血清饥饿条件下microRNA表达谱的变化及其功能研究

发布时间：2018-02-23 13:38

本文关键词： 微小RNA 靶基因细胞凋亡基因表达　出处：《天津医科大学》2007年硕士论文　论文类型：学位论文

【摘要】：目的： MicroRNA (miRNA)是一类新发现的能够调控其靶基因表达水平的小RNA分子。近几年已经发现并确定了上千种miRNA,目前1miRNA的功能研究正日益成为生物学一个非常活跃的研究领域。本研究旨在结合生物信息学、基因芯片技术检测血清饥饿状态下参与调控的miRNA并预测其靶基因,对血清饥饿状态下参与调控的miRNA对细胞活性的影响进行初步研究。方法：利用miRNA芯片技术对血清饥饿培养条件下(不含血清的培养基)和正常培养条件下(含10%血清的培养基)人类正常肝细胞系L02中miRNA的表达水平进行比较。对在血清饥饿条件下表达增高的miRNA(miR-27a、miR-320、 miR-338)使用miRNA反义寡聚核苷酸序列特异性抑制细胞内：niRNA (miR-27a、miR-320、miR-338)功能；对在血清饥饿条件下表达降低的miRNA (miR-181b1)使用小干扰RNA特异性增强细胞内miRNA (miR-181b1)功能；然后利用MTT法分析miRNA对细胞活性的影响。然后进行TUNEL法测定它们分别对细胞凋亡的影响。在此基础上结合靶基因预测程序和mRNA表达谱芯片的结果对miRNA的靶基因进行预测和筛选。结果：通过对miRNA表达谱基因芯片结果的分析发现在血清饥饿状态下人类正常肝细胞系L02中miR-27a、miR-320、miR-338的表达升高,miR-181b1的表达下降。随后利用MTT法和TUNEL法实验对miR-27a、miR-320、miR-338、 miR-181bl的功能进行筛选分析,发现在L02细胞中miR-27a、miR-320、miR-338功能受到抑制后细胞的活性均明显受到抑制,miR-181b1功能增强后细胞的活性明显被增强。在此基础上利用长寡聚核苷酸基因芯片(共7267个人类基因,包含凋亡相关基因、肿瘤相关基因、信号转导相关基因、肝酶代谢相关基因、细胞因子相关基因)分析血清饥饿状态下mRNA表达谱的变化,选取表达有差异基因,经基因芯片结果分析与靶基因预测程序相比较初步预测：adcy3、bc13、h1x1等是hsa-miR-27a的靶基因,ctnnb1、ywhaq、adcy3等是hsa-miR-320的靶基因,tps19、p4hb、atp5gl等是hsa-miR-338的靶基因,bcl-9、ddit4、dusp10等是hsa-miR-181b-1的靶基因。结论：本文首次确定了血清饥饿状态下参与调控的miRNA,并初步确定了miR-27a、miR-320、miR-338和miR-181b1在人类细胞内的靶基因。miR-27a、 miR-320、miR-338和miR-181b1可以分别介导其各自的靶mRNA的降解,调控血清饥饿状态下细胞的应答反应。这有助于我们深化对于细胞的生长和凋亡等生物学特性及其调控机制的理解。
[Abstract]:Objective:. MicroRNA simiRNAs are a new class of small RNA molecules which can regulate the expression level of their target genes. In recent years, thousands of miRNAs have been discovered and identified. At present, the functional study of 1miRNAs is becoming a very active research field in biology. The purpose of this study is to combine bioinformatics, The miRNA involved in the regulation of serum starvation was detected by gene chip technique and its target gene was predicted. The effect of miRNA on cell activity was studied preliminarily. Methods:. MiRNA microarray technique was used to compare the expression of miRNA in human normal liver cell line L02 under the condition of serum starvation (medium without serum) and normal culture (medium containing 10% serum). MiRNA antisense oligonucleotide sequence was used to specifically inhibit the function of miRNA antisense oligodeoxynucleotides (miRNA antisense oligodeoxynucleotides). Low expression of miRNA miR-181b1) was used to enhance intracellular miRNA miR-181b1 function with small interfering RNA. Then the effect of miRNA on cell activity was analyzed by MTT method, and then the effect of miRNA on cell apoptosis was determined by TUNEL method. Based on this, the target genes of miRNA were predicted and screened by combining the results of target gene prediction program and mRNA expression microarray. Results:. The results of miRNA gene chip analysis showed that the expression of miR-27a1 miR-320mmiR-338 increased in human normal liver cell line L02 under the condition of serum starvation, and the expression of miR-181b1 was decreased. Then the function of miR-27aF- 320miR-338, miR-181bl was screened by MTT and TUNEL assay. It was found that in L02 cells, the activity of miR-27aanfmiR-320miR-338 was obviously inhibited after the function of miR-181b1 was enhanced. Using oligonucleotide microarray (7267 human genes, including apoptosis-related genes, tumor-related genes, signal transduction related genes, liver enzyme metabolism-related genes, etc. Cytokine related genes (cytokine related genes) were used to analyze the changes of mRNA expression profile under the condition of serum starvation, and the differentially expressed genes were selected. The results of microarray analysis were compared with the program of target gene prediction. It was preliminarily predicted that the target gene of hsa-miR-27a is the target gene of hsa-miR-27a, ttnnb1, ywhaqadcy3 and so on. It is the target gene of hsa-miR-320, tps19, p4hbnatp5gl, etc. It is the target gene of hsa-miR-338, bcl-9DDIT4dusp10 and so on, which is the target gene of hsa-miR-181b-1. Conclusion:. In this paper, we first identified the miRNAs involved in the regulation under the condition of serum starvation, and preliminarily determined that the target genes. MiR-27a, miR-320miR-338 and miR-181b1 can mediate the degradation of their respective target mRNA, respectively. It is helpful to understand the biological characteristics of cell growth and apoptosis and its regulatory mechanism.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R3416

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