本文選題：PRR11 + SKA2　； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：腫瘤相關(guān)基因PRR11(proline-rich 11)和SKA2(spindle and kinetochore associated complex subunit 2),均定位于染色體17q22區(qū),兩者共享一個獨特的雙向啟動子,組成一個獨特的轉(zhuǎn)錄單元,是一個典型的“頭對頭”“基因?qū)Α?head-to-head gene pair)。我們課題組在前期研究中發(fā)現(xiàn),肺癌細胞中,PRR11和SKA2受到轉(zhuǎn)錄因子NF-Y和p53的調(diào)控,并與細胞周期調(diào)控和腫瘤發(fā)生發(fā)展均密切相關(guān)。肺癌組織中,PRR11和SKA2基因表達水平均顯著升高,且高水平的基因表達與肺癌病人的預(yù)后水平顯著負相關(guān);此外在肺癌細胞中,PRR11和SKA2的敲降能夠顯著抑制細胞的增殖、遷移、侵襲等生理過程。在此基礎(chǔ)上,本研究將對PRR11-SKA2“基因?qū)Α痹谌橄侔┲械墓δ芗氨磉_調(diào)控機制進行初步探索。(1)PRR11-SKA2“基因?qū)Α迸c乳腺癌預(yù)后的關(guān)聯(lián)性分析利用GEO數(shù)據(jù)庫,獲得乳腺癌芯片GSE3494及GSE4922中PRR11和SKA2的表達信息以及預(yù)后數(shù)據(jù),分析PRR11和SKA2在乳腺癌中的預(yù)后價值。即采用生物信息學(xué)方法,分析PRR11及SKA2的表達水平與乳腺癌預(yù)后的關(guān)聯(lián)性。結(jié)果表明,PRR11與SKA2低表達組的病人生存期明顯高于高表達組。(2)抑制PRR11與SKA2的表達對乳腺癌細胞增殖、遷移及侵襲能力的影響采用si RNA干擾技術(shù),分別在MCF-7、MDA-MB-231細胞中將PRR11與SKA2單獨沉默或聯(lián)合沉默。細胞表型分析結(jié)果表明,在兩種乳腺癌細胞系中,與陰性對照組細胞相比,PRR11與SKA2單獨沉默組和聯(lián)合沉默組細胞的增殖、遷移及侵襲能力均明顯降低;與單獨沉默組細胞相比,PRR11與SKA2聯(lián)合沉默后,細胞遷移及侵襲能力的降低程度更為顯著。由此可以推測,PRR11與SKA2在乳腺癌的發(fā)生發(fā)展中起到重要作用,且兩者在功能上可能具有一定的協(xié)同或互補效應(yīng)。定量RT-PCR驗證結(jié)果分析表明,PRR11與SKA2單獨沉默或聯(lián)合沉默后,多個與細胞增殖、遷移或侵襲相關(guān)基因的表達表現(xiàn)出不同程度的變化。該結(jié)果提示,PRR11與SKA2有可能通過影響這些基因的表達變化參與乳腺癌的發(fā)生發(fā)展過程。(3)NF-Y對PRR11-SKA2“基因?qū)Α钡霓D(zhuǎn)錄調(diào)控分析以前期構(gòu)建的啟動子熒光素酶報告基因重組體為模板,構(gòu)建不同長度的PRR11-SKA2啟動子截短突變重組體,將這些重組體單獨轉(zhuǎn)染或與NFYB真核表達質(zhì)粒共轉(zhuǎn)入MCF-7細胞,并檢測各重組體啟動子活性。分析結(jié)果發(fā)現(xiàn),PRR11-SKA2啟動子在乳腺癌細胞中具有雙向啟動子活性,NFYB對PRR11和SKA2方向的轉(zhuǎn)錄無明顯驅(qū)動作用;但是,在乳腺癌細胞中干擾NFYB表達,用定量RT-PCR檢測PRR11及SKA2的m RNA表達水平,結(jié)果發(fā)現(xiàn)NFYB基因沉默后可導(dǎo)致PRR11的轉(zhuǎn)錄下調(diào),但不影響SKA2的表達;進一步采用生物信息學(xué)方法分析乳腺癌組織中NFYB與PRR11-SKA2表達的相關(guān)性,發(fā)現(xiàn)NFYB與PRR11的表達相關(guān),而與SKA2的表達不相關(guān)。(4)轉(zhuǎn)錄因子p53對PRR11-SKA2“基因?qū)Α钡霓D(zhuǎn)錄調(diào)控及臨床意義分析將PRR11-SKA2啟動子報告基因系列截短體分別單獨轉(zhuǎn)染或與p53真核表達質(zhì)粒共轉(zhuǎn)染MCF-7細胞,并檢測各重組體啟動子活性。實驗結(jié)果顯示,p53能夠顯著抑制各截短體的啟動子活性。乳腺癌芯片數(shù)據(jù)分析發(fā)現(xiàn),與野生型p53組相比,突變型p53組乳腺癌組織中PRR11及SKA2的表達顯著升高。p53與PRR11-SKA2“基因?qū)Α钡穆?lián)合預(yù)后分析發(fā)現(xiàn),p53未突變、PRR11及SKA2低表達的病人生存期,明顯高于p53突變、PRR11及SKA2高表達的病人。綜上所述,本研究發(fā)現(xiàn)PRR11-SKA2“基因?qū)Α钡谋磉_與乳腺癌的預(yù)后負相關(guān),可作為乳腺癌預(yù)后因子,PRR11-SKA2“基因?qū)Α痹谌橄侔┑陌l(fā)生發(fā)展過程中具有重要作用,且轉(zhuǎn)錄因子NF-Y、p53可調(diào)控PRR11-SKA2“基因?qū)Α钡谋磉_。本研究豐富了對PRR11-SKA2“基因?qū)Α钡恼J識,為深入探索該“基因?qū)Α痹谀[瘤發(fā)生發(fā)展的作用和表達調(diào)控機制奠定了堅實的基礎(chǔ),對其在乳腺癌的分子診斷與靶向治療中的潛在應(yīng)用具有積極的理論和現(xiàn)實意義。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9

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本文編號：1819337