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前列腺癌细胞雄激素不依赖表型转换中 AR 的作用及其机制
Alternative TitleThe potential role and mechanisms of AR mediating conversion to androgen independence in prostate cancer cells
Thesis Advisor孙中生
Degree Grantor中国科学院研究生院
Place of Conferral北京
Degree Discipline心理学
Keyword前列腺癌 Lncap 进展细胞模型 Rna-seq Ar 基因 Cag 和 Ggc 重复序列 Ga 缺
Abstract前列腺癌是威胁老年男性泌尿生殖系统最常见的恶性肿瘤之一。临床上前列腺癌难治的最大障碍在于对前列腺癌雄激素不依赖表型转换机制的不明。对于这个难题,尽管已经做了大量的工作,但是所取得的研究成果仍不能很好的解释前列腺癌雄激素不依赖表型转换这一过程。我们利用前列腺癌体外细胞进展模型(LNCaP/LNCaP-AI-F)针对雄激素不依赖表型转换这个棘手的临床问题而展开研究,从而规避了临床肿瘤样本高度异质性的不利影响。利用RNA-seq对两个细胞系的转录组在基因表达水平、可变剪接和融合基因方面进行了比较分析,试图找到介导前列腺癌细胞雄激素不依赖表型转换中的潜在机制。结果我们筛选出 788 个差异表达的基因,通过基因 GO 注解分析,将其在功能上归结为 4 个主要方面,包括(1)与神经内分泌样细胞形态、结构和功能活动相关的;(2) 与染色质组装、脂质和类固醇激素代谢以及响应各种刺激相关的;(3)与细胞粘附、迁移、分化和增殖相关的;(4)与信号转导通路相关的(如 Notch、SAPK 和 IGF1 等信号转导通路)。进一步的差异表达基因 IPA 分析,确定了一个以 AR 和 PSA 为核心的功能涉及细胞和有机体发育、增殖前列腺癌是威胁老年男性泌尿生殖系统最常见的恶性肿瘤之一。临床上前列腺癌难治的最大障碍在于对前列腺癌雄激素不依赖表型转换机制的不明。对于这个难题,尽管已经做了大量的工作,但是所取得的研究成果仍不能很好的解释前列腺癌雄激素不依赖表型转换这一过程。我们利用前列腺癌体外细胞进展模型(LNCaP/LNCaP-AI-F)针对雄激素不依赖表型转换这个棘手的临床问题而展开研究,从而规避了临床肿瘤样本高度异质性的不利影响。利用RNA-seq对两个细胞系的转录组在基因表达水平、可变剪接和融合基因方面进行了比较分析,试图找到介导前列腺癌细胞雄激素不依赖表型转换中的潜在机制。结果我们筛选出 788 个差异表达的基因,通过基因 GO 注解分析,将其在功能上归结为 4 个主要方面,包括(1)与神经内分泌样细胞形态、结构和功能活动相关的;(2) 与染色质组装、脂质和类固醇激素代谢以及响应各种刺激相关的;(3)与细胞粘附、迁移、分化和增殖相关的;(4)与信号转导通路相关的(如 Notch、SAPK 和 IGF1 等信号转导通路)。进一步的差异表达基因 IPA 分析,确定了一个以 AR 和 PSA 为核心的功能涉及细胞和有机体发育、增殖重复序列。两株雄激素不依赖亚细胞系的分析结果显示:AR基因CAG和 GGC重复序列的多态性(长短)在雄激素不依赖表型转换过程中都发生了明显的变化,这突出了AR 基因 CAG和GGC重复序列在激素不依赖表型转换过程中发挥着重要作用。最后,我们还意外地在 AR基因GGC重复序列之后发现并确定了一个GA二核苷酸缺失的存在。此 GA 缺失推测可以产生一个高度截短仅保留了 N-端转录激活结构域的 AR,有可能在前列腺癌发病和雄激素不依赖表型转换中发挥着作用。总之,通过RNA-seq 比较分析前列腺癌进展细胞模型的转录组,我们获得了一个以 AR 为核心的,涉及 AR 分子多层面的变化,至少包括 AR 的表达水平、可变剪接体、ART877A 突变、CAG 和 GGC 多态性、GA 缺失等,在雄激素不依赖表性转换中协同发挥着作用。尤其是首次发现 AR 基因 CAG 和 GGC 重复序列的多态性在前列腺癌细胞雄激素不依赖表性转换前后发生了明显的改变;同时我们还在前列腺癌细胞中第一次鉴定出AR基因GA缺失,功能预测显示其可能在前列腺癌肿瘤发生和雄激素不依赖表型转换中发挥着作用。此外,进一步的临床样本验证有助于确认我们研究结果的有效性,为前列腺癌的临床诊治提供实验和理论证据,并有助于揭示前列腺癌雄激素不依赖表型转换的机制。 
Other AbstractProstate cancer is one of the most common malignant tumors in genitourinary system threatening the older men. The biggest obstacle of the treatment to clinical refractory prostate cancer is still unknown  to  the mechanisms  mediating the conversion to androgen independent prostate cancer. Despite a lot of work have been done, the existing research results still cannot well interpret the androgen independent phenotype conversion mechanism. To this thorny clinical problem, we carried out our research in an LNCaP progression cell model (LNCaP/LNCaP-AI-F), so that we can escape the adverse effects from highly tumor heterogeneity. Using RNA-seq and comparative analysis the transcriptomes of both cell lines in gene expression, alternative splicing and fusion genes, we  tried to find the potential mechanism mediating  the conversion  to  androgen independent phenotype.  The comparative analysis identified 788 differentially expressed genes (DEGs) and classed  them  into four categories according to  functional related  GO (gene ontology) annotation analysis, including: (1)  Involved in neuroendocrine-like cell morphology and the associated functional activities; (2) Involved in chromatin assembly, lipids and steroids metabolism and the response to various stimuli; (3) Cell adhesion, migration, differentiation and proliferation-associated; (4) Involved in signal transduction pathways. Such as Notch, SAPK and IGF1 signaling pathways seemed to play important roles. Further IPA analysis of DEGs identified an AR and KLK3-centred network functionally related to cellular development, cellular proliferation, and organismal development, which may contribute to the emergence of androgen independent phenotype. In addition, some characteristic changes are of concern in the conversion to androgen-independent phenotype, such as neuroendocrine phenotype associated structural and functional changes, the ability to endure stress and surpressed response to various stimuli, and tumor stem cell-like characteristics.  We identified 315 differentially alternative splicing events. Of particular note,  AR not only upregulates its expression level, but also changes its variants and expands its CAG repeat length significantly. We also found 22 potential fusion genes and 8 LNCaP-AI-F specific fusion genes. Among these fusion genes, EIF2AK1-ATR and GLYR1-SLC9A8 are predicted to be cell damaging and oncogenic on function.  Based on  the critical roles of  AR  in tumorigenesis and progression of prostate cancer in previous studies, as well as the prominent role in LNCaP progression cell model, we identified the core role of AR in the conversion to androgen dependence. In addition, we conducted an in-depth  analysis  of  AR CAG  repeat expansion  at mRNA level  in androgen independent phenotype conversion,  simultaneously  we also investigated the GGC repeat, another important regulatory factor on AR function. Two representative androgen independent sublines were analyzed, and the altered AR CAG and GGC repeat length were identified  in androgen independent phenotype conversion process, which highlight their potential roles in androgen independent phenotype conversion process. Finally, we have also unexpectedly identified a missing GA dinucleotide followed GGC repeat sequence, which presumably produce a highly truncated AR protein just retaining transcriptional activation domain and may play role in  the occurrence of prostate cancer and androgen independent phenotype conversion.  In conclusion, through comparative analysis of RNA-seq in an LNCaP progression cell model, we identified an AR-centered, involving in multiple changes of AR, including AR expression level, AR variants, AR T877A mutation, CAG and GGC repeat polymorphism and GA deletion, synergistically play role in androgen independent phenotype conversion. Especially,  we  firstly  found significant changes  of  AR gene CAG and GGC repeat sequences’ polymorphisms in androgen independent phenotype conversion. We also firstly identified a GA deletion in prostate cancer cells,and the functional speculation suggested that this mutant AR may play role in prostate cancer  tumorigenesis and androgen  independent phenotype conversion. Further clinical validation is helpful to confirm the validity of these results and provide experimental and theoretical evidences to clinical diagnosis and treatment, and is also helpful to reveal the  transformation mechanism  of androgen independent phenotype.
Subject Area行为遗传学
Document Type学位论文
Recommended Citation
GB/T 7714
王永庆. 前列腺癌细胞雄激素不依赖表型转换中 AR 的作用及其机制[D]. 北京. 中国科学院研究生院,2013.
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