本文關(guān)鍵詞： 乳腺癌 轉(zhuǎn)錄調(diào)節(jié) 驅(qū)動蛋白樣DNA結(jié)合蛋白 染色質(zhì)免疫沉淀 啟動子芯片 轉(zhuǎn)錄激活因子協(xié)調(diào)蛋白 細胞分裂周期素25C　出處：《天津醫(yī)科大學》2008年碩士論文　論文類型：學位論文

【摘要】：背景：驅(qū)動蛋白樣DNA結(jié)合蛋白Kid屬于驅(qū)動蛋白家族(kinesin family, KIF)。Kid分子結(jié)構(gòu)中N端的微管驅(qū)動區(qū)和ATP酶活性區(qū)及C端的DNA結(jié)合區(qū)和核定位區(qū)決定了該蛋白既具有Kinesin的微管驅(qū)動功能,又具有DNA結(jié)合蛋白(DNA binding protein, DBP)的轉(zhuǎn)錄因子功能。Kid作為微管驅(qū)動蛋白在有絲分裂的整個生物學過程中都起著關(guān)鍵作用,參與紡錘體的形成、染色體的運動以及調(diào)節(jié)細胞進入有絲分裂的末期；另外,Kid作為轉(zhuǎn)錄調(diào)節(jié)因子可能調(diào)節(jié)癌基因ErbB-2的表達。目前,國外關(guān)于Kid的研究僅限于對其分子結(jié)構(gòu)和基于分子結(jié)構(gòu)的功能描述,關(guān)于Kid在腫瘤發(fā)生發(fā)展中的作用機制尚無研究報道。本研究室前期研究發(fā)現(xiàn)Kid的編碼基因kinesin-like4(KNSL4)在乳腺癌的淋巴結(jié)轉(zhuǎn)移癌中的表達較其配對原發(fā)癌下調(diào),KNSL4mRNA在乳腺原發(fā)癌中的下調(diào)與乳腺癌患者差的預后相關(guān),提示Kid在乳腺癌的發(fā)生發(fā)展中發(fā)揮重要作用。 目的：通過篩選Kid下游的轉(zhuǎn)錄調(diào)節(jié)基因,并對篩選得到的候選靶基因進行生物學驗證,進一步探討Kid蛋白作為轉(zhuǎn)錄調(diào)節(jié)因子在乳腺癌發(fā)生發(fā)展中的作用機制。 方法：聯(lián)合染色質(zhì)免疫沉淀技術(shù)(chromatin immunoprecipitation, ChIP)和啟動子基因芯片(microarray)技術(shù)(ChIP-on chip),篩選Kid結(jié)合的候選靶基因。采用DNA選擇性連接(DNA selection and ligation, DSL)的方法標記Kid抗體富集的DNA,篩選其與啟動子芯片雜交的熒光強度與無抗體對照比較差異達2倍以上的基因作為候選的Kid調(diào)控靶基因;采用ChIP聯(lián)合實時定量PCR(ChIP-qPCR)方法驗證Kid與候選靶基因ATF7IP、CDC25、TM4SF3、PIP的啟動子區(qū)(-100bp～+400bp)的結(jié)合。 采用RNA干擾(RNA interference, RNAi)方法分別靶向KNSL4的第二和第三外顯子沉默,Kid在乳腺癌細胞MCF-7中的表達。采用實時定量RT-PCR和western blot方法檢測Kid的mRNA和蛋白的表達量,篩選Kid低表達的穩(wěn)定亞克隆,分別命名為MCF-7/exon2-siRNA和MCF-7/exon3-siRNA;采用實時定量RT-PCR (real time RT-PCR)方法比較ATF7IP、CDC25C、TM4SF3、PIP mRNA在MCF-7/exon3-siRNA與對照組MCF-7/vector細胞中mRNA表達量的差異。 結(jié)果：1.DSL標記方法用于啟動子芯片雜交的ChIP-DNA模板量為100ng,與連接介導PCR (ligation mediate PCR, LM-PCR)方法比較,DSL方法顯著提高了檢測轉(zhuǎn)錄因子靶基因的靈敏度,并減少了抗體用量和實驗成本。 2.共篩選出287個Kid調(diào)控的候選靶基因,其中26個與細胞周期調(diào)控相關(guān),27個與信號傳導相關(guān),40個與轉(zhuǎn)錄調(diào)節(jié)相關(guān),17個與細胞黏附相關(guān),16個與細胞運動相關(guān)。ATF7IP基因在Kid抗體免疫沉淀DNA中的富集比無抗體免疫沉淀的陰性對照高4.8倍,CDC25C為8.3倍,PIP為11倍,TM4SF3為27.9倍。 3.實時定量PCR檢測ChIP的實驗樣本與陰性對照樣本中ATF7IP、PIP CDC25C及TM4SF3啟動子區(qū)擴增產(chǎn)物的差異,結(jié)果在實驗樣本中ATF7IP擴增產(chǎn)物比陰性對照高4.9倍,CDC25C為10倍,PIP為3倍,TM4SF3為11.8倍。 4.與MCF-7/vector比較,MCF-7/exon3-siRNA中的KNSL4mRNA下調(diào)80%,Kid蛋白下調(diào)68%。MCF-7/exon2-siRNA中CNSL4mRNA下調(diào)76%,Kid蛋白下調(diào)52%。 5.與MCF-7/vecto·比較,MCF-7/exon3-siRNA細胞中ATF7IP mRNA表達上調(diào)3.5倍,CDC25C mRNA表達上調(diào)10倍,PIP和TM4SF3在MCF-7/vector和MCF-7/exon3-siRNA細胞中均無表達。 結(jié)論：1.本研究采用DSL標記法可以減少用于芯片雜交的ChIP-DNA模板量,檢測靈敏度高于]LM-PCR標記法。 2.本研究建立了Kid低表達的MCF-7細胞亞克隆。 3.Kid蛋白對ATF7IP和CDC25C基因的轉(zhuǎn)錄具有負向調(diào)節(jié)作用,Kid可能通過調(diào)節(jié)其靶基因的轉(zhuǎn)錄進而影響乳腺癌的生物學行為。
[Abstract]:Background: kinesin like DNA binding protein Kid belongs to the kinesin family (kinesin family, KIF) in the molecular structure of.Kid N end of the microtubule driving region and the activity of ATP and C terminal DNA binding region and nuclear localization region determines the protein is Kinesin and has driven microtubule function, DNA binding protein (DNA binding protein, DBP) the function of transcription factor.Kid in biology as a microtubule kinesin throughout the process of mitosis in plays a key role in the formation of the mitotic spindle, chromosome movement and regulation of cells into the telophase of mitosis; in addition, Kid as a transcription factor may regulate the expression of oncogene ErbB-2. At present, the research on the foreign Kid only on the molecular structure and molecular structure based on the function description about Kid in tumor development mechanism has not been reported. In our previous work. The discovery of the gene encoding kinesin-like4 Kid (KNSL4) expression in cancer metastasis is the primary cancer paired down regulated in breast cancer lymph KNSL4mRNA in primary breast cancer in the prognosis of patients with breast cancer by poor correlation, suggesting that Kid may play an important role in the development of breast cancer.
Objective: to screen transcriptional regulatory genes in downstream of Kid, and to screen candidate target genes for biological validation, and further explore the role of Kid protein as a transcription regulator in the occurrence and development of breast cancer.
Methods: combined chromatin immunoprecipitation (chromatin immunoprecipitation, ChIP) and the promoter of gene chip (microarray) technology (ChIP-on chip), screening of candidate target genes of Kid binding. Connected by DNA (DNA selection and ligation, selective DSL) method of labeled Kid antibody screening and enrichment of DNA, the fluorescence intensity of starting sub chip hybridization with no antibody control compared to more than 2 times the regulation of Kid gene as the candidate target genes; using ChIP combined with real-time quantitative PCR (ChIP-qPCR) method to verify the Kid and candidate target genes ATF7IP, CDC25, TM4SF3, PIP promoter (-100bp ~ +400bp) combination.
Using RNA interference (RNA interference RNAi) method respectively targeting KNSL4 second and exon third of silence, the expression of Kid in breast cancer MCF-7 cells. The expression of mRNA and protein by real-time quantitative RT-PCR and Western blot method to detect Kid, screening of Kid low expression of stable subclones were named as MCF-7/exon2-siRNA and MCF-7/exon3-siRNA; using quantitative real-time RT-PCR (real time RT-PCR) ATF7IP CDC25C, TM4SF3 method, PIP, mRNA in MCF-7/exon3-siRNA and control group mRNA expression difference in MCF-7/vector cells.
Results: the 1.DSL labeling method used for ChIP-DNA hybridization of promoters was 100ng. Compared with the method of PCR (ligation mediate PCR, LM-PCR), DSL method significantly improved the sensitivity of detecting target genes of transcription factors, and reduced the dosage of antibody and the cost of experiment.
A total of 2. screened 287 candidate target genes regulated by Kid, 26 of them associated with cell cycle regulation and signal transduction, 27, 40 and 17 and the related transcription regulation, cell adhesion, cell motility and 16 related gene.ATF7IP in DNA enriched than negative control antibody immunoprecipitation 4.8 times more precipitation in the Kid antibody, CDC25C 8.3 times, PIP 11 times, TM4SF3 27.9 times.
3., real-time quantitative PCR detection of ChIP in experimental samples and negative control samples showed differences in ATF7IP, PIP CDC25C and TM4SF3 promoter products. Results ATF7IP amplification products in experimental samples were 4.9 times higher than those in negative controls, CDC25C was 10 times, PIP was 3 times, TM4SF3 was 11.8 times.
4. compared with MCF-7/vector, KNSL4mRNA in MCF-7/exon3-siRNA decreased by 80%, Kid protein downregulated 68%.MCF-7/exon2-siRNA in 68%.MCF-7/exon2-siRNA by 76%, and Kid protein decreased 52%.
5. compared with MCF-7/vecto, the expression of ATF7IP mRNA in MCF-7/exon3-siRNA cells was increased by 3.5 times, the expression of CDC25C mRNA increased by 10 times, PIP and TM4SF3 were not expressed in MCF-7/vector and MCF-7/exon3-siRNA cells.
Conclusion: 1. the use of DSL labeling method can reduce the amount of ChIP-DNA template used in chip hybridization, and the sensitivity of detection is higher than that of]LM-PCR marker.
2. the low expression of Kid subcloning of MCF-7 cells was established in this study.
3.Kid protein plays a negative regulatory role in the transcription of ATF7IP and CDC25C genes. Kid may affect the biological behavior of breast cancer by regulating the transcription of its target genes.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R3416

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本文編號：1549090