【作者】 王爽；

【导师】 林蓓；

【作者基本信息】 中国医科大学 ， 妇产科学， 2021， 博士

【摘要】 目的:锌指蛋白703(ZNF703)作为一种转录因子,在多种肿瘤中表达升高,在卵巢癌中的功能尚不明确。本研究通过检测ZNF703在卵巢癌的表达及构建差异表达的细胞系,利用多种生物学方法结合ChIP-sequence高通量测序技术揭示ZNF703对卵巢癌生长和转移的影响及机制。方法:1、采用免疫组织化学的方法(即SP法)检查卵巢组织中ZNF703的表达情况,并分析评估ZNF703表达与临床病理参数的关系及对卵巢癌患者的总体存活率及预后的影响。检测四种卵巢癌细胞系中ZNF703的表达情况,分别构建ZNF703过表达和抑制表达的细胞模型,并通过Western Blot及qRT-PCR检测转染前后的ZNF703变化情况。MTT、细胞凋亡及细胞周期、侵袭实验及迁移实验用于评估ZNF703基因差异表达前后在体外对卵巢癌细胞恶性生物学行为的影响。GSEA方法用于对ZNF703进行GO和KEGG富集,并通过Western Blot检测pPI3K及p-AKT变化研究ZNF703对PI3K-AKT通路的作用。2、通过免疫共沉淀检测体外细胞中HE4与ZNF703结构上的相互作用关系,免疫荧光检测HE4与ZNF703在细胞中的共表达情况。从mRNA及蛋白角度分析ZNE703与HE4表达相关性(组化、WB、PCR),细胞浆细胞核分离实验及免疫荧光检测HE4差异表达前后ZNF703的核易位情况,检测ZNF703促进的卵巢癌恶性生物学行为是否受到HE4影响。3、将卵巢癌细胞甲醛交联并片段化处理后,使用抗ZNF703一抗进行染色质免疫共沉淀(chromatin immunoprecipitation,ChIP)实验,然后对富集得到的DNA进行定量、质检、测序,并将高通量测序结果定位到人类基因组(hg19)中进行分析。首先在ZNF703过表达或抑制表达的细胞中对潜在的靶基因进行qRT-PCR检测后,选取PEA15作为靶基因进行验证,根据PEA15对应的结合峰进行引物设计,ChIP-PCR检测ZNF703是否直接结合于PEA15的增强子区。构建双荧光素酶载体并转染293T细胞检测ZNF703对PEA15增强子区域的结合。Western Blot在蛋白水平检测ZNF703转染前后PEA15、p-PEA15(Ser 116)及pPEA15(Ser 104)的表达变化。临床标本检测PEA15的表达,分析PEA15和ZNF703的组化相关性,以及HE4与PEA15相关性,细胞实验分析HE4对PEA15mRNA、蛋白及磷酸化的影响。体内实验研究ZNF703过表达后对裸鼠皮下移植瘤增殖的影响,并利用HE染色及免疫组化检测移植瘤中ZNF703、Ki-67、PEA15及p-PEA15的水平。结果:1、ZNF703在卵巢癌组织中表达增加,其表达水平与卵巢癌患者临床病理参数无明显相关性,但ZNF703高表达的卵巢癌患者生存时间明显缩短、预后较差。CAOV3细胞中的ZNF703蛋白表达水平最高,依次为SKOV3和OVCAR3,在ES-2表达水平最低,分别进行siRNA的转染或过表达细胞系的构建,在蛋白和mRNA水平进行转染效率验证后实验。结果发现与对照组相比,过表达ZNF703后卵巢癌细胞增殖加快、细胞凋亡变少,细胞周期结果提示过表达后细胞向G2/S期转化。与此同时,过表达ZNF703后增强了卵巢癌细胞的侵袭和迁移能力。不仅如此,我们发现过表达ZNF703后相关蛋白PCNA、Bcl2/Bax、cyclin D1、MMP2、MMP9增加。在siRNA转染的细胞中得到了相反的结果。GSEA富集分析发现ZNF703富集于很多通路,其中ZNF703过表达后PI3K和AKT的磷酸化程度显著升高,在抑制表达后降低。2、免疫共沉淀结果显示在两种细胞系中存在ZNF703和HE4的相互作用。免疫荧光共定位显示ZNF703主要表达于细胞核,HE4主要表达于细胞质,二者存在共定位。对免疫组化结果分析显示ZNF703与HE4的表达具有相关性,但是在细胞系中的表达并无相关性。HE4过表达或抑制表达前后ZNF703总蛋白表达没有明显差异,核浆分离实验结果显示HE4过表达后ZNF703的核内表达增多,胞浆内表达减少,在HE4抑制表达后观察到相反的结果。免疫荧光实验对HE4抑制前后ZNF703进行检测,发现在HE4抑制表达后,核内ZNF703的荧光表达减弱,细胞浆增加。同时,ZNF703可以逆转HE4对卵巢癌细胞恶性生物学行为的影响,而HE4对ZNF703促进的卵巢癌细胞恶性生物学行为的影响并不显著。3、ChIP-Seq鉴定出多个ZNF703的调节靶点,这些靶点明显富集于很多通路。ChIP-PCR证实ZNF703可以直接与PEA15的增强子区结合,双荧光素酶报告基因也证明了这一结论。Western Blot结果说明过表达ZNF703后PEA15、p-PEA15、p-PEA15/PEA15均增加。在同样的临床标本中检测到PEA15在98例卵巢癌组织中高表达,PEA15高表达的患者预后更差。对组化结果进行相关性分析发现PEA15的表达与ZNF703、HE4均具有相关性。同时,PEA15 mRNA在siRNA抑制HE4表达时降低,在HE4过表达后增加。在蛋白水平HE4的差异表达会影响PEA15蛋白表达,但对PEA15磷酸化水平影响不明显。体内移植瘤实验结果显示相比于对照组,ZNF703过表达组瘤体的终体积更大、肿瘤质量更高、生长速度更快。且移植瘤组织中过表达组的Ki-67、PEA15、p-PEA15表达均增加。结论:1、ZNF703在卵巢癌组织及细胞中高表达,促使卵巢癌细胞增殖加快、细胞周期进展及侵袭转移能力增加、阻遏细胞凋亡。2、HE4和ZNF703的相互作用促进ZNF703核易位。3、ZNF703不仅通过直接结合PEA15的增强子促进PEA15的表达,还通过激活PI3K/AKT通路促进PEA15 Ser116位点的磷酸化,阻止卵巢癌细胞凋亡,进而促进卵巢癌进展。更多还原

【Abstract】 Objective: Zinc finger protein 703(ZNF703),acting as a transcription factor,was elevated expression in many tumors.But its functions in ovarian cancer are still unclear.In this study,we tested the expression of ZNF703 in ovarian cancer and constructed the differentially expressed cell lines.We used a variety of biological methods and highthroughput sequencing to reveal the effects and mechanisms of ZNF703 in the growth and metastasis of ovarian cancer.Methods: 1.Immunohistochemistry(SP)was applied for analyzing the ZNF703 protein levels in ovarian tissues.And we assessed the relationship between the ZNF703 levels and clinicopathological parameters and the overall prognosis of ovarian cancer patients.Besides,the basic levels of ZNF703 in cell lines of ovarian cancer were investigated.And we constructed the cell lines of overexpression and inhibition of ZNF703 level,and confirmed the transfection efficiency at mRNA and protein levels.The functions of ZNF703 on the malignant biological behavior of ovarian cancer cells in vitro was evaluated by MTT test,cell apoptosis and cell cycle by flow cytometry,invasion and migration experiments.The GSEA was used to enrich the KEGG pathway and GO functions of ZNF703,and the effects of ZNF703 on the PI3K-AKT pathway was tested by western blot.2.The structural interaction and the colocalization expression between HE4 and ZNF703 in vitro were analyzed by co-immunoprecipitation and immunofluorescence.The distribution ZNF703 protein after HE4 differential expression were evaluated by the cytoplasmic nuclear separation test.We evaluated the correlation between ZNE703 and HE4 expression,and detected whether the malignant biological behavior of ovarian cancer promoted by ZNF703 was affected by HE4.3.After crosslinking with formaldehyde and fragmenting of ovarian cancer cell,the anti-ZNF703 primary antibody was applied for ChIP experiment.Then the enriched DNA was quantified,quality checked and sequenced.The high-throughput sequencing was performed,of which the results were compared with the human genome(hg19)for analysis.First,the potential targets were screening in the cells overexpressed or inhibited by ZNF703.And then PEA15 was selected as the target gene for verification.Primers were designed based on the binding peaks corresponding to PEA15.ChIP-PCR was used to detect whether ZNF703 directly bound to the enhancer region of PEA15.The dual luciferase vectors were constructed and transfected into 293 T cells to detect the binding of ZNF703 to the PEA15 enhancer region.Western Blot detected protein levels of PEA15 and p-PEA15 after transfection of ZNF703.Clinical specimens were used to detect the expression of PEA15.We also analyzed the histochemical correlation between PEA15 and ZNF703,and the correlation between PEA15 and HE4,and the effect of HE4 on PEA15 mRNA,protein and phosphorylation in cell experiments.Experiments in nude mice were conducted to study the effect of ZNF703 overexpression on the proliferation of subcutaneous xenograft tumors in vivo.The expression levels of ZNF703,Ki-67,PEA15 and p-PEA15 in xenografts were identified by HE staining and immunohistochemistry.Results: 1.The ZNF703 expression increased in ovarian cancer tissues,and its expression level had no obvious correlation with clinicopathological parameters.However,the overall survival time was shorten and the prognosis was poor in the ovarian cancer patients with high ZNF703 level.It was proved that ZNF703 expressed highest in CAOV3 cell lines,and a lower expression in SKOV3 and OVCAR3,ES-2 cell lines was at the least.We obtained the cell lines with siRNA transfection or stable overexpression and verified in mRNA and protein levels.The results showed that compared with the control group,the proliferation of ovarian cancer cells was accelerated and apoptosis was reduced after ZNF703 overexpression.The cell cycle was accelerated the transition from the G0/G1 phase to G2/S phase after overexpression.At the same time,the ability of invasion and migration raised after ZNF703 overexpression.We also found that the levels of PCNA,Bcl2/Bax,cyclin D1,MMP2,MMP9 increased after overexpression.The opposite results were obtained in siRNA-transfected cells.GSEA enrichment found that ZNF703 was enriched in many pathways,including PI3K-AKT.We found that the phosphorylation levels of PI3 K and AKT increased after ZNF703 overexpression,and decreased after inhibiting the ZNF703 expression.2.Co-immunoprecipitation results showed that there was an interaction between ZNF703 and HE4 in the two cell lines. Immunofluorescence co-localization indicated that the nucleus was mainly ZNF703 and the cytoplasm was mainly HE4,and they could be co-localized.There was no difference in total protein expression of ZNF703 after HE4 overexpression or inhibition.The distribution of ZNF703 raised in the nucleus and reduced in the cytoplasm after HE4 overexpression in the cytoplasmic nuclear fractionation experiment.The contrary results were acquired after inhibiting HE4 expression.After inhibiting HE4 expression,ZNF703 fluorescence in the nucleus was weakened and the cytoplasmic fluorescence expression increased.Our study also showed that the expression of ZNF703 and HE4 was correlated in immunohistochemistry,but the expression in cell lines was not correlated.At the same time,ZNF703 could reverse the influences of HE4 on the malignant progress of ovarian cancer cells.But the inhibiting functions of HE4 siRNA on the malignant progress was not notable in the ovarian cancer cells promoted by ZNF703.3.ChIP-Seq identified multiple regulatory targets of ZNF703,which were obviously enriched in many pathways.ChIP-PCR confirmed that ZNF703 can directly bind to the enhancer region of PEA15.The dual luciferase reporter gene system also proved this conclusion.The expression of PEA15,p-PEA15,and p-PEA15/PEA15 also increased when ZNF703 was overexpressed.PEA15 was also highly expressed in clinical ovarian cancer tissues specimens.The high PEA15 expression patients had significantly shortened overall survival time.The analysis of histochemical relation revealed that PEA15 was related to ZNF703 and HE4.At the same time,PEA15 mRNA decreased when HE4 was downregulation by siRNA,and increased after HE4 overexpressed.The differential expression of HE4 affected the protein expression of PEA15,but there was no significant effects on its phosphorylation.The results of experiments in vivo showed that the growth rates,final volumes and mass of the transplanted tumors in the ZNF703 overexpression group were higher than those in the control group.In addition,the protein levels of ZNF703,Ki-67,PEA15,and p-PEA15 expressed in the overexpression group of transplanted tumor tissues were increased.Conclusions: 1.High expression of ZNF703 in ovarian cancer tissues and cells accelerated the cell proliferation and cell cycle progression,enhanced cell invasion,metastasis and repressed cell apoptosis in ovarian cancer.2.The interaction of HE4 and ZNF703 promoted the nuclear translocation of ZNF703.3.ZNF703 not only promoted the expression of PEA15 by directly binding to the enhancer of PEA15,but also facilitated the phosphorylation of PEA15 at ser116 through PI3K/AKT pathway,which obstructed the apoptosis of ovarian cancer cells and accelerated the progression of ovarian cancer.更多还原