Genetic and epigenomic studies of testicular tumor

睾丸肿瘤的遗传学和表观基因组研究

• 批准号: 7968740
• 负责人: Owen Rennert
• 金额: $ 20.56万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968740
• 关键词: Affect; Agar; Age; Bioinformatics; Biological Process; Biopsy; Cancer cell line; Cell Line; Cell Proliferation; Cells; Chromatin Structure; Chromosomes, Human, Pair 1; Clinical; DNA Methylation; DNA Methyltransferase Inhibitor; Data; Development; Disease; Down-Regulation; Embryonal Carcinoma; Embryonal Carcinoma Cell; Epigenetic Process; Functional RNA; Genetic; Genome Stability; Genomics; Germ Cell Cancers; Goals; Human Resources; In Situ Hybridization; Luciferases; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of testis; Mammals; Maps; Messenger RNA; Methylation; MicroRNAs; Minor; Mutation; Nature; Neoplasm Metastasis; Patients; Pattern; Primary Neoplasm; Promoter Regions; Proteins; RNA; Reporter; Role; Sample Size; Seeds; Seminoma; Sequence Analysis; Site; Subgroup; Testicular Embryonal Carcinoma; Testicular Germ Cell Tumor; Testicular Neoplasms; Testis; Time; Tissue Microarray; Tissues; Transfection; Tumor Cell Line; Tumor Suppressor Proteins; Untranslated Regions; base; bisulfite; cancer cell; demethylation; epigenomics; genome-wide; male; male health; novel; podocalyxin; tumorigenesis; vector

Epigenetic inactivation of microRNA in male germ cell cancer Personnel: Lee, Cheung, Chan, Rennert MicroRNAs (miRNAs) is a class of small non-coding RNAs that have been shown to be deregulated in many diseases including cancer. An intertwined connection between epigenetics and miRNAs has been supported by the recent identification of a specific subgroup of miRNAs called "epi-miRNAs" that can directly and indirectly modulate the activity of the epigenetic machinery. Using a genome-wide approach for studying differential methylation in testicular germ cell tumor cell line, we previously identified a novel hypermethylated locus on chromosome 1. Genomic mapping revealed that the hypermethylated region overlapped with a mircroRNA candidate, miR-199a and its upstream promoter region. Bisulfite sequencing and treatment with DNA methyltransferase inhibitor 5-aza in the Ntera-2 testis cancer cells confirmed the same observation in the tiling microarray data. To explore the role of miR-199s, we further examined its methylation and expression patterns in several testicular embryonal carcinoma cell lines (Ntera-2, 833K, NCCIT, Cates-1B and Tera-1), a cultured normal testis cell (HT) and 3 normal testis tissues from health males. Bisulfite sequencing showed that the normal cultured testis cell was unmethylated, while for normal testis tissues it is slightly methylated. Except Cates-1B, all examined testicular embryonal carcinoma cell lines were methylated. Expression of miR-199a, as revealed by real-time qPCR, showed that this miRNA was downregulated in all cancer cell lines as compared to either normal testis cell lines or tissues. A negative correlation between methylation and expression of miR-199a was observed in our study, indicating that DNA methylation is a critical regulator of miR-199a. Downregulation of miR-199a was not restricted in testicular cancer cell lines, but also in primary tissues. We collected total RNA from 9 normal testis biopsy, 8 embryonal carcinomas and 9 se-minomas from patients and analyzed the miR-199a expression by qPCR. After normalization, miR-199a-5p was found to be significantly down-regulated in both embryonal carcinomas and seminomas. Down-regulation in seminomas is more consistent in the analyzed cases, probably due to the more homogeneous nature of this poorly differentiated carcinoma cells. To expand the clinical sample size of testicular cancers, we are analyzing the expression of miR-199a in tissue arrays with LNA-based in situ hybridization. The result from tissue array will help us to understand whether miR-199a is significantly dysregulated in primary tumors. To examine the biological functions of miR-199a, we established stably transfected miR-199a cells (NT2-199a).We investigated the ability of NT2-199a cells to form colonies in soft agar medium. Cells of NT2-199a formed colonies similar to NT2-GFP cells. It was also demonstrated to inhibit cell proliferation. These data suggest that miR-199a may act as an anti-metastasis but not a tumor suppressor miRNA. Bioinformatic analysis on microarray expression data on Ntera-2 cells suggested podocalyxin-like (PODXL) might be one of the targets of miR-199a, which were significantly up-regulated in testicular cancer cell line. Transient transfection of miR-199a into Ntera-2 cells or stable expression of miR-199a suppressed the mRNA and protein level of PODXL. In addition, demethylation treatment of Ntera-2 cells by 5-aza restored expression of miR-199a and decreased PODXL expression. These data support that miR-199a-5p is a negative regulator of PODXL. Sequence analysis revealed two seed sequences of miR-199a in the 3-UTR of PODXL. One of them, denoted as UTR-A, is a highly conserved site in mammals. We cloned the two franking seed sequences into luciferase reporter vector. Co-transfection of luciferase reporter vector with miR-199a-5p significantly suppressed the activity of luciferase that carries the UTR-A site. miR-199a-5p has a minor effect on the non-conserved UTR-B site. Mutation of the seed sequence of UTR-A resulted in loss of suppression function of miR-199a-5p. These data indicate that miR-199a-5p suppresses PODXL through the conserved seed sequence in the 3-UTR.

男性生殖细胞癌中微小RNA的表观遗传失活 人员：Lee，Cheung，Chan，Rennert MicroRNA（miRNAs）是一类小的非编码RNA，在包括癌症在内的多种疾病中表达失调。表观遗传学和miRNA之间的相互交织的联系已经得到最近鉴定的称为“表观-miRNA”的miRNA的特定亚组的支持，所述表观-miRNA可以直接和间接地调节表观遗传机制的活性。利用全基因组方法研究睾丸生殖细胞肿瘤细胞系中的差异甲基化，我们先前在1号染色体上发现了一个新的高甲基化位点。基因组图谱显示，高甲基化区域与microRNA候选物miR-199 a及其上游启动子区域重叠。亚硫酸氢盐测序和用DNA甲基转移酶抑制剂5-aza处理Ntera-2睾丸癌细胞证实了平铺微阵列数据中的相同观察结果。 为了探讨miR-199 s的作用，我们进一步检测了其在几种睾丸胚胎癌细胞系（Ntera-2、833 K、NCCIT、Cates-1B和Tera-1）、培养的正常睾丸细胞（HT）和3种健康男性正常睾丸组织中的甲基化和表达模式。亚硫酸氢盐测序显示，正常培养的睾丸细胞未甲基化，而正常睾丸组织则轻微甲基化。除Cates-1B外，所有被检测的睾丸胚胎癌细胞系均发生甲基化。通过实时qPCR揭示的miR-199 a的表达表明，与正常睾丸细胞系或组织相比，该miRNA在所有癌细胞系中下调。在我们的研究中观察到甲基化与miR-199 a的表达之间呈负相关，表明DNA甲基化是miR-199 a的关键调节因子。miR-199 a的下调不仅限于睾丸癌细胞系，而且在原发性组织中也是如此。我们收集了9例正常睾丸组织、8例胚胎性睾丸癌和9例睾丸腺瘤患者的总RNA，并通过qPCR分析了miR-199 a的表达。标准化后，发现miR-199 a-5 p在胚胎癌和乳腺癌中均显著下调。在分析的病例中，腺瘤中的下调更一致，可能是由于这种低分化癌细胞的更同质性。为了扩大睾丸癌的临床样本量，我们正在用基于LNA的原位杂交分析组织阵列中miR-199 a的表达。组织芯片的结果将有助于我们了解miR-199 a在原发性肿瘤中是否显著失调。 为了研究miR-199 a的生物学功能，我们建立了稳定转染miR-199 a的细胞株（NT 2 - 199 a），观察了NT 2 - 199 a在软琼脂培养基中的集落形成能力。NT 2 - 199 a细胞形成类似于NT 2-GFP细胞的集落。它也被证明抑制细胞增殖。这些数据表明，miR-199 a可能作为一种抗转移，但不是一种肿瘤抑制miRNA。对Ntera-2细胞表达芯片数据的生物信息学分析表明，podocalyxin-like（PODXL）可能是miR-199 a的作用靶点之一，在睾丸癌细胞系中表达显著上调。瞬时转染或稳定表达miR-199 a可抑制PODXL的mRNA和蛋白水平。此外，通过5-aza对Ntera-2细胞进行去甲基化处理恢复了miR-199 a的表达并降低了PODXL的表达。这些数据支持miR-199 a-5 p是PODXL的负调控因子。序列分析显示，PODXL的3-UTR中有两个miR-199 a种子序列。其中之一，表示为UTR-A，是哺乳动物中高度保守的位点。我们将这两个标记种子序列克隆到荧光素酶报告载体中。荧光素酶报告载体与miR-199 a-5 p共转染显著抑制了携带UTR-A位点的荧光素酶的活性。miR-199 a-5 p对非保守UTR-B位点的影响较小。UTR-A种子序列的突变导致miR-199 a-5 p抑制功能的丧失。这些数据表明miR-199 a-5 p通过3-UTR中的保守种子序列抑制PODXL。