【作者】 关瑞；

【导师】 陈主初； Jim Hu；

【作者基本信息】 中南大学 ， 基础医学， 2012， 博士

【摘要】 锌指蛋白403（zinc finger protein403,ZNF403）,又称GGNBP2（gametogenetin binding protein2）,定位于人染色体17q12-17q21.1,该区位于乳腺癌,前列腺癌和喉癌等肿瘤的遗传易感区域内,其GenBank序列号为NM₀24835.3。ZNF403基因在生物物种进化中高度保守。目前已知人类ZNF403基因含有两种不同的RNA转录剪切本。其中一个截短型剪切本为本室李友军等采用RNA差异显示法从喉癌组织中所鉴定获得,称喉癌相关基因1（Laryngeal Carcinoma related gene1, LCRG1）,仅含有7个外显子,编码一个含288个氨基酸的蛋白。以往研究显示LCRG1在喉癌的发生、发展中发挥重要的抑瘤作用。另一个全长转录本,含有14个外显子,编码一个含696个氨基酸的蛋白质,其编码的蛋白目前功能尚不明确。因此本研究将以探讨ZNF403全长转录本在肿瘤细胞中的生物功能为目的,以期更全面的揭示该基因在肿瘤发生发展中的作用。本研究采用辅助依赖型腺病毒载体介导的RNA干扰技术对ZNF403进行全面的功能缺失型研究。首先采用Cre-loxp生产系统,成功构建生产了高纯度高效率的HD-Ad-ZNF403-shRNA病毒干扰表达系统,在获得特异高效的内源性表达沉默效果后,进一步对ZNF403表达缺失的肿瘤细胞进行生物学特性分析,在体内对细胞增殖,非停泊依赖性生长和细胞迁移活性进行探讨,并在体外对其细胞增殖影响进行了验证。为了揭示ZNF403影响细胞增殖生长的分子机制,同时采用碘化丙碇和BrdU标记的流式细胞分析术对ZNF403缺失在细胞周期中的影响进行分析,并采用高通量精确的人类细胞周期Realtime PCR array,全面比较ZNF403缺失后84个关键细胞周期相关调控基因的表达水平变化,并采用Western blot对部分重要基因的蛋白水平变化进行验证,以期明确ZNF403在细胞周期调控网络中的角色和位置。此外,本研究还对候选ZNF403基因的上游转录调控区进行了初步研究分析,成功克隆其5’-端启动子区域,并利用缺失突变、报告基因、转染技术对该基因启动子区域进行初步鉴定,同时采用TCDD药物处理分析特殊转录因子AHR结合位点的调控影响。本研究首次发现ZNF403基因为一个新的细胞周期调控蛋白,抑制ZNF403的表达能通过阻滞细胞周期的S期和G2/M检测点,显著抑制肿瘤的增殖,为肿瘤发生发展的分子机制提供了有价值的线索。[ZNF403基因结构,同源性分析及在肿瘤细胞系中的表达检测]ZNF403基因定位于肿瘤相关的遗传易感区17q12-17q21.1.区域内,该基因的全长转录本为2869bp,含有14个外显子,编码序列（CDS）位置为317-2410,编码一个包含696个氨基酸的蛋白质（蛋白序列登录号：Q9H3C7.1）。由于剪接方式不同目前已知形成至少2种mRNA剪接变异体,编码至少2种不同的蛋白质。除全长转录本ZNF403外,其剪切异构转录本LCRG1的全长为3448bp,包括7个外显子,编码一个由288个氨基酸组成的蛋白。利用比较基因组学分析发现人ZNF403基因在黑猩猩、猕猴、狗、牛及小鼠等多种动物中高度同源,提示该基因在进化中的的高度保守性。本研究采用特异性引物在不同细胞株中分别对ZNF403和LCRG1的表达进行Real time PCR检测,结果显示ZNF403的表达水平比LCRG1高大约10倍,而Western Blot方法仅能检测到ZNF403蛋白的表达,以上结果说明ZNF403为该基因的主要转录表达产物。[辅助依赖型腺病毒介导的人类ZNF403基因RNA干扰系统的设计,构建和鉴定]采用基于RNA聚合酶Ⅲ,鼠源性U6启动子的DNA载体构建表达shRNA。特异性针对ZNF403的目标序列如下：5’-GGGCAAATTCTGAAGAGAACGACA-3’。采用两对互补的DNA oligos构建一个含有U6promoter和ZNF403shRNA的辅助依赖型腺病毒（HD-Ad vector）质粒形式载体HD-Ad-ZNF403-shRNA。然后经PmeI酶切该质粒载体,暴露病毒末端倒置重复序列,再与辅助性病毒的共同感染116细胞,采用Cre/loxP系统进行辅助依赖型腺病毒的生产。经过拯救,扩增和大量扩增三大步骤后收集细胞,采用氯化铯不连续密度梯度离心及透析纯化病毒,经浓度和纯度检测得到了高浓度高纯度的HD-Ad-ZNF403-shRNA病毒载体。为检测该载体的RNA干扰效率,采用不同浓度的HD-Ad-ZNF403-shRNA和无效应HD-Ad-shRNA-control对ZNF403表达水平高的Hep-2和HEK293细胞进行感染,Realtime PCR检测结果显示HDAd-ZNF403-shRNA对ZNF403内源性mRNA表达水平有显著高效的干扰抑制效应。50MOI为用量最小且干扰效果最明显的载体浓度。Western blot检测说明在50MOI时,内源性ZNF403蛋白表达完全缺如,干扰效果好。同时结果显示,HDAd-ZNF403-shRNA不影响LCRG1的表达,证实了其RNA干扰的特异性。[ZNF403表达抑制后的肿瘤生物学特性研究]采用HD-Ad-ZNF403-shRNA和无效应HD-Ad-shRNA-control对Hep-2和HEK293细胞分别进行感染（50MOI）,24小时后,经过Realtime PCR验证其内源性ZNF403表达抑制效果后,进行后续肿瘤生物学功能研究实验。采用BrdU标记细胞增殖Elisa方法,结果显示ZNF403缺失在体内显著抑制细胞增殖活动,该结果得到了体外裸鼠成瘤实验的证实。此外,软琼脂集落形成实验提示ZNF403缺失对肿瘤细胞非停泊依赖性生长有明显损伤抑制效应；同时,细胞划痕试验显示ZNF403缺失亦对肿瘤细胞的迁移活动具有抑制效应。综合以上研究结果,初步揭示ZNF403缺失能在体内体外均具有抑制细胞增殖的效应,并能显著降低喉癌细胞的恶性表型。[7NF403在细胞周期中的作用机制研究]首先采用生物信息学软件ELM对ZNF403蛋白的功能结构域进行分析预测,发现存在多个与细胞周期相关的结构域,如RBLXCXE motif, Cyclins结合位点,PCNA结合位点等,提示ZNF403可能通过调节细胞周期对细胞增殖产生影响。继而采用碘化丙碇（PI）标记的流式细胞术检测ZNF403基因表达抑制后对细胞各周期的影响。结果表明,ZNF403基因表达抑制能导致G2/M阻滞,并轻度影响S期的进程。当采用不同剂量的HD-Ad-ZNF403-shRNA的感染细胞时,ZNF403的缺失导致的细胞周期G2/M期阻滞呈剂量相关效应。同时不同时间点的分析发现ZNF403的缺失对细胞周期的影响为非一过性的短期细胞反应,而是稳定的G2/M阻滞作用。此外,BrdU标记的动态细胞周期进程分析进一步验证了ZNF403的缺失导致的G2/M期阻滞。为了揭示ZNF403在细胞周期调控中的分子机制,采用高通量精确的人类细胞周期Realtime PCR array,全面比较ZNF403缺失后84个关键细胞周期相关调控基因的表达水平变化,提示以P21,MCM2,ATM, MRE11A为代表的多个细胞周期基因RNA水平发生显著改变。同时上述4个基因的表达变化在蛋白水平亦得到证实。该研究结果合理解释了ZNF403表达抑制对细胞增殖的抑制效应,首次报道了ZNF403在细胞周期中的重要功能,并初步对其在细胞周期中的调控分子机制进行了探讨。[ZNF403基因启动子区的预测,克隆,分离鉴定及其调控元件的初步分析]首先利用多个生物信息学软件预测ZNF403基因CpG岛及其启动子区域。经在线程序CpGplot, Methprimer, FirstEF, Gene2promoter和NNPP的启动子预测分析,并结合TSSs预测、CpG岛预测结果,初步认为ZNF403基因启动子位于（-1465,+199）较长区间内,并选取该1646bp片段构建一系列5’-或3’-端缺失的启动子片段荧光素酶报告基因载体,经瞬时转染细胞,双荧光素酶活性检测,结果表明ZNF403基因核心启动子定位于（-926～-682）区间。采用MatlnspectorV2.2和TESS软件搜索转录因子结合位点,显示核心启动子区存在AHR,E2F1等重要调控元件,同时保守进化足迹软件ECR发现AHR转录因子结合位点高度保守地同时存在于小鼠的5’端调控区和人ZNF403基因启动子区。采用AHR刺激物TCDD处理细胞,并检测启动子活性变化,结果显示TCDD处理的细胞启动子活性上升,这表明TCDD对ZNF403的表达调控可能是通过其启动子区域的AHR结合位点实现的,提示ZNF403可能为AHR致癌机制中一个重要的下游基因。更多还原

【Abstract】 ZNF403, also known as GGNBP2(gametogenetin bindingprotein2) is mapped to chromosome location,17q12-21.1, a region associated with cancers such as breast cancer, prostate cancer, and laryngeal cancer ZNF403is highly conserved from drosophila to human. The mouse and human ZNF403share96%amino acid sequence identity. In humans, there are two known RNA transcripts produced from ZNF403. The short transcript, laryngeal carcinoma-related gene1(LCRG1) was originally identified in human laryngeal carcinoma by mRNA differential display LCRG1encodes a nuclear protein of288amino acids, corresponding to a C-terminal truncation of ZNF403. It has been reported that over-expression of exogenous LCRG1suppresses the growth rate of human laryngeal cell lines.The full size ZNF403transcript is translated into a protein of696amino acids, which is the focus of the current study. However, the exact biological function of ZNF403is not clear.To investigate the role of ZNF403, we used the helper dependent adenoviral vector (HD-Ad) mediated RNA interference technology and conducted loss-of-function analysis of ZNF403in human tumor cells. U6promoter based vector was utilized to express ZNF403-targeted shRNA, which was then inserted it into HD-Ad vector. With the existence of helper virus, we successfully produced HD-Ad-ZNF403-shRNA of high purity and high efficiency by Cre-loxp system after rescue, amplification, large scale production and purification by CsCl gradient super centrifugation. Furthermore, we used HD-Ad-ZNF403-shRNA to knockdown expression of endogenous ZNF403and next characterized the impact of ZNF403knockdown on cell proliferation on cell proliferation, anchorage-independent growth and cell migration in human tumor cells. Moreover, we conducted tumor growth in nude mice and validated its role on cell proliferation in vivo. To elucidate the mechanism of ZNF403in cell proliferation, flow cytometric analyses of cellular DNA contents using Propidium Iodide and BrdU, indictive of DNA replication during S phase, were carried out to examine the role of ZNF403on cell progression. Subsequently, human cell cycle realtime PCR array with the advantages of high-throughput and accuracy were applied to further decipher the mechanism underlying the G2/M cell cycle arrest by ZNF403knockdown. We compared expression level of84cell-cycle genes between ZNF403knockdown group and control group in Hep-2and HEK293cells and validated several important altered genes by western blot analysis, which shed new light on the role of ZNF403in the regulation of cell cycle progression.Since it has been reported that ZNF403can be induced by TCDD through AHR pathway, in order to reveal the molecular mechanism underlying ZNF403’s induction by TCDD, we made use of bioinformatics analysis and investigated the transcription regulation of ZNF403. We first analyzed the5’upstream of ZNF403gene using bioinformatics and predicted the location of promoters and transcription binding sites. A series of promoter deletions within luciferase reporter genes were then constructed and dual-luciferase assay were performed in various cell lines to identify, localize and characterize the ZNF403promoter. Importantly, we accessed the transcription regulation of TCDD on its promoter. These findings suggest a previous unrecognized role of ZNF403in cell proliferation, which led to better understanding of the molecular mechanism on the role of ZNF403in tumorigenesis.[Gene structure,evolution and endogenous expression analysis of human ZNF403gene]ZNF403is mapped to chromosome location,17q12-21.1, a region associated with cancers such as breast cancer, prostate cancer, and laryngeal cancer. In human, there are two known RNA transcripts produced from ZNF403. The full size ZNF403transcript is translated into a protein of696amino acids, which is the focus of the current study. The length of the full size transcript contains2689bps, including14exons. The coding region spans from317-2410. The size of the short transcript, Laryngeal carcinoma related gene1(LCRG1) is3448bps including7exons and encodes a nuclear protein of288amino acids. Evolutionary comparisons of13vertebrate genomes by ECR browser, the human ZNF403gene shares highly phylogenetic homology to some extent with chimpanzee, rhesus monkey, dog, cow and mouse ZNF403genes, suggesting it is a highly conserved gene during evolution. To understand the relative expression level of ZNF403and LCRG1, we performed real-time PCR in different cell lines. The expression level of ZNF403is significantly higher (>10folds) than that of LCRG1in Hep-2, HeLa, Jurket, BEAS-2B, HEK293cell lines. At protein level, only ZNF403can be detected by western blot. Although the level of ZNF403varied in these cell lines, the expression pattern of ZNF403and LCRG1was similar. These data suggest that ZNF403is the major transcript produced from ZNF403gene.[Design, construction, identification and efficiency test of helper dependent adenoviral vector mediated ZNF403-targeted RNA interference system]RNA polymerase Ⅲ, U6promoter-based DNA vector was used to express shRNA. A ZNF403target region was chosen beginning with three guanines (51-GGGCAAATTCTGAAGAGAACGACA-3’). Two pairs of complementary DNA oligos were used to make the shRNA construct. The U6promoter and the ZNF403shRNA sequence was subcloned into the HD-Ad vector pC4HSU. The viral vector is linearized by Pme I to expose the viral ITRs and used to transfect116cells for virus rescue and amplification. The HDAd vectors were produced by Cre/loxP system. To rescue the HDAd, the linearized genome is transfected into116cells expressing Cre and infected with a helper virus (HV) providing a packaging signal flanked by loxP sites. After rescue, amplification and large scale production, producer cells containing a large amount of HD-Ads were harvested and digested by RNase A and DNase I. Finally, CsCl gradient super centrifugation and dialysis were performed to remove the helper virus and purify HD-Ad vectors. Southern blot and OD measurement showed that we achieved the production of highpurity HD-Ad-ZNF403-shRNA amounted to10^13.To test the silencing efficiency of the HD-Ad-ZNF403-shRNA, we transduced Hep-2and HEK293cell lines with HD-Ad-ZNF403-shRNA and HD-Ad-shRNA-control at various concentrations, real-time PCR analysis and western blot analysis showed that the expression of ZNF403was decreased dramatically at50MOI after HD-Ad-ZNF403-shRNA transduction. No difference was found in the expression of LCRG1between the cells treated with HD-Ad-ZNF403-shRNA and those with shRNA-control. Thus, HD-Ad delivery of shRNA effectively and specifically knocks down endogenous ZNF403and provides a powerful tool for loss-of-function analysis of ZNF403without interfering with expression of LCRG1.[Characterization impact of ZNF403knockdown on human laryngeal carcinoma Hep-2cells] HD-Ad-ZNF403-shRNA and none-effect HD-Ad-shRNA-control were used to transduce Hep-2and HEK293cells at50MOI. Cell proliferation assay showed that the amount of BrdU incorporated into ZNF403knockdown cells was-50%less compared to that of the control in both Hep-2and HEK293cells at the same time point, implicating that knockdown of ZNF403significantly suppressed cell proliferation. The inhibited cell proliferation by ZNF403knockdown was further supported and validated by tumor graft in nude mice. In addition, soft agar assay indicated that knockdown of ZNF403expression substantially impaired the anchorage-independent growth of Hep-2cells. Furthermore, wound-healing assay demonstrated that the ZNF403knockdown group exhibited an obvious reduction in migration ability as compared to the control groups.In summary, these findings revealed for the first time that ZNF403knockdown remarkably suppressed cell proliferation both in vitro and in vivo, as well as significantly reduced malignancy of laryngeal tumor Hep-2cells.[Function and mechanism analysis of ZNF403knockdown in cell cycle progressionAccording to the cell-cycle associated putative motifs in ZNF403using the software Eukaryotic Linear Motif Resource for Functional site in Proteins, we investigated the effect of ZNF403knockdown on cell cycle progression to elucidate the mechanism of ZNF403in cell proliferation. We showed by flow cytometric analysis of cellular DNA contents using propidium iodide that reduced expression of ZNF403led to the accumulation of cells at G2/M phase, suggesting that down-regulation of ZNF403promotes G2/M arrest in a dose-dependent manner. Moreover, cell cycle analysis at different time point (day2, day4, day6) demonstrated that the impact of ZNF403knockdown on cell cycle progression was not transient, but stable G2/M arrest. Additionally, BrdU was used to illustrate the role of ZNF403in DNA synthesis. To further decipher the mechanism underlying the G2/M cell cycle arrest by ZNF403knockdown, we compared expression level of84cell-cycle genes between ZNF403knockdown group and control group by Human Cell Cycle Profiler realtime PCR Array. Interestingly, the knockdown of ZNF403significantly increased the expression levels of p21, skp2, CDK5R1and CDKN2B, whereas repressed the expression of ATM, MCM2and MRE11A in both Hep-2and HEK293cells. Among them, the changes of P21, MCM2, ATM and MRE11A were further confirmed by western blot analysis.Altogether, these results suggest that the inhibited cell proliferation induced by ZNF403knockdown may due to the G2/M arrest which delayed progress towards mitosis and provide a new insight into the function of ZNF403in regulating the G2/M cell-cycle transition. [cloning and identification of the promoter of ZNF403and analysis of its transcription binding sites]Several bioinformatics programs were utilized to predict the candidate promoter region. Based on the bioinformatics analysis of CpG island, transcription start sites and candidate promoter location by CpGplot, Methprimer, FirstEF, Gene2promoter and NNPP, we selected the1646bps located at (-1465,+199) as the candidate promoter of ZNF403and constructed a series of promoter deletions within luciferase reportor vector (pGL3-Basic). We next performed dual luciferase assays and demonstrated that the core promoter was located at (-926～-682).Next, AHR was predicted to be a transcription factor by Matlnspector V2.2, TESS and ECR. Furthermore, combination of AHR ligand TCDD’s treatment and luciferase activity assay implied the induction of ZNF403by TCDD may be mediated by the transcription regulation of the AHR binding site within its promoter. This finding provides important support that ZNF403is a downstream gene regulated by AHR and also suggests its essential role in pollutant-related tumorigenesis.更多还原