PRR11-SKA2“基因对”在乳腺癌中的功能及表达调控机制初探

发布时间：2018-04-29 09:23

本文选题：PRR11 + SKA2　；参考：《重庆医科大学》2017年硕士论文

【摘要】：肿瘤相关基因PRR11(proline-rich 11)和SKA2(spindle and kinetochore associated complex subunit 2),均定位于染色体17q22区,两者共享一个独特的双向启动子,组成一个独特的转录单元,是一个典型的“头对头”“基因对”(head-to-head gene pair)。我们课题组在前期研究中发现,肺癌细胞中,PRR11和SKA2受到转录因子NF-Y和p53的调控,并与细胞周期调控和肿瘤发生发展均密切相关。肺癌组织中,PRR11和SKA2基因表达水平均显著升高,且高水平的基因表达与肺癌病人的预后水平显著负相关;此外在肺癌细胞中,PRR11和SKA2的敲降能够显著抑制细胞的增殖、迁移、侵袭等生理过程。在此基础上,本研究将对PRR11-SKA2“基因对”在乳腺癌中的功能及表达调控机制进行初步探索。(1)PRR11-SKA2“基因对”与乳腺癌预后的关联性分析利用GEO数据库,获得乳腺癌芯片GSE3494及GSE4922中PRR11和SKA2的表达信息以及预后数据,分析PRR11和SKA2在乳腺癌中的预后价值。即采用生物信息学方法,分析PRR11及SKA2的表达水平与乳腺癌预后的关联性。结果表明,PRR11与SKA2低表达组的病人生存期明显高于高表达组。(2)抑制PRR11与SKA2的表达对乳腺癌细胞增殖、迁移及侵袭能力的影响采用si RNA干扰技术,分别在MCF-7、MDA-MB-231细胞中将PRR11与SKA2单独沉默或联合沉默。细胞表型分析结果表明,在两种乳腺癌细胞系中,与阴性对照组细胞相比,PRR11与SKA2单独沉默组和联合沉默组细胞的增殖、迁移及侵袭能力均明显降低;与单独沉默组细胞相比,PRR11与SKA2联合沉默后,细胞迁移及侵袭能力的降低程度更为显著。由此可以推测,PRR11与SKA2在乳腺癌的发生发展中起到重要作用,且两者在功能上可能具有一定的协同或互补效应。定量RT-PCR验证结果分析表明,PRR11与SKA2单独沉默或联合沉默后,多个与细胞增殖、迁移或侵袭相关基因的表达表现出不同程度的变化。该结果提示,PRR11与SKA2有可能通过影响这些基因的表达变化参与乳腺癌的发生发展过程。(3)NF-Y对PRR11-SKA2“基因对”的转录调控分析以前期构建的启动子荧光素酶报告基因重组体为模板,构建不同长度的PRR11-SKA2启动子截短突变重组体,将这些重组体单独转染或与NFYB真核表达质粒共转入MCF-7细胞,并检测各重组体启动子活性。分析结果发现,PRR11-SKA2启动子在乳腺癌细胞中具有双向启动子活性,NFYB对PRR11和SKA2方向的转录无明显驱动作用;但是,在乳腺癌细胞中干扰NFYB表达,用定量RT-PCR检测PRR11及SKA2的m RNA表达水平,结果发现NFYB基因沉默后可导致PRR11的转录下调,但不影响SKA2的表达;进一步采用生物信息学方法分析乳腺癌组织中NFYB与PRR11-SKA2表达的相关性,发现NFYB与PRR11的表达相关,而与SKA2的表达不相关。(4)转录因子p53对PRR11-SKA2“基因对”的转录调控及临床意义分析将PRR11-SKA2启动子报告基因系列截短体分别单独转染或与p53真核表达质粒共转染MCF-7细胞,并检测各重组体启动子活性。实验结果显示,p53能够显著抑制各截短体的启动子活性。乳腺癌芯片数据分析发现,与野生型p53组相比,突变型p53组乳腺癌组织中PRR11及SKA2的表达显著升高。p53与PRR11-SKA2“基因对”的联合预后分析发现,p53未突变、PRR11及SKA2低表达的病人生存期,明显高于p53突变、PRR11及SKA2高表达的病人。综上所述,本研究发现PRR11-SKA2“基因对”的表达与乳腺癌的预后负相关,可作为乳腺癌预后因子,PRR11-SKA2“基因对”在乳腺癌的发生发展过程中具有重要作用,且转录因子NF-Y、p53可调控PRR11-SKA2“基因对”的表达。本研究丰富了对PRR11-SKA2“基因对”的认识,为深入探索该“基因对”在肿瘤发生发展的作用和表达调控机制奠定了坚实的基础,对其在乳腺癌的分子诊断与靶向治疗中的潜在应用具有积极的理论和现实意义。
[Abstract]:The tumor related genes PRR11 (proline-rich 11) and SKA2 (spindle and kinetochore associated complex subunit 2) are located in the chromosome 17q22 region. They share a unique bi-directional promoter to form a unique transcriptional unit. It is a typical "head pair" "gene pair" (head-to-head gene). In the previous study, PRR11 and SKA2 were regulated by the transcription factor NF-Y and p53, which were closely related to the regulation of cell cycle and the development of tumor. In lung cancer tissues, the expression level of PRR11 and SKA2 genes increased significantly, and the high level of gene expression was negatively correlated with the prognosis of lung cancer patients; moreover, the lung cancer patients were negatively correlated with the prognosis. In cancer cells, the knock down of PRR11 and SKA2 can significantly inhibit cell proliferation, migration, invasion and other physiological processes. On the basis of this, this study will explore the function and expression regulation mechanism of PRR11-SKA2 "gene pair" in breast cancer. (1) the correlation analysis of PRR11-SKA2 "gene pair" with the prognosis of breast cancer uses the GEO database To obtain the expression information of PRR11 and SKA2 in the breast cancer chip GSE3494 and GSE4922 and the prognostic data, analyze the prognostic value of PRR11 and SKA2 in breast cancer. That is, bioinformatics method is used to analyze the correlation between the expression level of PRR11 and SKA2 and the prognosis of breast cancer. The results show that the survival period of patients with low expression of PRR11 and SKA2 is significantly higher than that of SKA2. High expression group. (2) inhibition of the expression of PRR11 and SKA2 on the proliferation, migration and invasion of breast cancer cells using Si RNA interference technique, respectively in MCF-7, MDA-MB-231 cells, PRR11 and SKA2 in separate or combined silence. The results of cell phenotype analysis showed that in the two mammary gland cancer cell lines, PRR11 compared with the negative control group. The proliferation, migration and invasion ability of the cells with SKA2 alone and in the combined silencing group decreased significantly. Compared with the cells in a single silencing group, the cell migration and invasion ability decreased more significantly after the combined silence of PRR11 and SKA2. Thus, it is possible to speculate that PRR11 and SKA2 play an important role in the development of breast cancer. Function may have a certain synergistic or complementary effect. Quantitative RT-PCR verification results show that the expression of multiple cell proliferation, migration or invasion related genes changes in varying degrees after PRR11 and SKA2 are silent or combined. The results suggest that PRR11 and SKA2 may affect the changes in the expression of these genes. Participate in the development process of breast cancer. (3) NF-Y's transcriptional regulation and regulation of PRR11-SKA2 "gene pair" is based on the pre constructed promoter luciferase reporter gene recombinant as a template for the construction of a truncated recombinant of PRR11-SKA2 promoter with different lengths, and these recombinant bodies are transfected alone or with NFYB eukaryotic expression plasmids into MCF-7 The results showed that PRR11-SKA2 promoter had bi-directional promoter activity in breast cancer cells, NFYB had no obvious driving effect on the transcription of PRR11 and SKA2, but NFYB expression was disturbed in breast cancer cells and the m RNA expression of PRR11 and SKA2 was detected by quantitative RT-PCR, and N was found. FYB gene silencing can lead to the downregulation of PRR11, but does not affect the expression of SKA2; further bioinformatics method is used to analyze the correlation between NFYB and PRR11-SKA2 expression in breast cancer tissue, and it is found that NFYB is related to the expression of PRR11, but not related to the expression of SKA2. (4) transcriptional regulation of the transcription factor p53 to PRR11-SKA2 "gene pairs" and In clinical significance analysis, the PRR11-SKA2 promoter reporter gene series truncated or p53 eukaryotic expression plasmid was transfected to MCF-7 cells respectively, and the promoter activity of each recombinant was detected. The experimental results showed that p53 could significantly inhibit the promoter activity of each truncated body. The analysis of breast cancer chip data found that compared with the wild type p53 group, the analysis of the breast cancer chip data was found to be compared with the wild type p53 group. The expression of PRR11 and SKA2 in breast cancer tissues of the mutant p53 group increased significantly. The combined prognosis of.P53 and PRR11-SKA2 "gene pair" found that p53 was not mutated and the survival period of the patients with low expression of PRR11 and SKA2 was significantly higher than that of p53 mutation, PRR11 and SKA2. The prognosis of breast cancer is negatively correlated, which can be used as a prognostic factor in breast cancer. PRR11-SKA2 "gene pair" plays an important role in the development of breast cancer, and the transcription factor NF-Y, p53 can regulate the expression of PRR11-SKA2 "gene pair". This study enriches the understanding of the "gene pair" of PRR11-SKA2 and explores the "gene pair" in depth. It has laid a solid foundation for the role of tumor development and the mechanism of expression and regulation, and has a positive theoretical and practical significance for its potential application in the molecular diagnosis and target therapy of breast cancer.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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