探寻RNA结合蛋白QKI在前列腺癌演变过程中的作用及其分子机制

发布时间：2018-02-25 18:50

本文关键词： RNA结合蛋白QKI 前列腺癌慢病毒基因治疗　出处：《第四军医大学》2014年博士论文　论文类型：学位论文

【摘要】：前列腺癌（PCa）是在欧美国家最常见的非皮肤性癌症，在男性癌症死亡原因中位居第二。在中国随着生活方式的不断改变前列腺癌的发病率近年来也呈现出高增长的趋势[1,2]。目前主要的治疗方法是手术治疗，同时辅助放、化疗和内分泌治疗，治疗效果具有局限性。由于前列腺癌具有发病早期无明显症状等特点，很多患者被确诊时往往已经丧失了最佳的手术时机。突破传统治疗手段的局限性成为目前前列腺癌患者治疗的新的挑战。随着分子生物学技术的不断进步，基因治疗在越来越多的临床疾病诊疗中得到了利用，其针对靶点治疗和几乎无创伤性的特点能够显著的改善现有治疗手段的不足。因此探寻更多与前列腺癌相关的基因，针对这些基因开展靶向治疗就成为了前列腺癌治疗的一个新方向。现今，RNA结合蛋白和miRNA已经被证实是细胞生物学一个关键的调节点，，在转录后调控着RNA的稳定性、转录效率、转运和剪切。mRNA调节的缺失也被证实和许多人类疾病密切相关，其中就包括癌症[3]。RNA结合蛋白QKI隶属于STAR家族（evolutionarily conserved signal transduction and activation ofRNA family）是一个重要的转录后调控因子[4]。多种癌症相关基因都是QKI调控目标如：c-fos蛋白[6]，p27[7],β-catenin[8],新近研究表明，QKI的异常表达与人类许多肿瘤的发生和发展相关[9,10]。总之，所有这些都指出了QKI在多种肿瘤中的抑制作用。我们的研究是首次在前列腺癌中报道RNA结合蛋白QKI的表达情况，同时首次报道了QKI在前列腺癌发生发展中所起到的作用。通过免疫组化、Westernblot和qPCR方法在病理组织标本中筛查QKI表达水平以及表达趋势。通过对前列腺癌细胞系的筛选，选取过表达慢病毒以及基因沉默慢病毒分别对QKI的表达水平进行调整，再与对照组进行细胞生物学功能上的比对，以此阐述QKI在前列腺癌的发生发展过程中所起到的作用及其机制。具体实验分为以下2个部分： 1）RNA结合蛋白QKI在前列腺癌组织中的表达分析实验通过对前列腺癌组织标本进行免疫组化、qPCR和western blot以及启动子甲基化等检测方法，检测结果表明在前列腺癌组织中QKI的表达水平要低于癌旁对照组织，并且随着分化程度的不断减低QKI的表达水平也呈现降低状态。 2）探寻RNA结合蛋白QKI在前列腺癌中的作用及其机制实验选取了三株前列腺癌细胞株PC3,DU145,LNCaP并对这三株细胞进行了QKI表达水平的筛选，将选取出来QKI表达水平最低的PC3细胞用过表达慢病毒感染，将表达水平相对较高的DU145细胞用基因沉默慢病毒感染，并分别构建稳定感染细胞系。细胞模型构建成功后我们通过MTT、平板克隆实验对稳转细胞系的增殖能力进行检测，发现QKI能够明显的抑制前列腺癌细胞增殖（P＜0.05）。随后通过流式细胞仪对细胞周期和凋亡水平进行检测，发现QKI可以使前列腺癌细胞出现G1期阻滞，减少细胞S期的进入，同时QKI可以使癌细胞出现早期凋亡的呈倍数增长（P＜0.01）。在细胞侵袭能力检测中通过Transwell实验我们发现较对照组来言QKI可以明显的降低细胞侵袭能力（P＜0.05）。最后我们为了验证细胞实验中的结果又进行了动物实验，在随机分组的裸鼠中分别皮下注射了稳定转染QKI的细胞系，通过对瘤体体积，生长曲线绘制和瘤体重量的检测发现QKI能够明显的抑制肿瘤的生长。将荷瘤取出后对瘤体组织进行的TUNEL检测，结果提示QKI的过表达能够明显增加肿瘤的凋亡（P＜0.05）。综上，本次研究是国内外首次在前列腺癌中报道RNA结合蛋白QKI。实验得出的结论验证了研究初期对QKI在前列腺癌中可能发挥作用的推测，RNA结合蛋白在转录后水平调控前列腺癌细胞的增殖，并对前列腺癌细胞的周期产生了阻滞，导致了细胞的凋亡水平上升。因此我们认为针对QKI的前列腺癌靶向研究是有重要意义的。
[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.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

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