【作者】 罗清；

【导师】 黄常志；

【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2011， 博士

【摘要】 核诱导凋亡因子1(nuclear apoptosis-inducing factor 1, NAIF1)是一个新发现的核诱导凋亡蛋白,之前的研究表明其在宫颈癌细胞系HeLa人胚肾细胞系HEK293中过表达时能诱导凋亡,但具体的机制还尚未阐明。另有研究发现在HeLa细胞中NAIF11可以靠其Myb-like结构域(Myb-like domain)与HARBl结合并帮助后者入核,而HARB1足迄今唯以已知的与PIF/Harbinger转座元件同源的脊椎动物基因。因此,NAIF1可能在脊椎动物的转座方面发挥重要功能。但NAIF1在肿瘤与正常组织中的表达情况及在其肿瘤细胞中的功能相关研究还未见报道。本研究建立了人NAIF1蛋白的原核高效表达体系及纯化方法,并应用该方法获得一定数量和纯度的原核重组表达蛋白,同时用该蛋白作为抗原,制备了兔抗人NAIF1多克隆抗体,为后续工作提供了研究基础。.此外,我们利用该抗体对人NAIF1蛋白在正常和肿瘤组织中的表达进行了研究。组织芯片和病理切片免疫组化结果显示,NAIF1在人正常胃组织和正常乳腺组织中呈现高表达,而对应的胃癌组织和乳腺癌组织中NAIF1呈现低表达或者不表达(P<0.001)。我们还对NAIF1表达水平与临床参数如肿瘤分期、分级、性别、年龄等因索作了相关性分析。研究结果发现,NAIF1在高分化胃癌组织中的表达要高于中,、低分化胃癌组织(P=0.004)；并且NAIF1的表达量随着胃癌T分期从T1至T4期的发展而逐渐降低(P=0.024)。在胃癌细胞系MKN-45中,我们发现NAIF1能抑制MKN-45的生长,流式细胞术分析结果显示NAIF1的过表达能引起MKN-45细胞系出现sub-G1期峰,形态学分析MKN-45细胞出现典型的凋亡形态学特点,机制分析结果显示NAIF1在MKN-45细胞中高表达后,导致野生型P53和Bax表达上升,Bcl-2表达下降,并且通过活化procaspase-9而不活化procaspase-8进而激活procaspase-3和PARP从而诱导凋亡。另外,我们还构建了两个NAIF1的截短体,pEGFP-N1-NLS (1-90)和pEGFP-N1-GRR(1-117),并且通过研究发现NAIF1只需其N端片段(1-90aa)即可诱发凋亡。在乳腺癌细胞系MCF-7中,我们发现NAIF1高表达对其生长没有抑制作用,也不诱导凋亡(48 h),但能抑制细胞的体外迁移和体外侵袭能力(P<0.001),机制分析结果显示NAIF1可能是通过抑制FAK的Try397位、ERK的Thr202/Tyr204位磷酸化,进而抑制MMP-9而发挥其抑制作用的。综上所述,我们对NAIF1在胃癌和乳腺癌中的表达情况进行了研究,探讨并揭示乐NAIF1诱导胃癌细胞系MKN-45凋亡和抑制乳腺癌细胞系MCF-7运动、侵袭的可能机制,并且发现了NAIF1发挥作用的最小功能区。研究结果不仅对深入认识NAIF1的功能大有裨益,还具有潜在的临床应用前景。更多还原

【Abstract】 Nuclear Apoptosis-Inducing Factor 1 (NAIF1) was found to induce apoptosis when overexpressed in HeLa cells and HEK293 cells, but the mechanism was unclear. Another study showed NAIF1 can physically interact with HARB1 and promote its nuclear import by the Myb-like domain of NAIF1. As in vertebrates, the HARBI1 gene constitutes the only known example of domesticated genes derived from a PIF/Harbinger transposase, the NAIF1 directly interact with HARB1 may have important function in transposition. However, the expression and function of NAIF1 in normal and cancer tissue was lacked.In the present study, we first got an anti-NAIFl polyclonal antibody with high sensitivity and specificity. Then we found NAJF1 was widely expressed in many normal and cancer tissues using immunohistochemistry analysis. Especially, NAIF1 was highly expressed in normal gastric and breast tissue but down-regulated or lost in the cancer tissue respectively (P<0.001). Besides, the associations between NAIF1 protein levels and grade of differentiation, staging and other clinical indexes were also examined. The results showed NAIF1 expression was higher in well-differentiated than in moderately-or poorly-differentiated gastric cancer (p=0.004), and NAIF1 expression was associated with different T stages (p=0.024). In vitro, NAIF1 can inhibit tumor cell proliferation and induce GO/G1 phase cell cycle arrest in the MKN-45 cell line. NAIF1 can induce apoptosis through activation of procaspase-9 rather than procaspase-8 followed by activation of the caspase-3 pathway. Otherwise, we designed two truncation mutants, pEGFP-N1-NLS and pEGFP-N1-GRR. Then we identified the N-terminal 1-90 amino acid domain of NAIF1, which is a helix-turn-helix motif, was sufficient for inducing apoptosis. In breast carcinoma cell line MCF-7, we found NAIF1 can neither inhibit proliferation nor induce apoptosis, but it can inhibit the migration and invasion of MCF-7 cells. The mechanism study showed that NAIF1 can inhibit the phosphorylation of FAK(Try397) and ERK(Thr202/Thr204) pathway, therefore block the expression of MMP-9 to execute its inhibition role.In summary, we investigated the expression of NAIF1 in gastric and breast cancer tissue, explored the mechanism of NAIF1 inducing MKN-45 cells apoptosis and inhibiting the migration and invasion of MCF-7 cells, identified the minimal functional domain of NAIF1. Those would be beneficial for profound insights into the function of NAIF1, and will be helpful for potential clinic applications.更多还原