【作者】 李海霞；

【导师】 沈铿； 李夏璐；

【作者基本信息】 北京协和医学院 ， 妇产科学， 2014， 博士

【摘要】 研究背景卵巢癌是病死率最高的妇科恶性肿瘤,尽管在诊断和治疗上已有很多改进,但仍然有75%卵巢癌患者在诊断时已属晚期,五年生存率仍徘徊在30%。因此深入研究卵巢癌发生发展的分子机制,对于提高卵巢癌的早期诊断、治疗和改善预后具有重要意义。目前大多数研究都是着眼于与肿瘤细胞增殖、分化和代谢相关通路及过程,有mRNA加工在卵巢癌中的研究很少。：mRNA的加工是细胞转录的一个重要过程,3’端poly(A)尾是真核生物mRNA主要特征,在调节mRNA的稳定性、细胞内转运、翻译中发挥重要作用,是调控基因表达的重要机制。在本研究中我们从mRNA加工出发,着重研究mRNA可变加尾在卵巢癌发生发展过程中发挥的作用,探索卵巢癌的发生发展的分子机制,寻找与卵巢癌相关的基因,为临床卵巢癌诊断和治疗提供生物学基础依据。研究目的1.优化检测poly(A)位点的PAS-Seq (PolyA Site-Sequencing)方法。2.探讨mRNA加尾位点在正常卵巢上皮细胞和卵巢癌细胞中的分布和差异。3.探索引起卵巢癌细胞mRNA选择性加尾变化的分子机理。4.探索3’端poly(A)尾加工因子CstF-2T表达异常在卵巢癌发生、转移和复发中的临床意义。研究方法1.提取1株正常卵巢细胞和7株卵巢癌细胞系中的细胞总RNA,并对总RNA进行质检,通过纯化出mRNA并将其片段化,经逆转录,引入接头,扩增和回收片段等步骤,构建DNA测序文库,常规测序验证文库质量。2.采用高通量测序,鉴定卵巢癌细胞中特异的poly(A)加尾位点变化。3.Westernblot方法检测CstF-2T蛋白在正常卵巢细胞和卵巢癌细胞的表达水平。4.采用PAS-Seq方法比较在卵巢癌细胞shRNA沉默CstF-2T前后,细胞mRNA选择性加尾变化,探讨卵巢癌细胞中CstF-2T蛋白表达对mRNA poly(A)位点选择的影响。5.采用免疫组织化学方法,检测CstF-2T蛋白在正常卵巢上皮、卵巢浆液性乳头状囊腺癌原发灶、淋巴结转移灶和复发灶的表达情况。研究结果1.RNA经定量和电泳检测,质量符合PAS-Seq实验要求,DNA文库制备的各项参数达达到测序上机要求,文库制备成功。2.APA在卵巢细胞中发生的频率较高,大约有30%的基因在卵巢细胞中含有2个poly(A)位点,部分基因含有更多的poly(A)位点。与正常卵巢相比,有APA的基因与卵巢癌细胞增殖、粘附、血管形成等生物学特性有关。3.CstF-2T蛋白在卵巢癌细胞中的表达水平明显高于正常卵巢细胞。4.在卵巢癌细胞中shRNA沉默CstF-2T后,基因mRNA poly(A)尾位点发生改变,有poly(A)位点变化的基因参与PI3K/Akt、MAPK和Wnt等与肿瘤密切相关的信号转导通路。5.CstF-2T蛋白在卵巢浆液性乳头状囊腺癌中的表达水平明显高于正常卵巢组织,并且CstF-2T蛋白在卵巢浆液性乳头状囊腺癌复发病灶中的表达高于原发病灶。结论1.成功地优化了检测poly(A)位点的PAS-Seq方法。PAS-Seq方法可以从全基因组水平大通量地分析mRNA poly(lA)加尾位点的转换,为开展鉴定卵巢癌特异lnRNA选择性加尾事件和开展后续机理研究提供重要的保证。2.我们获得了正常卵巢细胞和卵巢癌细胞中mRNA poly(A)加尾谱,在细胞水平上根据mRNA选择性加尾位点不同进行了聚类分析。Poly(A)位点选择的差异能够有效地识别卵巢癌细胞和正常卵巢细胞,并且为卵巢癌细胞亚型分类提供了一个潜在的指标。3.在卵巢癌细胞系ES2、SKOV3和IGROV1中鉴定受CstF-2T调控的mRNApoly(A)位点,为后续理解卵巢癌细胞mRNA加尾变化的分子机理提供重要信息。4.CstF-2T蛋白在卵巢浆液性乳头状囊腺癌复发病灶中的表达水平高于原发病灶,有可能作为在卵巢癌复发过程中出现的肿瘤异质性分子指标。更多还原

【Abstract】 BackgroundOvarian cancer is the leading cause of death from gynecological cancers. Although considerable improvements have been made on the diagnosis and therapy, the majority of patients present with advanced stage disease, with an overall5-year rate of approximately30%. A better understanding of molecular pathogenesis of ovarian cancer will help to predict implications for early disease detection, develop strategies for rational intervention, and improve outcome. Currently, most researches focus on genetic events related to cell proliferation, differentiation and metabolism, the contribution of mRNA processing in ovarian cancer initiation and progression is poorly understood. The mRNA polyadenylation is an essential cellular process in eukaryotes, almost all eukaryotic mRNAs have a poly(A) tail at their3’ends. Poly(A) tails have shown to impact mRNA stability, translation, nuclear export and cytoplasmic localization. It is therefore that,3’end polyadenylation is a fundamental aspect for gene expression. Here we concentrate on mRNA polyadenylation in ovarian cancer and the insights that have emerged from this study may therefore hold diagnostic and therapeutic implications for ovarian cancer.Objectives1. To optimize the Poly(A) Site Sequencing technology.2. To explore the profiling of alternative polyadenylation(APA) sites in a human ovarian epithelial cell line and seven ovarian cancer cell lines.3. To investigate the complex mechanisms underlying APA sites switching in ovarian cancer cell lines.4. To estimate the possible significance of aberrant expression of CstF-2T protein in the pathogenesis, metastasis and recurrence of ovarian serous cystadenocarcinoma.Methods1. Libraries were constructed following several steps. Briefly, total RNA was isolated from a human normal ovarian epithelial cell and seven ovarian cancer cell lines respectively, then the polyadenylated mRNA was purified and fragmented, reverse transcription was conducted with an anchored oligo(d)T primer to generate the first-strand cDNA, then the improved PCR amplification was performed. The final PAS-Seq libraries were sequenced conventionally to ensure the libraries were feasible.2. The DNA libraries were sequenced on the Illumina HiSeq system. Bioinformatics analysis was used to investigate APA sites switching in ovarian cell lines.3. The expression of CstF-2T protein in human ovarian epithelial cells and ovarian cancer cells was detected by Westernblot.4. PAS-Seq technology was undertaken to investigate APA sites switching in ovarian cancer cells infected with shRNA CstF-2T virus, to verify whether expression of CstF-2T protein could affect APA sites switching.5. Immunohistochemical staining was applied to examine the expression of CstF-2T protein in normal ovary, serous cystadenocarcinoma, abdominal disseminated, lymphatic metastatic and recurrent specimens.Results1. The quality of total RNA was examined by qualification and electrophoresis. The DNA libraries were successfully constructed.2. APA sites switching events were prevalent in ovarian cell lines, over30%of all expressed genes produced alternatively polyadenylated mRNAs. Compared with human ovarian epithelial cells, genes with APA sites switching were associated with cell proliferation, adhesion, and angiogenesis et al.3. CstF-2T protein expressed higher in ovarian cancer cell lines.4. APA sites switching occurred in ovarian cancer cells infected with shRNA CstF-2T virus. Genes with APA switching were related to PI3K/Akt, MAPK, and Wnt signal pathways.5. The expression CstF-2T protein was overexpressed in ovarian serous cystadenocarcinoma specimens, with an increasing tendency in the recurrent lesions.Conclusions1. We successfully established the PAS-Seq method in our lab, which can detect APA sites switching at genome-wide levels.2. The profiling of APA switching was acquired which could help to construct heatmap analysis from ovarian cell lines. APA sites switching could distinguish human ovarian epithelial cells from ovarian cancer cell, also could help to subtype ovarian cancer cells.3. Elevation of CstF-2T protein could influence APA sites switching in ES2, SKOV3and IGROV1cell lines. That may provide new insights into the possible mechanisms underlying APA sites switching in ovarian cancer development and progression.4. CstF-2T protein was overexpressed in both ovarian cancer cell lines and human cystadenocarcinoma specimens. CstF-2T protein could be uses as a molecular biomarker of tumor heterogeneity in ovarian serous cystadenocarcionma.更多还原