發(fā)布時間：2018-02-25 18:50

  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》2014年博士論文　論文類型：學位論文

【摘要】：前列腺癌（PCa）是在歐美國家最常見的非皮膚性癌癥，在男性癌癥死亡原因中位居第二。在中國隨著生活方式的不斷改變前列腺癌的發(fā)病率近年來也呈現(xiàn)出高增長的趨勢[1,2]。目前主要的治療方法是手術(shù)治療，同時輔助放、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)病早期無明顯癥狀等特點，很多患者被確診時往往已經(jīng)喪失了最佳的手術(shù)時機。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)。隨著分子生物學技術(shù)的不斷進步，基因治療在越來越多的臨床疾病診療中得到了利用，其針對靶點治療和幾乎無創(chuàng)傷性的特點能夠顯著的改善現(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，針對這些基因開展靶向治療就成為了前列腺癌治療的一個新方向。 現(xiàn)今，RNA結(jié)合蛋白和miRNA已經(jīng)被證實是細胞生物學一個關(guān)鍵的調(diào)節(jié)點，，在轉(zhuǎn)錄后調(diào)控著RNA的穩(wěn)定性、轉(zhuǎn)錄效率、轉(zhuǎn)運和剪切。mRNA調(diào)節(jié)的缺失也被證實和許多人類疾病密切相關(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)控目標如：c-fos蛋白[6]，p27[7],β-catenin[8],新近研究表明，QKI的異常表達與人類許多腫瘤的發(fā)生和發(fā)展相關(guān)[9,10]�？傊�，所有這些都指出了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道RNA結(jié)合蛋白QKI的表達情況，同時首次報道了QKI在前列腺癌發(fā)生發(fā)展中所起到的作用。通過免疫組化、Westernblot和qPCR方法在病理組織標本中篩查QKI表達水平以及表達趨勢。通過對前列腺癌細胞系的篩選，選取過表達慢病毒以及基因沉默慢病毒分別對QKI的表達水平進行調(diào)整，再與對照組進行細胞生物學功能上的比對，以此闡述QKI在前列腺癌的發(fā)生發(fā)展過程中所起到的作用及其機制。 具體實驗分為以下2個部分： 1）RNA結(jié)合蛋白QKI在前列腺癌組織中的表達分析 實驗通過對前列腺癌組織標本進行免疫組化、qPCR和western blot以及啟動子甲基化等檢測方法，檢測結(jié)果表明在前列腺癌組織中QKI的表達水平要低于癌旁對照組織，并且隨著分化程度的不斷減低QKI的表達水平也呈現(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制 實驗選取了三株前列腺癌細胞株P(guān)C3,DU145,LNCaP并對這三株細胞進行了QKI表達水平的篩選，將選取出來QKI表達水平最低的PC3細胞用過表達慢病毒感染，將表達水平相對較高的DU145細胞用基因沉默慢病毒感染，并分別構(gòu)建穩(wěn)定感染細胞系。細胞模型構(gòu)建成功后我們通過MTT、平板克隆實驗對穩(wěn)轉(zhuǎn)細胞系的增殖能力進行檢測，發(fā)現(xiàn)QKI能夠明顯的抑制前列腺癌細胞增殖（P＜0.05）。隨后通過流式細胞儀對細胞周期和凋亡水平進行檢測，發(fā)現(xiàn)QKI可以使前列腺癌細胞出現(xiàn)G1期阻滯，減少細胞S期的進入，同時QKI可以使癌細胞出現(xiàn)早期凋亡的呈倍數(shù)增長（P＜0.01）。在細胞侵襲能力檢測中通過Transwell實驗我們發(fā)現(xiàn)較對照組來言QKI可以明顯的降低細胞侵襲能力（P＜0.05）。最后我們?yōu)榱蓑炞C細胞實驗中的結(jié)果又進行了動物實驗，在隨機分組的裸鼠中分別皮下注射了穩(wěn)定轉(zhuǎn)染QKI的細胞系，通過對瘤體體積，生長曲線繪制和瘤體重量的檢測發(fā)現(xiàn)QKI能夠明顯的抑制腫瘤的生長。將荷瘤取出后對瘤體組織進行的TUNEL檢測，結(jié)果提示QKI的過表達能夠明顯增加腫瘤的凋亡（P＜0.05）。 綜上，本次研究是國內(nèi)外首次在前列腺癌中報道RNA結(jié)合蛋白QKI。實驗得出的結(jié)論驗證了研究初期對QKI在前列腺癌中可能發(fā)揮作用的推測，RNA結(jié)合蛋白在轉(zhuǎn)錄后水平調(diào)控前列腺癌細胞的增殖，并對前列腺癌細胞的周期產(chǎn)生了阻滯，導致了細胞的凋亡水平上升。因此我們認為針對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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R737.25

【參考文獻】

相關(guān)期刊論文 前2條

1 胡燕;萬小潔;潘鎦鎦;章圣輝;鄭飛云;;鴉膽子油乳對HPV16亞型感染細胞的作用及機制研究[J];中國中西醫(yī)結(jié)合雜志;2013年11期

2 陳鍇,宋\

本文編號：1534739