發(fā)布時間：2018-04-11 01:20

  本文選題：前列腺癌 + GAS5��； 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：研究背景和目的前列腺癌是男性泌尿生殖系統(tǒng)最常見的惡性腫瘤之一,在西方國家中發(fā)病率尤其高,例如在美國其發(fā)病率居男性惡性腫瘤之首。近年來隨著生活方式的改變和醫(yī)療保健水平的提高,我國前列腺癌的發(fā)病率也顯著升高。實(shí)際工作中我們發(fā)現(xiàn),許多患者在就診時已經(jīng)是中晚期,錯過了手術(shù)治療的最佳時期。而在只能采用內(nèi)分泌治療的患者中,腫瘤由激素依賴性轉(zhuǎn)變成激素非依賴性或激素難治性的可能性非常大,這將導(dǎo)致患者的預(yù)后變差。因此,探索前列腺癌的發(fā)病機(jī)制、尋找新的方法來預(yù)防和治療前列腺癌就顯得尤為重要。長鏈非編碼RNA(lncRNA)是一類長度超過200個核苷酸的RNA,具有重要的生物學(xué)功能,能夠廣泛參與機(jī)體的生理和病理過程。研究發(fā)現(xiàn)lncRNA GAS5 (growth arrest-specific transcript 5)在乳腺癌、膀胱癌、胃癌等多種腫瘤組織與癌旁組織中存在差異性表達(dá),并且在腫瘤細(xì)胞侵襲遷移、增殖和凋亡等過程中發(fā)揮重要作用。目前關(guān)于GAS5在前列腺癌中的研究并不多,因此本課題的主要目的就是探討GAS5在前列腺癌發(fā)展過程中的作用及機(jī)制。研究方法采用qRT-PCR技術(shù)檢測并比較GAS5在正常前列腺細(xì)胞系RWPE-2以及前列腺癌細(xì)胞系PC3和DU145中的表達(dá)水平。在此基礎(chǔ)上,構(gòu)建GAS5過表達(dá)載體并設(shè)計針對GAS5的shRNA載體,分別轉(zhuǎn)染PC3和DU145細(xì)胞,通過功能缺失和功能獲得兩方面研究GAS5在前列腺癌細(xì)胞中的作用。首先,應(yīng)用MTT實(shí)驗(yàn)檢測細(xì)胞存活率,采用EdU實(shí)驗(yàn)檢測細(xì)胞增殖,使用流式細(xì)胞技術(shù)檢測前列腺癌細(xì)胞周期變化。接下來,使用Western blot檢測GAS5對靶蛋白的調(diào)控。此后,應(yīng)用雙熒光素酶、RIP、CHIP實(shí)驗(yàn)對GAS5的作用機(jī)制進(jìn)行探討。最后通過裸鼠皮下成瘤實(shí)驗(yàn)進(jìn)一步驗(yàn)證GAS5在體內(nèi)對前列腺癌細(xì)胞的影響并采用免疫組化驗(yàn)證靶蛋白在組織中的變化。研究結(jié)果一、GAS5在前列腺癌細(xì)胞系PC3和DU145中的表達(dá)低于在正常前列腺細(xì)胞系RWPE-2中的表達(dá),且差異具有顯著性。二、GAS5在前列腺癌細(xì)胞中的功能學(xué)研究。1.過表達(dá)GAS5能引起前列腺癌細(xì)胞G0/G1期阻滯,抑制細(xì)胞增殖。2.敲低GAS5能加快前列腺癌細(xì)胞的周期進(jìn)程、促進(jìn)前列腺癌細(xì)胞增殖。三、GAS5調(diào)控前列腺癌細(xì)胞增殖的機(jī)制研究。1.GAS5能正向調(diào)控P27Kip1蛋白及mRNA的表達(dá)。2.GAS5能夠增強(qiáng)P27Kip1的啟動子活性。3.轉(zhuǎn)錄因子E2F1能夠結(jié)合并正向調(diào)控P27Kip1的啟動子活性。4.GAS5能夠直接結(jié)合并增強(qiáng)E2F1對P27Kip1的調(diào)控作用。四、體內(nèi)實(shí)驗(yàn)驗(yàn)證GAS5對前列腺癌細(xì)胞的影響。1.過表達(dá)GAS5的PC3細(xì)胞形成的腫瘤體積、重量小于陰性對照組。、2.GAS5表達(dá)下調(diào)的PC3細(xì)胞形成的腫瘤體積、重量大于對照組。3.GAS5表達(dá)水平與P27Kip1蛋白在腫瘤組織的表達(dá)水平正相關(guān)。研究結(jié)論GAS5能夠直接結(jié)合轉(zhuǎn)錄因子E2F1并增強(qiáng)E2F1對P27Kip1啟動子的激活作用,導(dǎo)致前列腺癌細(xì)胞阻滯在G0/G1期,從而抑制前列腺癌細(xì)胞增殖。GAS5可能是前列腺癌治療的一個重要靶點(diǎn)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R737.25

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本文編號：1733802