miRNA-21在胆囊癌过表达通过抑制PTEN促进细胞侵袭与转移的机制研究

发布时间：2018-04-25 10:38

本文选题：胆囊癌 + microRNA-21　；参考：《安徽医科大学》2017年博士论文

【摘要】：研究背景:胆囊癌(Gallbladder Carcinoma,GBC)是常见的胆道系统肿瘤,早期无特异性的临床表现,发现时往往已到临床中晚期,预后较差。临床上GBC确诊时,多半已经发生了邻近器官浸润和/或通过血液系统发生远处转移,很难进行根治性手术,且手术效果较差,术后五年生存率不足5%。近年的流行病学调查调查显示我国大部分地区GBC发病率呈明显上升趋势。因此,提高GBC患者的远期生存率除了要在手术结合放化疗方面进一步规范和探索外,还应该在分子水平上进一步研究GBC的发生、发展及其侵袭的机制。通过在分子水平的研究、探索GBC的生物学行为,寻找特异性生物学标记提高早期诊断率,针对发病基因进行分子靶向治疗并在预后评估等方面进行探索。这也是目前GBC分子生物学研究的重点、热点方向之一。微小RNA(micro RNA,mi RNA)是一类内源性非编码短链RNA,约22个核苷酸大小,它主要通过与靶m RNA的3’UTR不完全性互补配对抑制m RNA的翻译,介导转录后基因调控。mi RNA不仅在细胞的增殖、分化和凋亡等生物学功能起到重要的调控作用,而且和肿瘤的发生发展、转移等生物学效应密切相关。PTEN是迄今为止发现的第一个具有双重磷酸酶活性的抑癌基因。PTEN基因编码的蛋白具有脂质磷酸酶和蛋白磷酸酶双重特异性磷酸酶活性,与多种肿瘤的发生、发展密切相关。本课题组发现在GBC-SD细胞系中mi RNA-21表达水平与PTEN表达密切相关,并进一步实验证实在GBC-SD细胞系中PTEN为mi RNA-21靶基因,初步认为mi RNA-21通过抑制PTEN基因影响了GBC发生、发展的可能机制;同时mi RNA-21与细胞紧密连接蛋白ZO-1和occludin表达密切相关,提示mi RNA-21可能参与GBC的侵袭与迁移。这为进一步研究mi RNA-21在GBC生物功能的机制拓宽了思路。方法:1.在GBC-SD细胞系沉默micro RNA 21;1)沉默mi RNA-21后分别在m RNA和蛋白水平检测PTEN、紧密连接相关蛋白Occludin、ZO-1蛋白表达水平;2)在GBC-SD细胞系过表达/抑制mi RNA-21表达,分别检测PTEN的蛋白水平和m RNA水平变化;2.Transwell assay检测GBC-SD细胞系沉默mi RNA-21后细胞迁移与侵袭能力;3.荧光素酶实验检测PTEN与mi RNA-21的相互作用机制;4.收集67例GBC临床标本,原位杂交技术检测mi RNA-21,免疫组化SP法检测PTEN、ZO-1和occludin表达水平;5.采用对mi RNA-21与临床参数、PTEN等指标进行相关性分析;Kaplan-Meier方法进行生存分析,并采用Log-rank检验比较两组生存率;采用Cox风险比例模型进行各个协变量的联合效应分析。P0.05为差异有统计学意义。结果:1.发现micro RNA 21与PTEN、紧密连接蛋白之间的关系:1)沉默micro RNA 21后,PTEN表达上调,Occludin、ZO-1蛋白水平升高;2)沉默micro RNA 21后,发现Occludin、ZO-1m RNA表达上升。2.证实mi RNA-21与PTEN在GBC中的表达负相关:1)GBC-SD细胞中过表达mi RNA-21可下调PTEN的m RNA和蛋白表达;2)GBC-SD细胞中抑制mi RNA-21可上调PTEN的m RNA和蛋白表达;3)GBC-SD细胞中抑制mi RNA-21后,细胞迁移与侵袭能力明显减弱;4)PTEN的3’UTR是mi RNA-21的靶序列;5)GBC患者术后生存结果分析显示,mi RNA-21表达与患者预后呈反比。结论:1.在GBC-SD细胞中mi RNA21可被沉默;体外实验证实mi RNA21基因表达与PTEN、紧密连接蛋白表达水平密切相关;在进一步的GBC-SD细胞和动物实验证明沉默mi RNA21后,GBC侵袭和转移能力减弱;2.初步认为PTEN的3’UTR是mi RNA-21在GBC细胞中作用的靶区域,mi RNA21可通过抑制靶基因PTEN的功能参与GBC的发生、发展;GBC病人癌组织mi RNA21表达上调,其表达与肿瘤的TNM分期相关,且高表达患者生存期短,预后差。mi RNA21或可成为GBC治疗的靶目标。
[Abstract]:Background: Gallbladder Carcinoma (GBC) is a common biliary system tumor. It has no specific clinical manifestations in the early stage. It is often found in the middle and late stages of the clinic, and the prognosis is poor. In the clinical GBC diagnosis, most of them have occurred adjacent organ infiltration and / or distant metastasis through the blood system, so it is difficult to perform radical operation. The effect of the operation is poor and the five year survival rate is less than five years after the operation. The epidemiological survey in recent years shows that the incidence of GBC in most areas of our country is obviously rising. Therefore, in addition to further standardization and exploration in the field of operation combined with radiotherapy and chemotherapy, the forward survival rate of GBC patients should be further studied at the molecular level of GBC. The mechanism of occurrence, development and invasion is explored by molecular level research, to explore the biological behavior of GBC, to find specific biological markers to improve early diagnosis rate, to explore the molecular targeting therapy and to evaluate the prognosis of the disease genes, which is also the focus of the current GBC molecular biology research. The small RNA (micro RNA, MI RNA) is a class of endogenous non coding short chain RNA, about 22 nucleotides size. It mainly mediates posttranscriptional gene regulation and regulates the biological functions of cell proliferation, differentiation and withering by inhibiting the translation of M RNA by complementary pairing with the 3 'UTR incompleteness of the target M RNA. The biological effects such as tumor development, metastasis and other biological effects are closely related to.PTEN, the first protein encoded by the.PTEN gene with double phosphatase activity, which has double specific phosphatase activity of the lipid phosphatase and protein phosphatase, is closely related to the development of a variety of tumors. The expression level of MI RNA-21 in the GBC-SD cell line is closely related to the expression of PTEN. Further experiments have proved that PTEN is the target gene of MI RNA-21 in the GBC-SD cell line. It is preliminarily believed that MI RNA-21 affects the occurrence of GBC and the possible mechanism of development by inhibiting PTEN gene. Mi RNA-21 may be involved in the invasion and migration of GBC. This is a way to further study the mechanism of MI RNA-21 in the biological function of GBC. Method: 1. in GBC-SD cell line silencing micro RNA 21; 1) mi RNA-21 after MI RNA-21, respectively, connected to the associated egg white, protein expression level; 2) in the cell To express / inhibit the expression of MI RNA-21, the protein level of PTEN and the level of M RNA were detected, and 2.Transwell assay was used to detect the cell migration and invasion ability of GBC-SD cell line after MI RNA-21. 3. luciferase test was used to detect the interaction mechanism between PTEN and MI. The expression level of PTEN, ZO-1 and occludin was detected by immunohistochemical SP method; 5. the correlation analysis between MI RNA-21 and clinical parameters, PTEN and other indexes was used. Kaplan-Meier method was used for survival analysis, and the Log-rank test was used to compare the survival rate of the two groups. The joint effect analysis of each co variable by Cox risk proportion model was different. Results: 1. the relationship between micro RNA 21 and PTEN, close connexin was found: 1) after silent micro RNA 21, PTEN expression was up-regulated, Occludin, ZO-1 protein level increased; 2) after silent micro RNA 21, Occludin, ZO-1m expressions were found to be negatively correlated with the expression in the expression of 1) A-21 can down regulate the m RNA and protein expression of PTEN; 2) the inhibition of MI RNA-21 in GBC-SD cells can up regulate m RNA and protein expression of PTEN; 3) the cell migration and invasiveness decreased significantly in GBC-SD cells; 4) 3 'is a target sequence; 5) the analysis of postoperative survival results showed that the prognosis of the patients was and the prognosis of the patients. Inversely. Conclusion: 1. mi RNA21 in GBC-SD cells can be silenced; in vitro experiments confirmed that the expression of MI RNA21 gene is closely related to the expression level of PTEN and close connexin; in further GBC-SD cells and animal experiments, the invasion and metastasis ability of GBC is weakened after the silencing of MI RNA21, and 2. preliminarily believed that PTEN 3 'UTR is in the cells. The target area, MI RNA21, can be involved in the development of GBC by inhibiting the function of the target gene PTEN. The expression of MI RNA21 in the cancer tissue of GBC patients is up regulated, its expression is related to the TNM staging of the tumor, and the high expression of the patient's survival time is short, the prognosis is poor.Mi RNA21 or can be the target target of GBC treatment.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.8

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