本文選題：組蛋白去甲基化酶　切入點：基因擴(kuò)增　出處：《吉林大學(xué)》2015年博士論文

【摘要】：研究背景 乳腺癌是來自乳腺終末導(dǎo)管小葉單元上皮的惡性腫瘤。發(fā)病率在過去幾十年緩慢上升，目前在女性惡性腫瘤中居第一位。常發(fā)生于40-60歲的女性。近年來，應(yīng)用基因芯片技術(shù)和免疫組化方法相結(jié)合，通過雌激素受體（estrogen receptor，ER）、孕酮受體（progesterone receptor,PR）、人類上皮生長因子（human epidermalgrowth factor2, HER2）和Ki-67的分子檢測，將乳腺癌分為5類分子亞型：LuminalA即管腔A型（ER+/PR+，HER2-）；Luminal B即管腔B型（ER+/PR+，HER2+）；HER2過表達(dá)型（ER-/PR-，HER2+）；Basal-like即基底樣型（ER-/PR-，HER2-，CK5/6+或EGFR+），另外還有一種Normal-like型即正常乳腺樣型（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體（ER）陽性。管腔A型預(yù)后較好，相比之下管腔B型預(yù)后較差�；准�(xì)胞樣型乳腺癌具有較高侵襲性，而且復(fù)發(fā)和轉(zhuǎn)移幾率高，預(yù)后最差。傳統(tǒng)上，乳腺癌的發(fā)展被視為基因改變逐步積累的一個復(fù)雜的、多因素的過程。然而，，包含組蛋白修飾的表觀遺傳學(xué)的改變在癌癥發(fā)生和發(fā)展中的基礎(chǔ)作用越來越明顯。 組蛋白賴氨酸去甲基化酶共有24種KDMs。這些酶能催化組蛋白和其他蛋白的賴氨酸殘基特異位點的去甲基化。它們在控制轉(zhuǎn)錄、染色質(zhì)結(jié)構(gòu)和表觀遺傳學(xué)信號傳導(dǎo)中起著關(guān)鍵的作用。在結(jié)構(gòu)上，KDMs能被大致分成兩種功能酶家族。第一個家族，包括兩個成員（KDM1A/LSD1和KDM1B/LSD2），只能通過胺基氧化反應(yīng)移除組蛋白的單甲基化和二甲基化（me1/me2）的賴氨酸殘基。第二個家族，簡稱為JumonjiC(Jmjc)結(jié)構(gòu)域去甲基化酶，用一種加氧酶機(jī)制去除單甲基、二甲基和三甲基化（me1/me2/me3）的賴氨酸殘基。在人體中，有32種蛋白酶屬于Jmjc結(jié)構(gòu)域蛋白家族，其中，22種蛋白酶已經(jīng)被證實在體內(nèi)和體外都能使組蛋白賴氨酸殘基去甲基化。根據(jù)序列的同源性和結(jié)構(gòu)的相似性，這22種Jmjc結(jié)構(gòu)域去甲基化酶能被分成7種功能上不同的亞家族（KDM2-8）。值得注意的是，Jmjc去甲基化酶的每個亞家族能抑制不同組蛋白賴氨酸殘基的不同的特異底物。例如， KDM2亞家族特異地底物是H3K36me2/me1， KDM4亞家族底物是H3K9me3/me2和H3K36me3/me2，KDM5亞家族底物是H3K4me3/me2。通常，H3K4和H3K36標(biāo)記與基因活化相關(guān)，H3K9和H3K27標(biāo)記與基因抑制相關(guān)聯(lián)。鑒于KDM2A底物的特異甲基標(biāo)記和基因轉(zhuǎn)錄狀態(tài)之間的相關(guān)性，KDMs的活化能導(dǎo)致不同轉(zhuǎn)錄和生物結(jié)果。 大量的證據(jù)表明，在癌細(xì)胞中基因和表觀遺傳事件執(zhí)行中是相互依存的。多種數(shù)據(jù)顯示KDMs的基因改變和失調(diào)與乳腺癌相關(guān)。KDM4C基因，最初被命名為GASC1(基因擴(kuò)增在鱗狀細(xì)胞癌1)，是從食管癌細(xì)胞系的9p24擴(kuò)增區(qū)域克隆的。我們之前的研究已證明KDM4C在侵襲性的基底細(xì)胞樣型乳腺癌中顯著地擴(kuò)增和過表達(dá)并且具有致癌基因的作用。KDM5A在乳腺癌中也是擴(kuò)增和過表達(dá)并且與藥物抵抗表型相關(guān)。然而，這些KDMs在乳腺癌中的基因組變化、與臨床預(yù)后的相關(guān)性及相關(guān)分子機(jī)制仍不明。相關(guān)知識的缺乏對開發(fā)新的治療性抑制劑是一個重大的阻礙。新的治療性抑制劑能選擇性地靶向這些組蛋白修飾酶，而這可為不同乳腺癌類型的患者提供顯著地益處。本研究綜合了KDMs在乳腺癌中的基因組、臨床和功能分析并確定了KDM2A中具有維持其惡性表型的亞型。 方法和結(jié)果 1．通過對TCGA數(shù)據(jù)庫中976例乳腺癌標(biāo)本中的24種KDMs進(jìn)行了Meta分析，確定DNA拷貝數(shù)改變、mRNA表達(dá)水平、乳腺癌亞型和臨床預(yù)后之間的相關(guān)性。結(jié)果顯示與其他KDMs相比較，KDM2A和KDM5B以不依賴亞型的方式具有較高頻率的基因擴(kuò)增和過表達(dá)。KDM5B和KDM2A分別在HER2+過表達(dá)型和管腔B型乳腺癌中具有較高頻率的基因擴(kuò)增。另外，結(jié)果表明KDM2A的mRNA高表達(dá)與乳腺癌患者的較短生存期之間有顯著地相關(guān)性。 2．在乳腺癌細(xì)胞系中，我們通過比較基因組雜交陣列數(shù)據(jù)分析、qRT-PCR和Western Blot的方法發(fā)現(xiàn)KDM2A的短亞型在DNA，mRNA和蛋白水平上含量均多于其長亞型。另外，我們用MTT、軟瓊脂克隆實驗、侵襲實驗等證實在具有KDM2A過表達(dá)的ZR75-1和T47D乳腺癌細(xì)胞中沉默KDM2A后可抑制細(xì)胞的增殖，但在非致瘤性的永生化的乳腺上皮細(xì)胞MCF10A中沉默KDM2A反而促進(jìn)細(xì)胞增殖，在沒有KDM2A擴(kuò)增和過表達(dá)的SUM52和SUM225細(xì)胞中沉默KDM2A對細(xì)胞生長沒有明顯地影響。 3．KDM2A有兩個亞型：長亞型和短亞型。我們用特異的siRNA轉(zhuǎn)染乳腺癌細(xì)胞分別地沉默KDM2A的長亞型和短亞型。結(jié)果顯示，KDM2A的短亞型具有維持乳腺癌惡性表型的作用，而長亞型沒有這種作用。 4．在乳腺癌細(xì)胞系ZR75-1中，沉默KDM2A后提取RNA后進(jìn)行了RNA測序，并通過qRT-PCR驗證，發(fā)現(xiàn)了幾個KDM2A潛在的靶標(biāo)基因如BRAF和FABP5。 結(jié)論 976例乳腺癌標(biāo)本中KDM2A和KDM5B具有較高頻率的基因擴(kuò)增和過表達(dá)。KDM2A的拷貝數(shù)和mRNA表達(dá)之間具有顯著正相關(guān)性，并且KDM2A的mRNA高表達(dá)與乳腺癌患者的較短生存期有顯著地相關(guān)性。大部分乳腺癌中KDM2A的短亞型在DNA、mRNA和蛋白水平上含量均多于長亞型。 KDM2A短亞型具有維持乳腺癌惡性表型的作用。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.9

【共引文獻(xiàn)】

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