长链非编码RNA ROR对胃癌干细胞增殖和侵袭能力以及特性的功能研究

发布时间：2018-07-04 21:35

本文选题：胃癌干细胞 + CD133　；参考：《山东大学》2017年博士论文

【摘要】：第一部分胃癌干细胞的分选与鉴定,以及细胞中lncRNAROR的表达情况。背景:胃癌是一种在全世界范围内发病率位居第四位的恶性肿瘤,在我国肿瘤发病率中,胃癌位列第二,并且肿瘤死亡率中,胃癌位列第三,给人们的健康带来了很大威胁。胃癌具有术后转移、容易复发、耐药等特性,而且至今还没有研究出其调控机制,在这几年中,肿瘤发生发展的相关研究中,研究学者提出了肿瘤干细胞学说。该学说认为肿瘤起源于小部分具有自我更新和多种分化潜能的肿瘤细胞,这部分细胞称为肿瘤干细胞。目前,利用肿瘤干细胞表面特异的标记分子分离和纯化肿瘤干细胞的方法已较为普遍,并且有研究通过利用特异性的细胞表面标志分子CD133,使用流式细胞仪从胃癌中成功分选出胃癌干细胞,并证明了CD133是胃癌干细胞的表面标志物。目的:使用MKN-45细胞构建胃癌干细胞细胞系,并检测其中lncRNA ROR的功能。方法:人MKN-45胃癌细胞用RMPI1640培养液常规培养,制作单细胞悬液,调整细胞浓度后加入CD133-FITC抗体进行分选,孵育后使用流式细胞仪收集被CD133-FITC抗体标记的细胞,即为CD133+MKN-45细胞(简称CD133+);收集余下的未被CD133-FITC抗体标记的细胞,即为CD133-MKN-45细胞(简称CD133-)。并使用免疫荧光法检测CD133+细胞中CD133与CD44v8-10的共表达定位情况;之后使用实时定量PCR法和免疫印迹法检测CD133+和CD133-细胞中OCT4,SOX2和NANOG等肿瘤干细胞标记物的表达。之后,我们使用实时定量PCR法对CD133-和CD133+细胞中lncRNA ROR的表达进行检测,并取患者胃癌组织标本,分析其中lncRNA ROR和CD133表达量之间的关系。结果:免疫荧光法检测结果显示,分选得到的CD133+细胞表面标记物CD133与CD44v8-10有非常好的共定位,这提示我们分选得到的细胞为胃癌肿瘤干细胞;实时定量PCR法和免疫印迹法结果则显示,与CD133-细胞相比,CD133+细胞中肿瘤干细胞标记物OCT4,SOX2和NANOG等出现明显的高表达,这进一步验证了CD133+细胞具有肿瘤干细胞的特质。同时,CD133+细胞中lncRNAROR的表达量较CD133-显著增多,并且患者的组织标本分析也显示lncRNA ROR与CD133+二者表达量呈现显著的正相关。结论:通过流式细胞分选技术,我们使用MKN-45细胞成功构建了胃癌肿瘤干细胞细胞系CD133+,并且测定胃癌干细胞中lncRNA ROR表达显著增高。第二部分lncRNA ROR对胃癌干细胞增殖、侵袭和凋亡能力的影响背景:近年来,lncRNA的研究领域如火如荼,长度超过200个核苷酸的非编码RNA就称之为lncRNA。而该类lncRNA是非编码蛋白质,所以在原来的研究中很多人的观点是其为转录噪音。不过随着研究水平的提高,发现lncRNA不管是表观遗传、翻译水平、转录水平等各个层次中都能够对基因表达产生调控影响,同时在多种肿瘤中都有其身影。Loewer等人员还在研究中得出lncRNA ROR与一些干细胞的关键转录因子存在着相互作用,在干细胞的重编程过程中发挥重要作用。Dewi、Lagadec等研究则发现与诱导干细胞相似,肿瘤干细胞参与相同的重编程过程来维持肿瘤的生长。KLF4和SOX11这两个基因与肿瘤干细胞的功能有关联,并且这两个基因与lncRNA ROR密切相关,因此lncRNA ROR可能是胃癌干细胞一个新的治疗靶点。综上所述,探究lncRNA ROR在胃癌干细胞中的功能势在必行,这为将lncRNA ROR作为治疗靶点提供理论基础。目的:探究lncRNA ROR对胃癌干细胞增殖、侵袭和凋亡能力的影响。方法:为了更好的探究lncRNA ROR在胃癌干细胞中的功能,我们将成功建立了lncRNA ROR的过表达系统以及敲低系统。我们将该过表达质粒转染至CD133-细胞中,外源性过表达lncRNAROR;同时,将能够特异性抑制lncRNA ROR的siRNA转染至CD133+细胞中,敲低内源性lncRNA ROR的表达。然后,在两组中,我们使用MTT法和EdU掺入法检测改变lncRNA ROR表达对细胞增殖能力的影响。随后,在处理细胞24 h后,我们使用Transwel1法检测改变lncRNA ROR表达对细胞侵袭能力的影响。最后,我们利用流式细胞术使用Annexin V-FITC/PI双染法检测改变lncRNA ROR表达对细胞凋亡能力的影响。结果:MTT法、EdU掺入法、Transwell法以及流式细胞术检测结果显示,在CD133-细胞中过表达lncRNA ROR能够显著提高细胞的增殖和侵袭能力,但对细胞凋亡能力影响并不大。与此相反,在CD133+细胞中抑制内源性lncRNAROR则能够明显抑制细胞的增殖和侵袭能力,并且能够促进细胞的凋亡能力。结论:内源性lncRNA ROR对胃癌干细胞增殖、侵袭以及抑制凋亡能力至关重要。第三部分lncRNA ROR能够调控维持胃癌干细胞特性的转录因子的表达背景:研究结果显示,干细胞基因的表达不正常,和恶性肿瘤发生有密切关系。而且对于胃癌的出现、发展、复发、转移过程中都有关键性影响。干细胞转录因子会使得有关基因簇2(sexdeterminingregionY-box2,SOX2)表达不正常时,致使胃粘膜上皮细胞分化失衡紊乱,引发肿瘤出现;在细胞多能性的特点中,能够维持该功能的标志物是八聚体结合蛋白-4(octamerbindingfactor4,OCT4),该蛋白能够和细胞分化有密切关系;Nanog与体细胞实体瘤的关系也日益受到关注,NANOG具有激发肿瘤形成的干细胞样特性。因此,本研究以胃癌干细胞为研究对象,改变内源性lncRNA ROR表达后,探讨lncRNA ROR对维持胃癌干细胞特性的重要转录因子的作用,为胃癌的临床治疗和预后判断提供可能的参考依据。目的:探究lncRNA ROR对胃癌干细胞特性的影响。方法:我们将该过表达质粒转染至CD133-细胞中,外源性过表达lncRNAROR;同时,将能够特异性抑制lncRNA ROR的siRNA转染至CD133+细胞中,敲低内源性lncRNAROR的表达。然后,在两组中,我们使用实时定量PCR法和免疫印迹法检测维持胃癌干细胞特性的关键转录因子OCT4、SOX2和NANOG的表达水平。结果:实时定量PCR法和免疫印迹法检测结果显示,在CD133-细胞中过表达lncRNA ROR能够有效的上调OCT4、SOX2和NANOG的表达水平。与此相反,在CD133+细胞中抑制内源性lncRNA ROR则能够明显抑制OCT4、SOX2和NANOG的表达水平。结论:胃癌干细胞中,内源性lncRNA ROR的高表达能够通过上调OCT4、SOX2和NANOG等的表达水平,有效的维持胃癌干细胞特性。
[Abstract]:The first part is the separation and identification of gastric cancer stem cells and the expression of lncRNAROR in the cells. Background: gastric cancer is one of the fourth malignant tumors in the world. In the incidence of cancer, gastric cancer ranks second in our country, and the number of gastric cancer is third, which poses a great threat to people's health. Gastric cancer has the characteristics of postoperative metastasis, recurrence, drug resistance, and so far the regulatory mechanism has not been studied. In these years, the researchers have proposed the theory of tumor stem cells in the development of tumor. This theory suggests that the tumor originates in a small part of tumor cells with self renewal and multiple differentiation potential. The cells are called tumor stem cells. At present, the method of separating and purifying cancer stem cells with specific marker molecules on the surface of cancer stem cells is more common, and the research has been done by using the specific cell surface marker molecule CD133, using flow cytometry to separate the gastric cancer stem cells from gastric cancer, and prove that CD133 is the stomach. The surface markers of cancer stem cells. Objective: to construct the cell line of gastric cancer stem cells using MKN-45 cells and detect the function of lncRNA ROR. Methods: human MKN-45 gastric cancer cells were cultured in RMPI1640 culture medium, made single cell suspension, and adjusted the cell concentration after adding CD133-FITC anti body, and then used the flow cytometry to collect the cells. The cells labeled by CD133-FITC, namely, CD133+MKN-45 cells (CD133+), collect the remaining cells that are not labeled with CD133-FITC antibodies, that is, CD133-MKN-45 cells (called CD133-). The co expression of CD133 and CD44v8-10 in CD133+ cells is detected by immunofluorescence; and then the real-time quantitative PCR method and immunoblotting are used. The expression of OCT4, SOX2, NANOG and other tumor stem cell markers in CD133+ and CD133- cells was detected by method. The expression of lncRNA ROR in CD133- and CD133+ cells was detected by real-time quantitative PCR method, and the relationship between lncRNA ROR and the amount of expression was analyzed. Results: immunofluorescence assay The results showed that the selected CD133+ cell surface markers CD133 and CD44v8-10 were well Co located, which suggested that the selected cells were cancer stem cells of gastric cancer. Real-time quantitative PCR and immunoblotting results showed that the markers of OCT4, SOX2 and NANOG in CD133+ cells were compared with CD133- cells. Obvious high expression, which further verified the characteristics of CD133+ cells with tumor stem cells. At the same time, the expression of lncRNAROR in CD133+ cells was significantly higher than that of CD133-, and the tissue specimen analysis of the patients showed that the expression of lncRNA ROR was positively correlated with the expression of CD133+ two. Conclusion: we use flow cytometry, we use MKN-45 cells successfully constructed the cancer stem cell line CD133+, and the expression of lncRNA ROR in gastric cancer stem cells was significantly increased. Second the impact of lncRNA ROR on the proliferation, invasion and apoptosis of gastric cancer stem cells: in recent years, the research field of lncRNA is like a raging fire, and the non coded RNA with a length of more than 200 nucleotides is known as a non coded RNA. It is lncRNA. and this class of lncRNA is a non coding protein, so in the previous study, many people have the view that they are transcriptional noise. However, as the level of research is raised, it is found that lncRNA can regulate the expression of genes at all levels, regardless of epigenetic, translation and transcriptional levels, as well as in many kinds of tumors. .Loewer and other people have also found that lncRNA ROR has interaction with key transcription factors of some stem cells, plays an important role in the reprogramming of stem cells,.Dewi, Lagadec and other studies have found that the stem cells are similar to induced stem cells, and the tumor stem cells participate in the same reprogramming process to maintain the growth of the tumor.KL The two genes of F4 and SOX11 are associated with the function of cancer stem cells, and these two genes are closely related to lncRNA ROR. Therefore, lncRNA ROR may be a new target for the treatment of gastric cancer stem cells. To sum up, it is imperative to explore the function of lncRNA ROR in gastric cancer stem cells, which provides the theoretical basis for the lncRNA ROR as a target for treatment. Objective: To explore the effect of lncRNA ROR on the proliferation, invasion and apoptosis of gastric cancer stem cells. Methods: in order to better explore the function of lncRNA ROR in gastric cancer stem cells, we will successfully establish the overexpression system of lncRNA ROR and the knockout system. We transfect the overexpressed particles into CD133- cells and express the exogenous lncRNA. In the two groups, we used MTT and EdU incorporation to detect the effect of lncRNA ROR expression on cell proliferation ability in the two groups. Then, after processing cell 24 h, we used the method to detect the change of siRNA after 24 h. In the two groups, we used the MTT method and EdU incorporation method to detect the effect of the expression of lncRNA ROR on the cell proliferation. The effect of OR expression on cell invasiveness. Finally, we used Annexin V-FITC/PI double staining to detect the effect of lncRNA ROR expression on the cell apoptosis ability. Results: MTT, EdU incorporation, Transwell and flow cytometry showed that the overexpression of lncRNA ROR in CD133- cells could be significantly improved. Cell proliferation and invasion ability, but not to cell apoptosis, in contrast, the inhibition of endogenous lncRNAROR in CD133+ cells can significantly inhibit cell proliferation and invasion ability, and can promote cell apoptosis. Conclusion: endogenous lncRNA ROR proliferation, invasion and inhibition of apoptosis in gastric cancer stem cells The third part, the third part, can regulate the expression background of the transcription factors that maintain the characteristics of gastric cancer stem cells. The results show that the expression of stem cell genes is abnormal and has a close relationship with the occurrence of malignant tumors. It also has a critical effect on the occurrence, development, recurrence and metastasis of gastric cancer. Stem cell transcription factors will be important. When the expression of the gene cluster 2 (sexdeterminingregionY-box2, SOX2) is abnormal, the differentiation of the epithelial cells of the gastric mucosa is unbalanced and causes the appearance of the tumor. In the characteristics of cell pluripotent, the marker that can maintain the function is the eight polymer binding protein -4 (octamerbindingfactor4, OCT4), which can be closely related to the differentiation of cells. The relationship between Nanog and somatic solid tumor is becoming more and more concerned, and NANOG has the stem cell like characteristics to stimulate the formation of tumor. Therefore, this study takes gastric cancer stem cells as the research object, changes the endogenous lncRNA ROR expression, and explores the role of lncRNA ROR in maintaining the important transcription factors of gastric cancer stem cell specificity for the clinical treatment of gastric cancer. Objective: to provide a possible reference for the treatment and prognosis. Objective: To explore the effect of lncRNA ROR on the characteristics of gastric cancer stem cells. Methods: We transfected the overexpressed plasmid into CD133- cells and overexpressed lncRNAROR; meanwhile, we transfected the siRNA of lncRNA ROR to CD133+ cells and knocked down the expression of low endogenous lncRNAROR. Then, in the two groups, we used real-time quantitative PCR and immunoblotting to detect the expression level of key transcription factors, OCT4, SOX2 and NANOG, to maintain the characteristics of gastric cancer stem cells. Results: real-time quantitative PCR and Western blot detection results showed that the overexpression of lncRNA ROR in CD133- cells could effectively increase the OCT4, SOX2 and NANOG tables. In contrast, inhibition of endogenous lncRNA ROR in CD133+ cells can significantly inhibit the expression level of OCT4, SOX2 and NANOG. Conclusion: the high expression of endogenous lncRNA ROR in gastric cancer stem cells can effectively maintain the characteristics of gastric cancer stem cells by up regulation of the expression level of OCT4, SOX2 and NANOG.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2

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