综合KDMs的基因组和功能分析确定乳腺癌中可致癌的KDM2A亚型

发布时间：2018-04-03 22:26

本文选题：组蛋白去甲基化酶　切入点：基因扩增　出处：《吉林大学》2015年博士论文

【摘要】：研究背景乳腺癌是来自乳腺终末导管小叶单元上皮的恶性肿瘤。发病率在过去几十年缓慢上升，目前在女性恶性肿瘤中居第一位。常发生于40-60岁的女性。近年来，应用基因芯片技术和免疫组化方法相结合，通过雌激素受体（estrogen receptor，ER）、孕酮受体（progesterone receptor,PR）、人类上皮生长因子（human epidermalgrowth factor2, HER2）和Ki-67的分子检测，将乳腺癌分为5类分子亚型：LuminalA即管腔A型（ER+/PR+，HER2-）；Luminal B即管腔B型（ER+/PR+，HER2+）；HER2过表达型（ER-/PR-，HER2+）；Basal-like即基底样型（ER-/PR-，HER2-，CK5/6+或EGFR+），另外还有一种Normal-like型即正常乳腺样型（ER-/PR-，HER2-，CK5/6-或EGFR-）。其中管腔A型和管腔B型乳腺癌都是雌激素受体（ER）阳性。管腔A型预后较好，相比之下管腔B型预后较差。基底细胞样型乳腺癌具有较高侵袭性，而且复发和转移几率高，预后最差。传统上，乳腺癌的发展被视为基因改变逐步积累的一个复杂的、多因素的过程。然而，，包含组蛋白修饰的表观遗传学的改变在癌症发生和发展中的基础作用越来越明显。组蛋白赖氨酸去甲基化酶共有24种KDMs。这些酶能催化组蛋白和其他蛋白的赖氨酸残基特异位点的去甲基化。它们在控制转录、染色质结构和表观遗传学信号传导中起着关键的作用。在结构上，KDMs能被大致分成两种功能酶家族。第一个家族，包括两个成员（KDM1A/LSD1和KDM1B/LSD2），只能通过胺基氧化反应移除组蛋白的单甲基化和二甲基化（me1/me2）的赖氨酸残基。第二个家族，简称为JumonjiC(Jmjc)结构域去甲基化酶，用一种加氧酶机制去除单甲基、二甲基和三甲基化（me1/me2/me3）的赖氨酸残基。在人体中，有32种蛋白酶属于Jmjc结构域蛋白家族，其中，22种蛋白酶已经被证实在体内和体外都能使组蛋白赖氨酸残基去甲基化。根据序列的同源性和结构的相似性，这22种Jmjc结构域去甲基化酶能被分成7种功能上不同的亚家族（KDM2-8）。值得注意的是，Jmjc去甲基化酶的每个亚家族能抑制不同组蛋白赖氨酸残基的不同的特异底物。例如， KDM2亚家族特异地底物是H3K36me2/me1， KDM4亚家族底物是H3K9me3/me2和H3K36me3/me2，KDM5亚家族底物是H3K4me3/me2。通常，H3K4和H3K36标记与基因活化相关，H3K9和H3K27标记与基因抑制相关联。鉴于KDM2A底物的特异甲基标记和基因转录状态之间的相关性，KDMs的活化能导致不同转录和生物结果。大量的证据表明，在癌细胞中基因和表观遗传事件执行中是相互依存的。多种数据显示KDMs的基因改变和失调与乳腺癌相关。KDM4C基因，最初被命名为GASC1(基因扩增在鳞状细胞癌1)，是从食管癌细胞系的9p24扩增区域克隆的。我们之前的研究已证明KDM4C在侵袭性的基底细胞样型乳腺癌中显著地扩增和过表达并且具有致癌基因的作用。KDM5A在乳腺癌中也是扩增和过表达并且与药物抵抗表型相关。然而，这些KDMs在乳腺癌中的基因组变化、与临床预后的相关性及相关分子机制仍不明。相关知识的缺乏对开发新的治疗性抑制剂是一个重大的阻碍。新的治疗性抑制剂能选择性地靶向这些组蛋白修饰酶，而这可为不同乳腺癌类型的患者提供显著地益处。本研究综合了KDMs在乳腺癌中的基因组、临床和功能分析并确定了KDM2A中具有维持其恶性表型的亚型。方法和结果 1．通过对TCGA数据库中976例乳腺癌标本中的24种KDMs进行了Meta分析，确定DNA拷贝数改变、mRNA表达水平、乳腺癌亚型和临床预后之间的相关性。结果显示与其他KDMs相比较，KDM2A和KDM5B以不依赖亚型的方式具有较高频率的基因扩增和过表达。KDM5B和KDM2A分别在HER2+过表达型和管腔B型乳腺癌中具有较高频率的基因扩增。另外，结果表明KDM2A的mRNA高表达与乳腺癌患者的较短生存期之间有显著地相关性。 2．在乳腺癌细胞系中，我们通过比较基因组杂交阵列数据分析、qRT-PCR和Western Blot的方法发现KDM2A的短亚型在DNA，mRNA和蛋白水平上含量均多于其长亚型。另外，我们用MTT、软琼脂克隆实验、侵袭实验等证实在具有KDM2A过表达的ZR75-1和T47D乳腺癌细胞中沉默KDM2A后可抑制细胞的增殖，但在非致瘤性的永生化的乳腺上皮细胞MCF10A中沉默KDM2A反而促进细胞增殖，在没有KDM2A扩增和过表达的SUM52和SUM225细胞中沉默KDM2A对细胞生长没有明显地影响。 3．KDM2A有两个亚型：长亚型和短亚型。我们用特异的siRNA转染乳腺癌细胞分别地沉默KDM2A的长亚型和短亚型。结果显示，KDM2A的短亚型具有维持乳腺癌恶性表型的作用，而长亚型没有这种作用。 4．在乳腺癌细胞系ZR75-1中，沉默KDM2A后提取RNA后进行了RNA测序，并通过qRT-PCR验证，发现了几个KDM2A潜在的靶标基因如BRAF和FABP5。结论 976例乳腺癌标本中KDM2A和KDM5B具有较高频率的基因扩增和过表达。KDM2A的拷贝数和mRNA表达之间具有显著正相关性，并且KDM2A的mRNA高表达与乳腺癌患者的较短生存期有显著地相关性。大部分乳腺癌中KDM2A的短亚型在DNA、mRNA和蛋白水平上含量均多于长亚型。 KDM2A短亚型具有维持乳腺癌恶性表型的作用。
[Abstract]:Research background
Breast cancer is terminal ductal lobular unit from breast epithelial malignant tumors. The incidence rate increased slowly in the past few decades, the current in the female malignant tumors in the first place. Often occurred in a 40-60 year old female. In recent years, combined with the application of gene chip technique and immunohistochemical method, through the estrogen receptor (estrogen receptor. ER), progesterone receptor (progesterone receptor, PR), human epidermal growth factor (human epidermalgrowth, FACTOR2, HER2) molecular detection and Ki-67, will be divided into 5 types of breast cancer molecular subtypes: LuminalA A (ER +/PR+, luminal HER2-); Luminal B (ER+/PR+ HER2+, luminal B); over expression of HER2 (ER-/PR-, HER2+); Basal-like is the base type (ER-/PR-, HER2-, CK5/6+ or EGFR+), in addition to a Normal-like type: normal breast like subtype (ER-/PR-, HER2-, CK5/6- or EGFR-). The lumen type A and luminal B breast cancer Estrogen receptor (ER) positive. The prognosis of luminal A better, compared the prognosis of luminal B is poor. Basal like breast cancer with high invasiveness, recurrence and metastasis and high probability, the worst prognosis. Traditionally, the development of breast cancer is regarded as a gradual accumulation of genetic changes complex. The process of multi factors. However, including histone modifications of epigenetic changes in cancer occurrence and basic role in the development of more and more obvious.
Histone lysine demethylase, a total of 24 KDMs. of these enzymes can catalyze the histones and other proteins of the lysine residue Keith demethylation sites. They are in the control of transcription, chromatin structure and epigenetic genetics plays a key role in signal transduction. In the structure, KDMs can be roughly divided into two kinds of functional enzyme family. The first family, including two members (KDM1A/LSD1 and KDM1B/LSD2), only through the amine oxidation removal of histone methylation and two methylation (me1/me2) lysine residue. Second families, referred to as JumonjiC (Jmjc) domain of demethylation remove the single enzyme, a methyl oxygenase mechanism, two methyl and trimethylation (me1/me2/me3) lysine residues. In humans, there are 32 kinds of proteases belonging to the Jmjc domain protein family, among them, 22 proteases have been shown to have the histone lysine in vivo and in vitro Acid residues of demethylation. According to homology and structural similarity of the sequence, the 22 domain of Jmjc demethylase can be divided into 7 sub family functions on different (KDM2-8). It is worth noting that each subfamily Jmjc demethylase can inhibit different histone lysine acid residues of different specific substrate. For example, the KDM2 subfamily specific substrate is H3K36me2/me1, KDM4 is H3K9me3/me2 and H3K36me3/me2 subfamily substrate, KDM5 subfamily is usually H3K4 H3K4me3/me2. substrate, and H3K36 markers and gene activation, H3K9 and H3K27 markers and gene suppression associated. In view of the correlation between methylation specific markers and gene transcription state KDM2A substrate, the activation of KDMs can lead to different transcription and biological results.
A lot of evidence that genes in cancer cells and epigenetic events in the implementation are interdependent. Many data showed that KDMs gene change and disorder associated with breast cancer.KDM4C gene, was originally named GASC1 (gene amplification in squamous cell carcinoma, 1) from esophagus cancer cell line 9p24 amplified region cloning. Our previous studies have demonstrated that KDM4C in invasive basal like breast carcinoma was amplified and overexpressed and.KDM5A carcinogenic gene in breast cancer is amplified and overexpressed and phenotype associated with drug resistance. However, genomic changes in breast cancer and the correlation between these KDMs. The clinical prognosis and the related molecular mechanism is still unknown. The lack of related knowledge on the development of new therapeutic inhibitors is one of the major obstacles. Treatment of new inhibitors that selectively target these histone modification With enzyme, which can provide significant benefits for different types of breast cancer patients. The study of KDMs in breast cancer genome analysis and clinical function were determined with the maintenance of the malignant phenotype of KDM2A subtype.
Methods and results
1. of the 24 kinds of KDMs TCGA database in 976 cases of breast cancer specimens were analyzed by Meta, determine the DNA copy number changes, the expression level of mRNA, the correlation between the subtypes of breast cancer and clinical prognosis. The results showed that compared with other KDMs, KDM2A and KDM5B do not depend on the subtypes of type with high frequency the gene amplification and overexpression of.KDM5B and KDM2A with high frequency and expression of luminal B breast cancer gene respectively in HER2+ amplification. In addition, the results show that there are significant correlation between the short period of survival and the high expression of KDM2A and mRNA in patients with breast cancer.
In 2. breast cancer cell lines, we through array comparative genomic hybridization data analysis method, qRT-PCR and Western Blot found that KDM2A short isoforms in DNA, mRNA and protein levels were more than the long isoform. In addition, we use MTT, soft agar cloning experiments, invasion experiments confirmed in proliferation the silence of KDM2A could inhibit ZR75-1 cells and T47D breast cancer cells KDM2A expression in mammary epithelial cells but MCF10A tumorigenicity in non immortalized in silence but KDM2A promote cell proliferation in the absence of KDM2A amplification and overexpression of SUM52 and SUM225 cells in KDM2A silencing on cell growth was not significantly affected.
3.KDM2A has two isoforms: long isoform and short long isoform subtypes. We respectively used to silence KDM2A specific siRNA was transfected into breast cancer cells and short isoforms. The results showed that the short isoform of KDM2A can maintain the malignant phenotype of breast cancer, while the long isoform was without effect.
In 4. breast cancer cell lines ZR75-1, KDM2A silencing RNA was extracted for RNA sequencing, and verified by qRT-PCR, KDM2A found several potential target genes such as BRAF and FABP5.
conclusion
KDM2A and KDM5B in 976 cases of breast cancer specimens with high frequency of gene amplification and overexpression of.KDM2A copy number and mRNA had a significant positive correlation between the expression of KDM2A, and the high expression of mRNA was significantly associated with breast cancer patients with shorter survival. Most of the KDM2A in breast cancer subtypes in short DNA, mRNA and the protein level were more than the long isoform. KDM2A short isoforms have maintained the malignant phenotype of breast cancer.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

【共引文献】