MicroRNA-582在膀胱癌中表達(dá)的生物學(xué)功能及調(diào)控機(jī)制
發(fā)布時間：2018-09-18 11:52
【摘要】：背景與目的膀胱癌是目前泌尿系統(tǒng)常見的惡性腫瘤之一,也是我國發(fā)病率最高的泌尿系統(tǒng)腫瘤,且呈逐年增高趨勢。膀胱癌的發(fā)病原因較復(fù)雜,主要有遺傳和環(huán)境兩大因素。吸煙和長期接觸化工藥品是目前兩大環(huán)境致病危險(xiǎn)因素。其中,吸煙是最為肯定的危險(xiǎn)致病因素。研究表明,吸煙可以使膀胱癌發(fā)病率增加2-4倍,且危險(xiǎn)系數(shù)與吸煙強(qiáng)度及吸煙時間成正比。目前,膀胱癌根據(jù)浸潤深度的不同,臨床上多采用不同的治療方案。淺表性膀胱癌多采用經(jīng)尿道膀胱腫瘤電切術(shù)聯(lián)合術(shù)后膀胱灌注治療,但術(shù)后復(fù)發(fā)率較高,約60-70%,且復(fù)發(fā)后有惡性程度增加的趨勢。而對于肌層浸潤性膀胱癌,臨床上目前的標(biāo)準(zhǔn)治療方法是根治性全膀胱切除術(shù)。但是,此手術(shù)創(chuàng)傷較大,術(shù)后多并發(fā)癥,且極大的影響患者生活治療。因此,泌尿外科醫(yī)生希望明確膀胱癌發(fā)生、發(fā)展的分子生物學(xué)過程,并以此找到能夠有效早期診斷、治療膀胱癌的新方法。MicroRNA是一類小分子非蛋白編碼RNA(長度約22個堿基)。它們雖然不能直接編碼合成蛋白,但可以通過與靶基因3'UTR部分或完全配對結(jié)合抑制其翻譯,或造成其特異性降解,以此起到調(diào)控基因轉(zhuǎn)錄后表達(dá)的作用。我們根據(jù)前期基因芯片研究發(fā)現(xiàn),miR-582在膀胱尿路上皮癌細(xì)胞中呈低表達(dá),通過體外細(xì)胞實(shí)驗(yàn),探索miR-582在人膀胱癌細(xì)胞中的作用,為膀胱癌的診斷和治療提供新的靶點(diǎn)。材料、方法及結(jié)果第一章miR-582在人膀胱尿路上皮癌中的表達(dá)差異方法:1.收集根治性全膀胱切除術(shù)后,經(jīng)病理確認(rèn)為尿路上皮癌的新鮮腫瘤組織標(biāo)本為腫瘤(陽性)組及對應(yīng)的癌旁正常上皮組織,提取組織RNA。2.利用QPCR技術(shù),檢測膀胱癌組織及細(xì)胞中,miR-582的表達(dá)水平。結(jié)果:1.與正常癌旁組織相比,膀胱尿路上皮癌組織中,miR-582的表達(dá)水平明顯下調(diào)。2.膀胱尿路上皮癌的組織及細(xì)胞中,miR-582呈現(xiàn)穩(wěn)定低表達(dá),且其表達(dá)量與腫瘤的病理分級、臨床分期密切相關(guān)。第二章miR-582在膀胱癌致病中生物學(xué)功能研究方法:1.利用lipofectation 2000將miR-582 mimic瞬時轉(zhuǎn)染進(jìn)人膀胱癌24、5637細(xì)胞中,造成miR-582表達(dá)下調(diào)。設(shè)立對照組(轉(zhuǎn)染miR-NC組)和實(shí)驗(yàn)組(轉(zhuǎn)染 miR-582 mimic)。2.轉(zhuǎn)染后的細(xì)胞,通過QPCR方法評估轉(zhuǎn)染效率;通過劃痕實(shí)驗(yàn),評估膀胱癌細(xì)胞的遷移能力;通過Transwell實(shí)驗(yàn),評估膀胱癌細(xì)胞的侵襲能力;通過流式細(xì)胞技術(shù),評估膀胱癌細(xì)胞的干性能力;通過Western Blot實(shí)驗(yàn),評估膀胱癌細(xì)胞表征細(xì)胞侵干性能力的CD44、S0X2蛋白表達(dá)情況。結(jié)果:1.轉(zhuǎn)染miR-582mimic后,T24、5637細(xì)胞中miR-582的表達(dá)明顯上調(diào),差異存在統(tǒng)計(jì)學(xué)意義(P0.05)。2.劃痕實(shí)驗(yàn)中,轉(zhuǎn)染20h后,miR-582 mimic組的傷口愈合時間明顯延長,差異存在統(tǒng)計(jì)學(xué)意義(P0.05);Transwell實(shí)驗(yàn)中,轉(zhuǎn)染miR-582mimic組的T24、5637細(xì)胞穿膜細(xì)胞數(shù)遠(yuǎn)小于miR-NC mimic組處理細(xì)胞,差異存在統(tǒng)計(jì)學(xué)意義(P0.05);通過流式細(xì)胞技術(shù),證實(shí)miR-582 mimic組的膀胱癌T24、5637細(xì)胞,細(xì)胞干性明顯下降。第三章miR-582靶向負(fù)調(diào)控FOXG1表達(dá)及FOXG1的作用和意義方法:1.利用miRNA靶基因在線預(yù)測軟件,預(yù)測miR-582下游靶基因,并通過熒光酶素報(bào)告基因加以證實(shí)。2.在膀胱癌T24、5637細(xì)胞中共轉(zhuǎn)染miR-582和過表達(dá)F0XG1質(zhì)粒后進(jìn)行拯救實(shí)驗(yàn),利用Transwell實(shí)驗(yàn)檢測膀胱癌細(xì)胞侵襲能力的變化,Western blot實(shí)驗(yàn)檢測蛋白表達(dá)變化。3.利用si-FOXG1轉(zhuǎn)染處理人膀胱癌T24、5637細(xì)胞株,通過Western blot方法評估轉(zhuǎn)染效率;通過Transwell實(shí)驗(yàn),評估膀胱癌細(xì)胞的侵襲能力。結(jié)果:1.miRNA靶基因在線預(yù)測軟件與熒光素酶報(bào)告實(shí)驗(yàn)結(jié)果提示:miR-582在F0XG1基因的3'UTR存在miR-582的互補(bǔ)結(jié)合位點(diǎn)。2.轉(zhuǎn)染miR-582于膀胱癌細(xì)胞后,膀胱癌細(xì)胞侵襲能力下降,而隨著外源性F0XG1的加入,侵襲能力增強(qiáng);MMP2、MMP9等蛋白也在miR-582的影響下表達(dá)下調(diào),而隨著外源性F0XG1的加入而表達(dá)增強(qiáng)。3.Transwell實(shí)驗(yàn)中,轉(zhuǎn)染si-FOXG1組的T24、5637細(xì)胞穿膜細(xì)胞數(shù)遠(yuǎn)小于si-NC組處理細(xì)胞,差異存在統(tǒng)計(jì)學(xué)意義(P0.05)。此結(jié)果提示,F0XG1表達(dá)下調(diào)能夠有效抑制膀胱癌細(xì)胞的侵襲能力。結(jié)論和意義miR-582在膀胱癌組織中表達(dá)下調(diào),且其表達(dá)量與腫瘤的病理分級、臨床分期密切相關(guān),病理分級越高、臨床分期越晚的病人,表達(dá)量越低。miR-582在膀胱癌細(xì)胞中,靶向負(fù)調(diào)控下游靶基因F0XG1,從而實(shí)現(xiàn)起抑制膀胱癌細(xì)胞的遷移、侵襲、細(xì)胞干性的功能,發(fā)揮抑癌基因的作用。本項(xiàng)研究,可以為膀胱癌的分子靶向治療,提供實(shí)驗(yàn)依據(jù)。
[Abstract]:BACKGROUND & OBJECTIVE Bladder cancer is one of the most common malignant tumors in the urinary system, and it is also the most common one in China. The incidence of bladder cancer is increasing year by year. Smoking is the most definite risk factor. Studies have shown that smoking can increase the incidence of bladder cancer by 2-4 times, and the risk factor is proportional to the intensity of smoking and smoking time. But the recurrence rate is high, about 60-70%, and the malignant degree is increasing after the recurrence. For myometrial invasive bladder cancer, the current standard treatment is radical cystectomy. Thus, urologists hope to clarify the molecular biological processes involved in the development of bladder cancer, and to find new ways to effectively diagnose and treat bladder cancer. We found that the expression of microRNAs in bladder urothelial carcinoma cells was low. Through in vitro cell experiments, we explored the role of microRNAs in human bladder cancer cells. Materials, Methods and Results Chapter 1 Differential expression of microRNA582 in human bladder urothelial carcinoma Methods: 1. Fresh tumor specimens confirmed by pathology after radical cystectomy were collected as tumor (positive) group and corresponding normal epithelial tissue adjacent to the tumor, and RNA was extracted. Results: 1. Compared with normal adjacent tissues, the expression of microRNAs in bladder urothelial carcinoma tissues was significantly down-regulated. 2. In bladder urothelial carcinoma tissues and cells, the expression of microRNAs-582 was stable and low, and the expression level was correlated with the pathological grade and clinical significance of the tumor. Chapter 2: Biological function of microRNAs in the pathogenesis of bladder cancer: 1. Transient transfection of microRNAs into human bladder cancer 24,5637 cells by lipofection 2000 resulted in the down-regulation of microRNAs-582 expression. Control group (transfected with microRNAs-NC group) and experimental group (transfected with microRNAs-582 mimic) were established. Methods To evaluate the transfection efficiency, to evaluate the migration ability of bladder cancer cells by scratch test, to evaluate the invasion ability of bladder cancer cells by Transwell test, to evaluate the dry ability of bladder cancer cells by flow cytometry, and to evaluate the expression of CD44 and S0X2 protein in bladder cancer cells by Western Blot test. Results: 1. After transfection of Mi-582 mic, the expression of Mi-582 was significantly up-regulated in T24,5637 cells, and the difference was statistically significant (P 0.05). 2. In scratch test, the wound healing time of Mi-582 mic group was significantly prolonged after 20 hours of transfection, and the difference was statistically significant (P 0.05); In Transwell experiment, the transfection of Mi-582 mic group had fine membrane penetration. The number of cells treated with microRNAs was much smaller than that of microRNAs (P 0.05), and the difference was statistically significant (P 0.05). Flow cytometry showed that the cell trunkness of T24,5637 cells in microRNAs group was significantly decreased. Chapter 3: The role and significance of microRNAs targeting negative regulation of FOXG1 expression and FOXG1: 1. Mi-582 downstream target gene was confirmed by luciferase reporter gene. 2. Mi-582 and over-expressed F0XG1 plasmid were co-transfected into bladder cancer T24,5637 cells. Transwell assay was used to detect the invasion ability of bladder cancer cells. Western blot assay was used to detect the expression of protein. 3. Human bladder cancer cells were transfected with si-FOXG1. The transfection efficiency of bladder cancer cell line T24,5637 was evaluated by Western blot, and the invasiveness of bladder cancer cell line T24,5637 was evaluated by Transwell assay. Results: 1. MiRNA target gene online prediction software and luciferase report results indicated that there were complementary binding sites of Mi-582 in the 3'UTR of F0XG1 gene. 2. Mi-582 was transfected in bladder. The invasion ability of bladder cancer cells decreased with the addition of exogenous F0XG1, and increased with the addition of exogenous F0XG1. The expression of MMP2, MMP9 and other proteins was down-regulated under the influence of Mi-582, but increased with the addition of exogenous F0XG1. 3. Transwell experiment showed that the number of transmembrane cells of T24,5637 cells transfected with si-FOXG1 was much smaller than that of si-NC group. The results suggest that the down-regulation of F0XG1 expression can effectively inhibit the invasion of bladder cancer cells. Conclusion The down-regulation of microRNA582 expression in bladder cancer tissue is closely related to the pathological grade and clinical stage of the tumor. The higher the pathological grade, the later the clinical stage, the lower the expression of microRNA582. In bladder cancer cells, microRNA-582 targets the downstream target gene F0XG1, thereby inhibiting the migration, invasion and stem cell function of bladder cancer cells and exerting the role of tumor suppressor genes.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.14
，
本文編號：2247824
[Abstract]:BACKGROUND & OBJECTIVE Bladder cancer is one of the most common malignant tumors in the urinary system, and it is also the most common one in China. The incidence of bladder cancer is increasing year by year. Smoking is the most definite risk factor. Studies have shown that smoking can increase the incidence of bladder cancer by 2-4 times, and the risk factor is proportional to the intensity of smoking and smoking time. But the recurrence rate is high, about 60-70%, and the malignant degree is increasing after the recurrence. For myometrial invasive bladder cancer, the current standard treatment is radical cystectomy. Thus, urologists hope to clarify the molecular biological processes involved in the development of bladder cancer, and to find new ways to effectively diagnose and treat bladder cancer. We found that the expression of microRNAs in bladder urothelial carcinoma cells was low. Through in vitro cell experiments, we explored the role of microRNAs in human bladder cancer cells. Materials, Methods and Results Chapter 1 Differential expression of microRNA582 in human bladder urothelial carcinoma Methods: 1. Fresh tumor specimens confirmed by pathology after radical cystectomy were collected as tumor (positive) group and corresponding normal epithelial tissue adjacent to the tumor, and RNA was extracted. Results: 1. Compared with normal adjacent tissues, the expression of microRNAs in bladder urothelial carcinoma tissues was significantly down-regulated. 2. In bladder urothelial carcinoma tissues and cells, the expression of microRNAs-582 was stable and low, and the expression level was correlated with the pathological grade and clinical significance of the tumor. Chapter 2: Biological function of microRNAs in the pathogenesis of bladder cancer: 1. Transient transfection of microRNAs into human bladder cancer 24,5637 cells by lipofection 2000 resulted in the down-regulation of microRNAs-582 expression. Control group (transfected with microRNAs-NC group) and experimental group (transfected with microRNAs-582 mimic) were established. Methods To evaluate the transfection efficiency, to evaluate the migration ability of bladder cancer cells by scratch test, to evaluate the invasion ability of bladder cancer cells by Transwell test, to evaluate the dry ability of bladder cancer cells by flow cytometry, and to evaluate the expression of CD44 and S0X2 protein in bladder cancer cells by Western Blot test. Results: 1. After transfection of Mi-582 mic, the expression of Mi-582 was significantly up-regulated in T24,5637 cells, and the difference was statistically significant (P 0.05). 2. In scratch test, the wound healing time of Mi-582 mic group was significantly prolonged after 20 hours of transfection, and the difference was statistically significant (P 0.05); In Transwell experiment, the transfection of Mi-582 mic group had fine membrane penetration. The number of cells treated with microRNAs was much smaller than that of microRNAs (P 0.05), and the difference was statistically significant (P 0.05). Flow cytometry showed that the cell trunkness of T24,5637 cells in microRNAs group was significantly decreased. Chapter 3: The role and significance of microRNAs targeting negative regulation of FOXG1 expression and FOXG1: 1. Mi-582 downstream target gene was confirmed by luciferase reporter gene. 2. Mi-582 and over-expressed F0XG1 plasmid were co-transfected into bladder cancer T24,5637 cells. Transwell assay was used to detect the invasion ability of bladder cancer cells. Western blot assay was used to detect the expression of protein. 3. Human bladder cancer cells were transfected with si-FOXG1. The transfection efficiency of bladder cancer cell line T24,5637 was evaluated by Western blot, and the invasiveness of bladder cancer cell line T24,5637 was evaluated by Transwell assay. Results: 1. MiRNA target gene online prediction software and luciferase report results indicated that there were complementary binding sites of Mi-582 in the 3'UTR of F0XG1 gene. 2. Mi-582 was transfected in bladder. The invasion ability of bladder cancer cells decreased with the addition of exogenous F0XG1, and increased with the addition of exogenous F0XG1. The expression of MMP2, MMP9 and other proteins was down-regulated under the influence of Mi-582, but increased with the addition of exogenous F0XG1. 3. Transwell experiment showed that the number of transmembrane cells of T24,5637 cells transfected with si-FOXG1 was much smaller than that of si-NC group. The results suggest that the down-regulation of F0XG1 expression can effectively inhibit the invasion of bladder cancer cells. Conclusion The down-regulation of microRNA582 expression in bladder cancer tissue is closely related to the pathological grade and clinical stage of the tumor. The higher the pathological grade, the later the clinical stage, the lower the expression of microRNA582. In bladder cancer cells, microRNA-582 targets the downstream target gene F0XG1, thereby inhibiting the migration, invasion and stem cell function of bladder cancer cells and exerting the role of tumor suppressor genes.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.14
，
本文編號：2247824
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