本文關(guān)鍵詞： RNA結(jié)合蛋白QKI 前列腺癌 慢病毒 基因治療　出處：《第四軍醫(yī)大學(xué)》2014年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：前列腺癌（PCa）是在歐美國(guó)家最常見(jiàn)的非皮膚性癌癥，在男性癌癥死亡原因中位居第二。在中國(guó)隨著生活方式的不斷改變前列腺癌的發(fā)病率近年來(lái)也呈現(xiàn)出高增長(zhǎng)的趨勢(shì)[1,2]。目前主要的治療方法是手術(shù)治療，同時(shí)輔助放、化療和內(nèi)分泌治療，治療效果具有局限性。由于前列腺癌具有發(fā)病早期無(wú)明顯癥狀等特點(diǎn)，很多患者被確診時(shí)往往已經(jīng)喪失了最佳的手術(shù)時(shí)機(jī)。突破傳統(tǒng)治療手段的局限性成為目前前列腺癌患者治療的新的挑戰(zhàn)。隨著分子生物學(xué)技術(shù)的不斷進(jìn)步，基因治療在越來(lái)越多的臨床疾病診療中得到了利用，其針對(duì)靶點(diǎn)治療和幾乎無(wú)創(chuàng)傷性的特點(diǎn)能夠顯著的改善現(xiàn)有治療手段的不足。因此探尋更多與前列腺癌相關(guān)的基因，針對(duì)這些基因開(kāi)展靶向治療就成為了前列腺癌治療的一個(gè)新方向。 現(xiàn)今，RNA結(jié)合蛋白和miRNA已經(jīng)被證實(shí)是細(xì)胞生物學(xué)一個(gè)關(guān)鍵的調(diào)節(jié)點(diǎn)，，在轉(zhuǎn)錄后調(diào)控著RNA的穩(wěn)定性、轉(zhuǎn)錄效率、轉(zhuǎn)運(yùn)和剪切。mRNA調(diào)節(jié)的缺失也被證實(shí)和許多人類(lèi)疾病密切相關(guān)，其中就包括癌癥[3]。RNA結(jié)合蛋白QKI隸屬于STAR家族（evolutionarily conserved signal transduction and activation ofRNA family）是一個(gè)重要的轉(zhuǎn)錄后調(diào)控因子[4]。多種癌癥相關(guān)基因都是QKI調(diào)控目標(biāo)如：c-fos蛋白[6]，p27[7],β-catenin[8],新近研究表明，QKI的異常表達(dá)與人類(lèi)許多腫瘤的發(fā)生和發(fā)展相關(guān)[9,10]�？傊羞@些都指出了QKI在多種腫瘤中的抑制作用。 我們的研究是首次在前列腺癌中報(bào)道RNA結(jié)合蛋白QKI的表達(dá)情況，同時(shí)首次報(bào)道了QKI在前列腺癌發(fā)生發(fā)展中所起到的作用。通過(guò)免疫組化、Westernblot和qPCR方法在病理組織標(biāo)本中篩查QKI表達(dá)水平以及表達(dá)趨勢(shì)。通過(guò)對(duì)前列腺癌細(xì)胞系的篩選，選取過(guò)表達(dá)慢病毒以及基因沉默慢病毒分別對(duì)QKI的表達(dá)水平進(jìn)行調(diào)整，再與對(duì)照組進(jìn)行細(xì)胞生物學(xué)功能上的比對(duì)，以此闡述QKI在前列腺癌的發(fā)生發(fā)展過(guò)程中所起到的作用及其機(jī)制。 具體實(shí)驗(yàn)分為以下2個(gè)部分： 1）RNA結(jié)合蛋白QKI在前列腺癌組織中的表達(dá)分析 實(shí)驗(yàn)通過(guò)對(duì)前列腺癌組織標(biāo)本進(jìn)行免疫組化、qPCR和western blot以及啟動(dòng)子甲基化等檢測(cè)方法，檢測(cè)結(jié)果表明在前列腺癌組織中QKI的表達(dá)水平要低于癌旁對(duì)照組織，并且隨著分化程度的不斷減低QKI的表達(dá)水平也呈現(xiàn)降低狀態(tài)。 2）探尋RNA結(jié)合蛋白QKI在前列腺癌中的作用及其機(jī)制 實(shí)驗(yàn)選取了三株前列腺癌細(xì)胞株P(guān)C3,DU145,LNCaP并對(duì)這三株細(xì)胞進(jìn)行了QKI表達(dá)水平的篩選，將選取出來(lái)QKI表達(dá)水平最低的PC3細(xì)胞用過(guò)表達(dá)慢病毒感染，將表達(dá)水平相對(duì)較高的DU145細(xì)胞用基因沉默慢病毒感染，并分別構(gòu)建穩(wěn)定感染細(xì)胞系。細(xì)胞模型構(gòu)建成功后我們通過(guò)MTT、平板克隆實(shí)驗(yàn)對(duì)穩(wěn)轉(zhuǎn)細(xì)胞系的增殖能力進(jìn)行檢測(cè)，發(fā)現(xiàn)QKI能夠明顯的抑制前列腺癌細(xì)胞增殖（P＜0.05）。隨后通過(guò)流式細(xì)胞儀對(duì)細(xì)胞周期和凋亡水平進(jìn)行檢測(cè)，發(fā)現(xiàn)QKI可以使前列腺癌細(xì)胞出現(xiàn)G1期阻滯，減少細(xì)胞S期的進(jìn)入，同時(shí)QKI可以使癌細(xì)胞出現(xiàn)早期凋亡的呈倍數(shù)增長(zhǎng)（P＜0.01）。在細(xì)胞侵襲能力檢測(cè)中通過(guò)Transwell實(shí)驗(yàn)我們發(fā)現(xiàn)較對(duì)照組來(lái)言QKI可以明顯的降低細(xì)胞侵襲能力（P＜0.05）。最后我們?yōu)榱蓑?yàn)證細(xì)胞實(shí)驗(yàn)中的結(jié)果又進(jìn)行了動(dòng)物實(shí)驗(yàn)，在隨機(jī)分組的裸鼠中分別皮下注射了穩(wěn)定轉(zhuǎn)染QKI的細(xì)胞系，通過(guò)對(duì)瘤體體積，生長(zhǎng)曲線繪制和瘤體重量的檢測(cè)發(fā)現(xiàn)QKI能夠明顯的抑制腫瘤的生長(zhǎng)。將荷瘤取出后對(duì)瘤體組織進(jìn)行的TUNEL檢測(cè)，結(jié)果提示QKI的過(guò)表達(dá)能夠明顯增加腫瘤的凋亡（P＜0.05）。 綜上，本次研究是國(guó)內(nèi)外首次在前列腺癌中報(bào)道RNA結(jié)合蛋白QKI。實(shí)驗(yàn)得出的結(jié)論驗(yàn)證了研究初期對(duì)QKI在前列腺癌中可能發(fā)揮作用的推測(cè)，RNA結(jié)合蛋白在轉(zhuǎn)錄后水平調(diào)控前列腺癌細(xì)胞的增殖，并對(duì)前列腺癌細(xì)胞的周期產(chǎn)生了阻滯，導(dǎo)致了細(xì)胞的凋亡水平上升。因此我們認(rèn)為針對(duì)QKI的前列腺癌靶向研究是有重要意義的。
[Abstract]:Prostate cancer (PCa) is the most common in Europe and the United States non skin cancer, ranked second in the cause of male cancer deaths in Chinese. With the way of life is changing the incidence of prostate cancer in recent years is also showing high growth trend of [1,2]. is currently the main treatment is surgery and adjuvant radiotherapy and chemotherapy. Endocrine therapy, the treatment effect is limited. The incidence of early prostate cancer has the characteristics of no obvious symptoms, many patients are diagnosed often have lost the best time for surgery. To break the limitations of traditional therapy is a new challenge for the patients in the treatment of prostate cancer. With the progress of molecular biology, gene therapy in more and more clinical disease diagnosis and treatment are utilized, the target for the treatment of traumatic and almost no features can significantly improve the existing means of treatment Therefore, exploring more genes related to prostate cancer and targeting these genes have become a new direction for the treatment of prostate cancer.
Today, RNA binding protein and miRNA has been proved to be a key regulator of cell biology in the post transcriptional regulation of RNA transcription efficiency, stability, lack of transport and shear.MRNA regulation has also been confirmed and is closely related to many human diseases, including cancer [3].RNA binding protein QKI belongs to the STAR family (evolutionarily conserved signal transduction and activation ofRNA family) is an important transcription regulatory factor [4]. many cancer related genes are QKI control objectives such as: c-fos protein [6], p27[7], beta -catenin[8], recent studies show that the abnormal expression of QKI and a lot of human tumor occurrence and development of [9,10]. in short, all of these are pointed out. Inhibition of QKI in tumors.
Our study is the first report of RNA in prostate cancer with the expression of QKI protein, and QKI are reported for the first time in prostate cancer plays a role in the development. By immunohistochemistry, Westernblot and qPCR screening method in pathological specimens in the expression level of QKI and the expression trend. Through the screening of prostate cancer cell line the adjusted selected lentivirus expressing and gene silencing lentivirus respectively on the expression level of QKI, compared the biological function of the cells with the control group, so as to explain the QKI in prostate cancer development mechanisms by which to process.
The experiment is divided into 2 parts:
1) expression of RNA binding protein QKI in prostate cancer
Through the experiment of immunohistochemistry on prostate cancer tissue samples, qPCR and Western blot and promoter methylation detection method, the detection results show that the expression of QKI in prostate cancer tissue is lower than that of the adjacent normal tissues, and the expression level decreased QKI also showed a lower degree of differentiation.
2) explore the role and mechanism of RNA binding protein QKI in prostate cancer
The experiment selected three strains of prostate cancer cell lines PC3, DU145, LNCaP and the level of screening on the expression of QKI in these three cell lines, will be selected out of the lowest level of the QKI expression of PC3 cells with overexpression of lentivirus infection, the expression of DU145 cells with a relatively high level of gene silencing lentivirus infection, and build a stable infected cells. Cell model was constructed successfully by our MTT tablet cloning experiment to detect stabletransfection cell lines proliferation, we found that QKI can inhibit proliferation of prostate cancer cells significantly (P < 0.05). Then by flow cytometry to detect the cell cycle and apoptosis level, found that QKI can make the prostate cancer cells were arrested in G1 phase, decrease into the cells in S phase, while QKI can cause cancer cells to early apoptosis exponentially (P < 0.01). The cell invasion assay by Transwell experiment They found that compared with the control group, it can significantly reduce the invasive ability of QKI cells (P < 0.05). Finally, we in order to verify the results of the experiments and the cells of animal experiments in nude mice were randomly divided into subcutaneous injection of the stable transfected QKI cell lines, the tumor volume growth curve detection and the tumor weight found that QKI can obviously suppress tumor growth. TUNEL detection of the tumor after the removal of the tumor tissue, the results indicated that overexpression can significantly increase tumor cell apoptosis in QKI (P < 0.05).
In summary, this study is the first report of RNA in prostate cancer with the protein QKI. experiment verified the conclusion at the beginning of the study on speculation QKI may play a role in prostate cancer, RNA combined with the proliferation of protein in the post transcriptional regulation of prostate cancer cells, and the block cycle on prostate cancer cells, leading to the level of apoptosis cells increased. Therefore we think that the targeting of QKI for prostate cancer research is of great significance.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.25

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相關(guān)期刊論文 前2條

1 胡燕;萬(wàn)小潔;潘鎦鎦;章圣輝;鄭飛云;;鴉膽子油乳對(duì)HPV16亞型感染細(xì)胞的作用及機(jī)制研究[J];中國(guó)中西醫(yī)結(jié)合雜志;2013年11期

2 陳鍇,宋\

本文編號(hào)：1534739