长链非编码RNA GAS5在前列腺癌中的作用及机制研究

发布时间：2018-04-11 01:20

本文选题：前列腺癌 + GAS5　；参考：《华中科技大学》2016年博士论文

【摘要】：研究背景和目的前列腺癌是男性泌尿生殖系统最常见的恶性肿瘤之一,在西方国家中发病率尤其高,例如在美国其发病率居男性恶性肿瘤之首。近年来随着生活方式的改变和医疗保健水平的提高,我国前列腺癌的发病率也显著升高。实际工作中我们发现,许多患者在就诊时已经是中晚期,错过了手术治疗的最佳时期。而在只能采用内分泌治疗的患者中,肿瘤由激素依赖性转变成激素非依赖性或激素难治性的可能性非常大,这将导致患者的预后变差。因此,探索前列腺癌的发病机制、寻找新的方法来预防和治疗前列腺癌就显得尤为重要。长链非编码RNA(lncRNA)是一类长度超过200个核苷酸的RNA,具有重要的生物学功能,能够广泛参与机体的生理和病理过程。研究发现lncRNA GAS5 (growth arrest-specific transcript 5)在乳腺癌、膀胱癌、胃癌等多种肿瘤组织与癌旁组织中存在差异性表达,并且在肿瘤细胞侵袭迁移、增殖和凋亡等过程中发挥重要作用。目前关于GAS5在前列腺癌中的研究并不多,因此本课题的主要目的就是探讨GAS5在前列腺癌发展过程中的作用及机制。研究方法采用qRT-PCR技术检测并比较GAS5在正常前列腺细胞系RWPE-2以及前列腺癌细胞系PC3和DU145中的表达水平。在此基础上,构建GAS5过表达载体并设计针对GAS5的shRNA载体,分别转染PC3和DU145细胞,通过功能缺失和功能获得两方面研究GAS5在前列腺癌细胞中的作用。首先,应用MTT实验检测细胞存活率,采用EdU实验检测细胞增殖,使用流式细胞技术检测前列腺癌细胞周期变化。接下来,使用Western blot检测GAS5对靶蛋白的调控。此后,应用双荧光素酶、RIP、CHIP实验对GAS5的作用机制进行探讨。最后通过裸鼠皮下成瘤实验进一步验证GAS5在体内对前列腺癌细胞的影响并采用免疫组化验证靶蛋白在组织中的变化。研究结果一、GAS5在前列腺癌细胞系PC3和DU145中的表达低于在正常前列腺细胞系RWPE-2中的表达,且差异具有显著性。二、GAS5在前列腺癌细胞中的功能学研究。1.过表达GAS5能引起前列腺癌细胞G0/G1期阻滞,抑制细胞增殖。2.敲低GAS5能加快前列腺癌细胞的周期进程、促进前列腺癌细胞增殖。三、GAS5调控前列腺癌细胞增殖的机制研究。1.GAS5能正向调控P27Kip1蛋白及mRNA的表达。2.GAS5能够增强P27Kip1的启动子活性。3.转录因子E2F1能够结合并正向调控P27Kip1的启动子活性。4.GAS5能够直接结合并增强E2F1对P27Kip1的调控作用。四、体内实验验证GAS5对前列腺癌细胞的影响。1.过表达GAS5的PC3细胞形成的肿瘤体积、重量小于阴性对照组。、2.GAS5表达下调的PC3细胞形成的肿瘤体积、重量大于对照组。3.GAS5表达水平与P27Kip1蛋白在肿瘤组织的表达水平正相关。研究结论GAS5能够直接结合转录因子E2F1并增强E2F1对P27Kip1启动子的激活作用,导致前列腺癌细胞阻滞在G0/G1期,从而抑制前列腺癌细胞增殖。GAS5可能是前列腺癌治疗的一个重要靶点。
[Abstract]:Background and objective Prostate cancer is one of the most common malignant tumors in the male genitourinary system.In recent years, with the change of lifestyle and the improvement of health care, the incidence of prostate cancer in China has increased significantly.In practice, we found that many patients at the time of treatment is late, missed the best period of surgical treatment.In patients who can only be treated with endocrine therapy, it is very likely that the tumor will change from hormone dependence to hormone independent or intractable hormone, which will lead to poor prognosis.Therefore, it is very important to explore the pathogenesis of prostate cancer and find new methods to prevent and treat prostate cancer.Long chain noncoding RNAs (LNRNAs) are a class of RNAs with a length of more than 200 nucleotides, which have important biological functions and can be widely involved in the physiological and pathological processes of the body.It has been found that lncRNA GAS5 growth arrest-specific transcript 5 has different expression in breast cancer, bladder cancer, gastric cancer and adjacent tissues, and plays an important role in the invasion, migration, proliferation and apoptosis of tumor cells.At present, there are few studies on GAS5 in prostate cancer, so the main purpose of this paper is to explore the role and mechanism of GAS5 in the development of prostate cancer.Methods qRT-PCR technique was used to detect and compare the expression of GAS5 in normal prostate cell line RWPE-2 and prostate cancer cell line PC3 and DU145.On this basis, the GAS5 overexpression vector was constructed and the shRNA vector for GAS5 was designed to transfect PC3 and DU145 cells, respectively. The function of GAS5 in prostate cancer cells was studied by functional loss and function acquisition.First, MTT assay was used to detect cell survival rate, EdU assay was used to detect cell proliferation, and flow cytometry was used to detect cell cycle changes.Next, Western blot was used to detect the regulation of GAS5 on target proteins.After that, the mechanism of GAS5 action was studied by using double luciferase rippon chip experiment.Finally, the effect of GAS5 on prostate cancer cells in vivo was further verified by subcutaneous tumorigenesis in nude mice, and the changes of target proteins in tissues were verified by immunohistochemistry.Results the expression of GAS5 in prostate cancer cell line PC3 and DU145 was lower than that in normal prostate cell line RWPE-2, and the difference was significant.Functional study of GAS5 in Prostate Cancer cells. 1.Overexpression of GAS5 could induce G0/G1 phase arrest and inhibit proliferation of prostate cancer cells.Knockdown of GAS5 can accelerate the cell cycle progression and promote the proliferation of prostate cancer cells.Study on the Mechanism of GAS5 regulating Prostate Cancer Cell Proliferation. 1. GAS5 can positively regulate P27Kip1 protein and mRNA expression. 2. GAS5 can enhance the promoter activity of P27Kip1.Transcription factor E2F1 can bind and positively regulate the promoter activity of P27Kip1. 4. GAS5 can directly bind and enhance the regulation of P27Kip1 by E2F1.Fourth, in vivo experiments to verify the effect of GAS5 on prostate cancer cells. 1.The tumor volume of PC3 cells with overexpression of GAS5 was smaller than that of PC3 cells with down-regulated GAS5 expression in negative control group, and the weight was higher than that of control group .3.GAS5 expression level was positively correlated with the expression level of P27Kip1 protein in tumor tissue.Conclusion GAS5 can directly bind to the transcription factor E2F1 and enhance the activation of P27Kip1 promoter by E2F1, which leads to the arrest of prostate cancer cells in G0/G1 phase, thus inhibiting the proliferation of prostate cancer cells. GAS5 may be an important target of prostate cancer therapy.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25

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