驱动蛋白样DNA结合蛋白下游调节基因的筛选与验证

发布时间：2018-02-28 21:33

本文关键词： 乳腺癌转录调节驱动蛋白样DNA结合蛋白染色质免疫沉淀启动子芯片转录激活因子协调蛋白细胞分裂周期素25C　出处：《天津医科大学》2008年硕士论文　论文类型：学位论文

【摘要】：背景：驱动蛋白样DNA结合蛋白Kid属于驱动蛋白家族(kinesin family, KIF)。Kid分子结构中N端的微管驱动区和ATP酶活性区及C端的DNA结合区和核定位区决定了该蛋白既具有Kinesin的微管驱动功能,又具有DNA结合蛋白(DNA binding protein, DBP)的转录因子功能。Kid作为微管驱动蛋白在有丝分裂的整个生物学过程中都起着关键作用,参与纺锤体的形成、染色体的运动以及调节细胞进入有丝分裂的末期；另外,Kid作为转录调节因子可能调节癌基因ErbB-2的表达。目前,国外关于Kid的研究仅限于对其分子结构和基于分子结构的功能描述,关于Kid在肿瘤发生发展中的作用机制尚无研究报道。本研究室前期研究发现Kid的编码基因kinesin-like4(KNSL4)在乳腺癌的淋巴结转移癌中的表达较其配对原发癌下调,KNSL4mRNA在乳腺原发癌中的下调与乳腺癌患者差的预后相关,提示Kid在乳腺癌的发生发展中发挥重要作用。目的：通过筛选Kid下游的转录调节基因,并对筛选得到的候选靶基因进行生物学验证,进一步探讨Kid蛋白作为转录调节因子在乳腺癌发生发展中的作用机制。方法：联合染色质免疫沉淀技术(chromatin immunoprecipitation, ChIP)和启动子基因芯片(microarray)技术(ChIP-on chip),筛选Kid结合的候选靶基因。采用DNA选择性连接(DNA selection and ligation, DSL)的方法标记Kid抗体富集的DNA,筛选其与启动子芯片杂交的荧光强度与无抗体对照比较差异达2倍以上的基因作为候选的Kid调控靶基因;采用ChIP联合实时定量PCR(ChIP-qPCR)方法验证Kid与候选靶基因ATF7IP、CDC25、TM4SF3、PIP的启动子区(-100bp～+400bp)的结合。采用RNA干扰(RNA interference, RNAi)方法分别靶向KNSL4的第二和第三外显子沉默,Kid在乳腺癌细胞MCF-7中的表达。采用实时定量RT-PCR和western blot方法检测Kid的mRNA和蛋白的表达量,筛选Kid低表达的稳定亚克隆,分别命名为MCF-7/exon2-siRNA和MCF-7/exon3-siRNA;采用实时定量RT-PCR (real time RT-PCR)方法比较ATF7IP、CDC25C、TM4SF3、PIP mRNA在MCF-7/exon3-siRNA与对照组MCF-7/vector细胞中mRNA表达量的差异。结果：1.DSL标记方法用于启动子芯片杂交的ChIP-DNA模板量为100ng,与连接介导PCR (ligation mediate PCR, LM-PCR)方法比较,DSL方法显著提高了检测转录因子靶基因的灵敏度,并减少了抗体用量和实验成本。 2.共筛选出287个Kid调控的候选靶基因,其中26个与细胞周期调控相关,27个与信号传导相关,40个与转录调节相关,17个与细胞黏附相关,16个与细胞运动相关。ATF7IP基因在Kid抗体免疫沉淀DNA中的富集比无抗体免疫沉淀的阴性对照高4.8倍,CDC25C为8.3倍,PIP为11倍,TM4SF3为27.9倍。 3.实时定量PCR检测ChIP的实验样本与阴性对照样本中ATF7IP、PIP CDC25C及TM4SF3启动子区扩增产物的差异,结果在实验样本中ATF7IP扩增产物比阴性对照高4.9倍,CDC25C为10倍,PIP为3倍,TM4SF3为11.8倍。 4.与MCF-7/vector比较,MCF-7/exon3-siRNA中的KNSL4mRNA下调80%,Kid蛋白下调68%。MCF-7/exon2-siRNA中CNSL4mRNA下调76%,Kid蛋白下调52%。 5.与MCF-7/vecto·比较,MCF-7/exon3-siRNA细胞中ATF7IP mRNA表达上调3.5倍,CDC25C mRNA表达上调10倍,PIP和TM4SF3在MCF-7/vector和MCF-7/exon3-siRNA细胞中均无表达。结论：1.本研究采用DSL标记法可以减少用于芯片杂交的ChIP-DNA模板量,检测灵敏度高于]LM-PCR标记法。 2.本研究建立了Kid低表达的MCF-7细胞亚克隆。 3.Kid蛋白对ATF7IP和CDC25C基因的转录具有负向调节作用,Kid可能通过调节其靶基因的转录进而影响乳腺癌的生物学行为。
[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.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R3416

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