示，XIAP^-/-（vector）细胞形成克隆的数量较WT（vector）细胞明显减少；向XIAP^-/-细胞中转染HA-XIAP后，克隆形成数量明显增加。但是向XIAP^-/-细胞中转染HA-△BIR和HA-XIAP H467A表达质粒后，与XIAP^-/-（vector）细胞相比克隆形成数量没有明显改变，XIAP^-/-（HA-△RING）细胞克隆形成数量和大小明显增加。（2）ATP酶活性检测细胞增殖能力，结果与软琼脂内克隆形成实验趋势一致。这一结果说明，XIAP调节肿瘤细胞克隆形成能力是通过影响细胞增殖速率发挥作用的。2．XIAP的BIR结构域影响NFAT1转录因子的蛋白稳定性及其生物学功能（1）Western Blot显示在XIAP^-/-（vector）、XIAP^-/-（HA-△BIR）细胞内Akt激酶第308和473位的磷酸化水平高于其他细胞。（2）转染Akt激酶的显性负性突变质粒（Dominant negative mutant Akt, DN-Akt）抑制Akt磷酸化发现XIAP^-/-（HA-△BIR）细胞在软琼脂内的克隆形成恢复正常。（3）Western blot结果显示在XIAP^-/-（vector）和XIAP^-/-（HA-BIR）细胞中NFAT1蛋白表达低于正常，荧光素酶报告基因检测显示NFAT依赖的转录活性低于XIAP^-/-（HA-XIAP）细胞。（4）在XIAP^-/-（vector）和XIAP^-/-（HA-BIR）细胞中NFAT1蛋白的降解速度比WT细胞中快。（5）XIAP BIR结构域缺乏时MDM2的磷酸化增加，XIAP BIR结构域调节MDM2的磷酸化与Akt磷酸化增高有关。以上结果显示，XIAP介导的细胞凋亡信号通路和Akt介导的细胞生存信号通路之间存在相互影响。3．XIAP RING结构域缺失影响cyclin E基因转录及其生物学功能（1）流式细胞检测显示XIAP RING结构域缺失影响肿瘤细胞G1/S期过渡，G0/G1期比例明显减少。（2）Western blot结果显示，XIAP RING结构域缺失的细胞内cyclin E表达增高。（3）RT-PCR和启动子报告基因分析显示cyclin E表达发生在转录水平。（4）使用cyclin E特异的shRNA转染XIAP^-/-（HA-△RING）细胞，抑制了细胞异常增殖的能力。（5）启动子报告基因分析显示，XIAP RING结构域缺失影响cyclin E表达与E2F1转录因子转录活性增强有关。（6）免疫共沉淀显示，XIAP通过BIR结构域与E2F1结合。（7）蛋白亚定位分析显示，XIAP的RING结构域影响XIAP蛋白的细胞内亚定位。以上结果显示，XIAP能够通过BIR结构域与E2F1结合增强其对下游基因的转录活性，当RING结构域缺失的情况下，XIAP在核内异常表达，影响了肿瘤细胞的增殖能力。4．XIAP通过其RING结构域的E3连接酶活性调节cyclin D1基因转录及其生物学功能（1）XIAP^-/-（vector）细胞处于G0/G1期的比例较WT（vector）细胞明显增加，达到84.58%；转染了HA-XIAP的XIAP^-/-细胞处于G0/G1期的比例为69.31%，与WT（vector）细胞（71.51%）相比无明显区别；转染了HA-XIAP H467A的XIAP^-/-细胞处于G0/G1期的比例为78.94%，与XIAP^-/-（vector）细胞接近。（2）Western blot结果显示，XIAP E3连接酶活性能够影响cyclin D1蛋白表达，对其他细胞周期调节蛋白的表达没有影响。（3）RT-PCR和启动子基因报告检测证实XIAP的E3连接酶活性影响cyclin D1转录。转录因子转位分析结果显示，c-Jun磷酸化和AP-1转录因子可能参与了XIAP E3连接酶活性调节的cyclin D1转录。（4）荧光素酶报告基因分析结果显示，XIAP的E3连接酶活性可以影响AP-1转录活性。（5）c-Jun的显性负性突变体TAM67能够改变XIAP E3连接酶活性调节的cyclin D1表达。（6）免疫共沉淀证实PP2A能够与c-Jun在细胞内结合。（7）抑制PP2A活性能够使c-Jun磷酸化和cyclin D1表达增高。以上结果提示，XIAP的E3连接酶活性通过PP2A/c-Jun/cyclin D1通路影响肿瘤细胞G1/S期过渡和细胞增殖。以上结果提示， XIAP的E3连接酶活性通过PP2A/c-Jun/cyclin D1通路影响肿瘤细胞G1/S期过渡和细胞增殖。【结论】XIAP蛋白除了调节细胞凋亡，还可以调节细胞增殖，而且其分子结构中的RING结构域、BIR结构域及E3连接酶活性在调节肿瘤细胞生长过程中发挥了不同的作用。XIAP的E3连接酶活性影响肿瘤细胞增殖能力与调节cyclin D1表达有关；XIAP的E3连接酶活性能够抑制PP2A磷酸化，进而影响PP2A对c-Jun磷酸化的调节，从而调节cyclin D1的转录。XIAP的RING结构域缺引起肿瘤细胞中cyclin E高表达，其中BIR结构域能够与E2F1结合促进其转录活性，进而影响cyclin E转录和肿瘤细胞增殖。XIAP的BIR结构域缺失引起肿瘤细胞中Akt磷酸化活性增强，进而影响MDM2磷酸化，NFAT1蛋白稳定性和肿瘤细胞增殖能力。以上结果提示，E3连接酶活性是肿瘤治疗的潜在靶标；以XIAP为靶标的肿瘤治疗需要考虑不同结构域的不同生物学功能；XIAP和Akt介导的细胞增殖调节通路之间存在复杂的调节联系，需要进一步研究其精确的分子调控机制。更多还原

【Abstract】 【Background】As we all know, XIAP is involved in the regulation of apoptosis by inhibiting caspaseactivity[1]. XIAP expression increases in a variety of malignant tumors, including prostatecancer[2], acute and chronic leukemia[3,4], and other malignancies[5-7]. Although involvedin apoptosis regulation[8,9], it also has other non-apoptosis biological functions, such asparticipating in signal transduction, being involved in copper metabolism, being involvedin ubiquitination[11]. In the inhibitor of apoptosis protein （IAP） family members, XIAP isthe most potential cancer therapeutic target. Chemical inhibitors or small interfering RNA（siRNA） inhibition of XIAP expression can inhibit the proliferation of tumor cells[12,13],but these studies did not clarify whether XIAP is a direct or indirect inhibitor of tumor cellproliferation. XIAP protein contains three BIRs （baculovirus IAP repeats） located in the N-terminaldomain and the C-terminal RING （Really Interesting New Gene） domain[14]. XIAP cancombine caspase3/7/9to regulate their function in apoptosis[14]. XIAP RING domain’s E3ubiquitin ligase activity connects labeled target proteins to ubiquitin molecules, andpromote protein ubiquitination and degradation. By adjusting the type of the target proteindegradation pathway, domain of XIAP RING structure plays different biological functions,including cell death （14）, inflammation （15）, and migration （16）. Our recent studiesconfirmed the the XIAP knockout or low expression can inhibit HCT116cell migrationand invasion, and restore the expression of XIAP can reverse this change （17）. This is thefirst report that XIAP has the ability to regulate migration and invasion of malignant cells.Our further studies confirmed XIAP’s binding with RhoGDIα, inhibition the stable ofRhoGDIα protein through the SUMOylation at138histidine, thereby affecting the activityof small Rho GTP, tumor cell beta-actin polymerization, cytoskeletal rearrangements, andcell migration （17,18）. Therefore, the discovery and identification of XIAP non-cellapoptosis biological functions have important biological significance.【Objective】The purpose of the study is to investigate the role of XIAP in regulating themolecular mechanisms of tumor cell proliferation based on our previous studies.【Method】（1） By liposome transfection, HA-XIAP, HA-XIAP ΔRING, HA-XIAP ΔBIR,HA-XIAP H467A, and pcDNA3.0empty plasmids were transfected into HCT116XIAP^-/-cells, and antibiotic selection was used to obtain stably transfected cell lines;（2） By softagar colony formation assay, we investigated colony formation ability ofstably-transfected cell lines;（3） By ATP activity detection assay, we investigated cellproliferation rate in different stably-transfected cell lines;（4） PI staining flow cytometryassay was used to determine cell cycle distribution;（5） Western blot assay was used todetermine the expression of cell cycle regulatory proteins;（6） RT-PCR assay was carried out to determine the transcription of NFAT gene;（7） By luciferase activity assay, weinvestigated NFAT dependent transcription activity;（8） Stable transfection withdomin-negative mutant DN-Akt plasmid was used to inhibit the activity of AKT andobserve its effect in cell colony formation;（9） The translocation of transcription factorswere determined after the separation of nuclear and cytoplasmic proteins;（10） The role ofc-Jun in XIAP-mediated cyclin D1transcription was determined by liposome transfectionwith a c-Jun dominant-negative mutant, TAM67;（11） Co-immunoprecipitation was usedto determine the binding of c-Jun and PP2A proteins in cells;（12） Liposomal transfectionof cyclin E-specific shRNA plasmid was performed to reduce the expression of cyclin E;（13） Soft agar colony formation assay was carried out to determine the effect of cyclin Eexpression inhibition on cell colony formation;（14） To detect the role of E2F1in cyclin Etranscription regulated by XIAP, cells were transfected cyclin E promoter reporter plasmidwith E2F1mutant, and luciferase activity was detected;（15） Co-immunoprecipitationexperiment was used to detect the binding capacity between XIAP BIR structure domainand E2F1protein.【Results】1. XIAP’ different domains and E3ligase activity regulated tumor cell proliferation（1） Soft agar colony formation experimental results show that the quantity of coloniesformed in XIAP^-/-（vector） cells was significantly lower than that of wt （vector） cells.Transfection of HA-XIAP into XIAP^-/-cells increased the quantity of colonies. However,the transfection of HA-XIAP H467A into XIAP^-/-had no observed effect on colonyformation. XIAP^-/-（HA-△RING） colony formation increased significantly.（2） The resultsof cell proliferation assayed by ATPase activity determination were consistent with theresults of the soft agar clone formation assay. The results showed that XIAP regulatedtumor cell clone forming ability by affecting cell proliferation rate.2. XIAP BIR domain regulated NFAT1transcription factor protein stability and itsbiological function （1） Western blot showed that the phosphorylation level of Akt at308and473washigher in XIAP^-/-（vector） and XIAP^-/-cells （HA-△BIR） than other cells.（2） Thesuppression of Akt phosphorylation by transfection with the Akt kinase dominant negativemutant plasmid （Dominant negative mutant Akt, DN-Akt） lead the clone formation abilityof XIAP^-/-（HA-△BIR） cells back to normal.（3） Western Blot results showed that theprotein expression level of NFAT1in XIAP^-/-（vector） and XIAP^-/-cells （the HA-ΔBIR）was lower than WT cells, the luciferase reporter gene assay showed that NFAT-dependenttranscriptional activity was less than XIAP^-/-（HA-XIAP） cells.（4） In XIAP^-/-（vector） andXIAP^-/-cells （HA-ΔBIR）, NFAT1protein degradation rate was faster than that of the WTcells.（5） The XIAP lack of BIR domain had the increased MDM2phosphorylation. Theregulatory function of XIAP BIR domain on MDM2phosphorylation was dependent onincreased phosphorylation of Akt phosphorylation. The above results showed that there isinteraction between the apoptosis mediated by XIAP signal pathway and cell survivalsignaling pathways mediated by Akt.3. XIAP RING domain deletion affected cyclin E gene transcription and its biologicalfunction（1） Flow cytometry assay showed that the XIAP RING structure domain deletionaffected G1/S phase transition in tumor cells, leadting to significantly reduced number ofcells in G0/G1phase.（2） Western blot results showed that cyclin E expression wasincreased in cells deficient of XIAP RING structure domain.（3） RT-PCR and promoterreporter gene analysis showed that the expression of cyclin E was at the transcriptionallevel.（4） The transfection of cyclin E-specific shRNA into XIAP^-/-（HA-△RING） cellsinhibited the abnormal cell proliferation ability.（5） Promoter reporter gene analysisshowed that the deletion of XIAP RING structure domain affected the expression of cyclinE through increased transcription activativity of E2F1transcription factor.（6） InCo-immunoprecipitation, XIAP was found to bind to E2F1through its BIR domain.（7）Sub-celluar protein localization analysis showed that the RING domain of XIAP affects itsown cell localization. The above results showed that XIAP can enhance its transcription activity on its downstream genes through the binding of BIR domain to E2F1. Theabnormal expression of XIAP without BIR structure may affect the cell proliferation oftumor cells.4. XIAP regulated cyclin D1gene transcription and its biological function through itsE3ligase activity（1） The ratio of cells in G0/G1phase in XIAP^-/-（vector） cells was significantlyincreased compared with that in wt （vector） cells reaching84.58%; transfection ofHA-XIAP into XIAP^-/-cells reduce this ratio to69.31%, with no significant differencecompared with that of WT （vector） cells （71.51%）; the G0/G1phase ratio in XIAP^-/-cellstransfected with HA-XIAP H467A was78.94%, similar to that of of XIAP^-/-（vector） cells.（2） Western blot results showed that XIAP E3ligase activity can affect the expression ofcyclin D1expression, with no effect on other cell cycle regulatory proteins.（3） RT-PCRand gene promoter report confirmed the effect of XIAP E3ligase enzyme activity oncyclin D1transcription. Transcription factor translocation analysis showed that both c-Junphosphorylation and AP-1transcription factor may be involved in XIAP E3ligase activityregulation on cyclin D1transcription.（4） Luciferase reporter gene analysis showed thatXIAP E3ligase activity can affect the activity of AP-1transcription.（5） The dominantnegative mutant of c-Jun TAM67was able to change the regulatory effect of XIAP E3ligase activity on cyclin D1expression.（6） Co-immunoprecipitation confirmed thecombination between PP2A and c-Jun in the cell.（7） Inhibition of PP2A activity enabledincreased c-Jun phosphorylation and cyclin D1expression. The above results indicatedthat XIAP E3ligase activity affected G1/S phase transition and proliferation of tumor cellsthrough a PP2A/c-Jun/cyclin D1pathway.【Conclusion】In addition to its regulation on apoptosis, XIAP protein could regulate cancer cellproliferation, and the RING domain, BIR domains, and E3ligase activity played differentroles in regulating tumor cell growth. XIAP E3ligase activity affected tumor cell proliferation through its regulation on cyclin D1expression. The E3ligase activity ofXIAP can inhibit the phosphorylation of PP2A, thereby affecting the regulation of PP2Aon c-Jun phosphorylation, and the further regulation on the transcription of cyclin D1. Thedeletion of RING domain in XIAP can cause the high expression of cyclin E in tumor cells,in which the BIR domain can be combined with E2F1to promote its transcriptionalactivity, thereby affecting the transcription of cyclin E and tumor cell proliferation. XIAPBIR domain deletion induced enhanced Akt phosphorylation activity in the tumor cells,thereby affecting the MDM2phosphorylation, NFAT1protein stability, and tumor cellproliferation. These results suggest that XIAP’s E3ligase activity is a potential cancertherapy target, and that XIAP-targeted tumor therapy need to consider the differentbiological functions of different structural domains, and that there are complicatedrelationship between XIAP and AKT-mediated celluar proliferation-regulatory pathways.更多还原