Sema3C在神经胶质瘤中的功能及分子调控机制研究
发布时间：2019-04-27 14:35
【摘要】：目的:探讨在胶质瘤中调控Semaphorin3c表达调控机制。方法:1、生物信息学软件分析和预测可能调控Sema3C表达的microRNA:(1)GeneBank中输入Sema3C基因,找出该基因mRNA的3'UTR序列。(2)采用TargetScan和miRanDa软件,将Sema3C mRNA的3'UTR序列输入其中,评分选择可能调控Sema3C表达的5-10个候选microRNA。(3)通过PubMed软件进行文献检索,在2中选择的5-10个候选microRNA中进一步缩小范围,将待检测目的microRNA缩小至2-3个。2、检测microRNA对Sema3C表达的调控:(1)订制针对人microRNA的模拟物(mimics)和抑制剂。(2)体外常规培养人胶质瘤细胞系(A172和U251),采用Lipotamine2000转染法将目的microRNA的模拟物(mimics)和抑制剂,及其相应的对照(模拟物对照和抑制剂对照)分别转染入以上细胞系,提取总蛋白和总RNA,Westernblot和荧光定量PCR法检测Sema3C的表达。3、检测待选microRNA是否可影响Sema3C mRNA的3'UTR的活性:(1)提取人胶质瘤细胞系A172的总RNA,进行cDNA的反转录,扩增软件中预测出可能有microRNA结合的序列,将其连接到pmirGLo质粒上。(2)将microRNA的模拟物和抑制剂及其对照、含有Sema3C mRNA的3'UTR的pmirGLo载体等分别转染入HEK293T细胞中,对细胞样品进行裂解后检测各组荧光信号值。4、收集胶质瘤组织(约30例)和周边正常脑组织(约30例)后,进行如下实验:(1)提取组织总RNA和总蛋白,Western blot和实时荧光定量PCR法检测Sema3C和microRNA的表达。(2)统计学分析Sema3C和microRNA的表达在胶质瘤和周边癌组织中是否存在相关性。结果:1、Sema3C的组织学表达特点免疫组织化学法检测发现Sema3C在胶质瘤中的过表达的频率(78.2%)明显高于周边正常组织(20.0%);低表达Sema3C的胶质瘤患者预后较好(p=0.017);临床指标分析发现,其表达量与组织学分型(p=0.008)和病理分级(p=0.002)相关;在Sema3C表达量较高的组织中IDH1(可溶性异柠檬酸脱氢酶1)突变的发生率低(p=0.0001);此外,还与Ki67指数相关(p=0.02)。2、Sema3C的细胞学功能沉默Sema3C表达后,细胞的增殖和侵袭能力明显降低;对增殖能力的调控是通过影响细胞周期实现的;Sema3C可通过改变细胞的上皮-间质转换影响细胞的侵袭能力等。3、Sema3C致病机制的研究Sema3C在胶质瘤细胞中受mi R-142-5p的表达调控,miR-142-5p是通过影响Sema3C m RNA的翻译水平实现的;miR-142-5p也可调控胶质瘤细胞的增殖和侵袭能力,并且该作用的发挥也可能通过影响细胞的上皮-间质细胞转化而发生。4、Sema3C和miR-142-5p组织表达相关性miR-142-5p在胶质瘤组织的表达随着级别的升高而降低;Sema3C和miR-142-5p在胶质瘤组织中的表达成负相关。结论:1、Sema3C在胶质瘤中可作为不良预后指标用于临床诊断方面的继续研究;2、Sema3C在胶质瘤中发挥癌基因的作用,该作用的发挥可能通过调控上皮-间质细胞转化而实现;3、miR-142-5p在胶质瘤中负向调控Sema3C的表达,提示在胶质瘤病变过程中可能存在miR-142-5p/Sema3C致病途径。
[Abstract]:Objective: to investigate the regulation mechanism of Semaphorin3c expression in glioma. Methods: 1. Bioinformatics software was used to analyze and predict the input of Sema3C gene into microRNA: (1) GeneBank, which might regulate the expression of Sema3C. (2) the 3'UTR sequence of Sema3C mRNA was inputted into it by TargetScan and miRanDa software. The 5 / 10 candidate microRNA. (3), which may regulate the expression of Sema3C, were retrieved by PubMed software and further narrowed in the 5 / 10 candidate microRNA selected in 2, and the target microRNA to be detected was reduced to 2. 2. To detect the regulation of Sema3C expression by microRNA: (1) (mimics) and inhibitors targeting human microRNA were made. (2) Human glioma cell lines (A172 and U251) were cultured routinely in vitro. The target microRNA mimicants (mimics) and inhibitors were transfected by Lipotamine2000. And the corresponding control (mock control and inhibitor control) were transfected into the above cell lines respectively. The total protein and total RNA,Westernblot were extracted and the expression of Sema3C was detected by fluorescence quantitative PCR. To detect whether microRNA could affect the activity of 3'UTR of Sema3C mRNA: (1) the total RNA, of human glioma cell line A172 was extracted for reverse transcription of cDNA, and the sequence of microRNA binding was predicted in the amplification software. (2) the mimics and inhibitors of microRNA and their control, pmirGLo vector containing Sema3C mRNA 3'UTR and so on were transfected into HEK293T cells respectively, and the fluorescent signal values of each group were detected after the cell samples were lysed. After collecting glioma tissues (about 30 cases) and peripheral normal brain tissues (about 30 cases), the following experiments were carried out: (1) Total RNA and total protein were extracted from the tissues. Western blot and real-time fluorescence quantitative PCR were used to detect the expression of Sema3C and microRNA. (2) the correlation between the expression of Sema3C and microRNA in glioma and peripheral carcinoma was analyzed statistically. Results: (1) Immunohistochemistry showed that the frequency of Sema3C overexpression in glioma (78.2%) was significantly higher than that in peripheral normal tissues (20.0%). The prognosis of glioma patients with low expression of Sema3C was better (p < 0. 017), and the expression level was correlated with histological type (p < 0. 008) and pathological grade (p < 0. 002), and the expression level was correlated with histological type (p < 0. 008). The incidence of IDH1 (soluble isocitrate dehydrogenase 1) mutation was lower in the tissues with higher expression of Sema3C (p < 0.0001). In addition, it was also correlated with Ki67 index (p = 0.02). 2. After silencing Sema3C expression by cytological function of Sema3C, the ability of cell proliferation and invasion was significantly decreased, and the regulation of proliferation ability was realized by affecting the cell cycle. Sema3C can affect the invasiveness of glioma cells by changing the epithelial-interstitial transformation. 3. The study of the pathogenesis of Sema3C to investigate the regulation of Sema3C expression in glioma cells by the expression of mi R _ 2 ~ 2 ~ 2 ~ (5) p, P < 0.01, P < 0.05, P < 0.05. MiR-142-5p is realized by influencing the translation level of Sema3C m RNA; MiR-142-5p can also regulate the proliferation and invasion of glioma cells, and this effect may also occur by affecting epithelial-stromal cell transformation. 4, The expression of Sema3C and miR-142-5p-related miR-142-5p in glioma tissues decreased with the increase of grade. There was a negative correlation between the expression of Sema3C and miR-142-5p in glioma. Conclusion: (1) Sema3C can be used as an indicator of poor prognosis for further study in clinical diagnosis, and Sema3C may play an oncogene role in gliomas, which may be achieved by regulating epithelial-stromal cell transformation. 3. The negative regulation of the expression of Sema3C in gliomas suggests that miR-142-5p/Sema3C may be involved in the pathogenesis of glioma.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.4
本文编号：2467058
[Abstract]:Objective: to investigate the regulation mechanism of Semaphorin3c expression in glioma. Methods: 1. Bioinformatics software was used to analyze and predict the input of Sema3C gene into microRNA: (1) GeneBank, which might regulate the expression of Sema3C. (2) the 3'UTR sequence of Sema3C mRNA was inputted into it by TargetScan and miRanDa software. The 5 / 10 candidate microRNA. (3), which may regulate the expression of Sema3C, were retrieved by PubMed software and further narrowed in the 5 / 10 candidate microRNA selected in 2, and the target microRNA to be detected was reduced to 2. 2. To detect the regulation of Sema3C expression by microRNA: (1) (mimics) and inhibitors targeting human microRNA were made. (2) Human glioma cell lines (A172 and U251) were cultured routinely in vitro. The target microRNA mimicants (mimics) and inhibitors were transfected by Lipotamine2000. And the corresponding control (mock control and inhibitor control) were transfected into the above cell lines respectively. The total protein and total RNA,Westernblot were extracted and the expression of Sema3C was detected by fluorescence quantitative PCR. To detect whether microRNA could affect the activity of 3'UTR of Sema3C mRNA: (1) the total RNA, of human glioma cell line A172 was extracted for reverse transcription of cDNA, and the sequence of microRNA binding was predicted in the amplification software. (2) the mimics and inhibitors of microRNA and their control, pmirGLo vector containing Sema3C mRNA 3'UTR and so on were transfected into HEK293T cells respectively, and the fluorescent signal values of each group were detected after the cell samples were lysed. After collecting glioma tissues (about 30 cases) and peripheral normal brain tissues (about 30 cases), the following experiments were carried out: (1) Total RNA and total protein were extracted from the tissues. Western blot and real-time fluorescence quantitative PCR were used to detect the expression of Sema3C and microRNA. (2) the correlation between the expression of Sema3C and microRNA in glioma and peripheral carcinoma was analyzed statistically. Results: (1) Immunohistochemistry showed that the frequency of Sema3C overexpression in glioma (78.2%) was significantly higher than that in peripheral normal tissues (20.0%). The prognosis of glioma patients with low expression of Sema3C was better (p < 0. 017), and the expression level was correlated with histological type (p < 0. 008) and pathological grade (p < 0. 002), and the expression level was correlated with histological type (p < 0. 008). The incidence of IDH1 (soluble isocitrate dehydrogenase 1) mutation was lower in the tissues with higher expression of Sema3C (p < 0.0001). In addition, it was also correlated with Ki67 index (p = 0.02). 2. After silencing Sema3C expression by cytological function of Sema3C, the ability of cell proliferation and invasion was significantly decreased, and the regulation of proliferation ability was realized by affecting the cell cycle. Sema3C can affect the invasiveness of glioma cells by changing the epithelial-interstitial transformation. 3. The study of the pathogenesis of Sema3C to investigate the regulation of Sema3C expression in glioma cells by the expression of mi R _ 2 ~ 2 ~ 2 ~ (5) p, P < 0.01, P < 0.05, P < 0.05. MiR-142-5p is realized by influencing the translation level of Sema3C m RNA; MiR-142-5p can also regulate the proliferation and invasion of glioma cells, and this effect may also occur by affecting epithelial-stromal cell transformation. 4, The expression of Sema3C and miR-142-5p-related miR-142-5p in glioma tissues decreased with the increase of grade. There was a negative correlation between the expression of Sema3C and miR-142-5p in glioma. Conclusion: (1) Sema3C can be used as an indicator of poor prognosis for further study in clinical diagnosis, and Sema3C may play an oncogene role in gliomas, which may be achieved by regulating epithelial-stromal cell transformation. 3. The negative regulation of the expression of Sema3C in gliomas suggests that miR-142-5p/Sema3C may be involved in the pathogenesis of glioma.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.4
【参考文献】
相关期刊论文 前1条
1 曾冉;况建国;;胶质瘤发病机制的研究进展[J];广东医学;2013年06期
，本文编号：2467058
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