The Role of Centromeres in the Molecular Biology of Prostate Cancer

项目总结/摘要 着丝粒是细胞机械的一个重要组成部分，需要忠实地分离的蛋白质。 在有丝分裂过程中，染色体的有丝分裂是一个在癌症中显著失调的过程。虽然精确的着丝粒 组装对于维持基因组的稳定性和确保准确的细胞分裂至关重要， 在癌症生物学方面的研究还不充分。构成基因组的高度重复的潜在序列 景观使得着丝粒区域的注释非常困难， 基因组学和表观基因组学是寻求更全面理解 人类基因组 因此，需要能够导航着丝粒序列的重复性质的新方法， 有效地研究癌症背景下的着丝粒遗传学和分子生物学。我们开发了新颖的 生物信息学工具，使我们能够在大型数据集中调查基因表达，以确定癌症特异性 目的基因的表达。我们小组还开发了一种定量PCR检测方法， 染色体特异性着丝粒序列，重点是检测α-卫星重复，这是 着丝粒的标志此外，我们已经确定了着丝粒H3组蛋白的显著过表达， 前列腺癌的CENPA变异。初步研究表明，CENPA在前列腺癌细胞中的敲低 限制了它们的繁殖能力。已知着丝粒α-卫星RNA（CeASaR）调节CENPA 功能，并且我们可以证明在比较前列腺癌和良性前列腺癌时， 前列腺组织使用我们的染色体特异性qPCR测定法在全细胞RNA中验证了这种富集 前列腺癌和前列腺上皮细胞系的提取物。鉴于上述情况，我们假设， 着丝粒代表了一个功能重要的分子特征，可以驱动前列腺癌 生物学我们将详细研究CENPA和CeASaRs在前列腺癌背景下的功能， 通过深度测序、生物信息学方法、细胞培养技术和小鼠模型。具体地说， 在前列腺癌细胞系中通过过表达和敲低研究调节CENPA表达， 细胞培养和异种移植将使我们能够证明CENPA与前列腺的生物学相关性 癌此外，通过PacBio RS II，我们可以使用北方印迹、RACE和长读技术 这个平台将帮助我们在着丝粒序列的高度重复景观中导航。的重要性 然后将探索这些特异性CeASaR与CENPA一起沿着驱动前列腺细胞增殖。这些 这些研究将揭示生物学和癌症研究中一个基本上未探索的领域，并有可能提供信息， 新的治疗方法。

Project Summary/Abstract The centromere is an essential component of the cellular machinery required for the faithful segregation of chromosomes during mitosis, a process that is significantly dysregulated in cancer. Though accurate centromeric assembly is vital for maintaining genomic stability and ensuring accurate cell division, the role centromeres play in cancer biology remains understudied. The highly repetitive underlying sequence that makes up the genomic landscape has made annotation of the centromeric region very difficult, leaving the study of centromere genomics and epigenomics as one of the last frontiers in the quest towards a more complete understanding of the human genome. New methodologies that can navigate the repetitive nature of centromeric sequences are thus required to effectively study centromere genetics and molecular biology in the context of cancer. We have developed novel bioinformatics tools that allow us to survey gene expression across large data sets to ascertain cancer specific expression of target genes. Our group has additionally developed a quantitative PCR assay to detect chromosome-specific centromeric sequences, with a focus on detecting α-satellite repeats, which are the hallmark of centromeres. Further, we have identified significant overexpression of the centromeric H3 histone variant CENPA in prostate cancer. Preliminary studies suggest that CENPA knockdown in prostate cancer cells limits their ability to proliferate. Centromeric α-satellite RNAs (CeASaRs) are known to modulate CENPA function, and we can demonstrate enrichment of specific CeASaRs when comparing prostate cancer to benign prostatic tissue. This enrichment was validated using our chromosome specific qPCR assay in whole cell RNA extracts from prostate cancer and prostatic epithelial cell lines. In view of the above, we hypothesize that centromeres represent a functionally important molecular signature that can drive prostate cancer biology. We will investigate in detail the functions of CENPA and CeASaRs in the context of prostate cancer, through deep sequencing, bioinformatics approaches, cell culture techniques and mouse models. Specifically, modulating CENPA expression in prostate cancer cell lines through overexpression and knockdown studies in cell culture and xenograft transplants will enable us to demonstrate the biological relevance of CENPA to prostate cancer. Additionally, northern blots, RACE, and long read technology available to us through the PacBio RS II platform will assist us in navigating the highly repetitive landscape of centromeric sequences. The importance of these specific CeASaRs in driving prostate cell proliferation, along with CENPA, will then be explored. These studies will shed light on a largely unexplored area in biology and in cancer research, with the potential to inform novel therapeutic approaches.