Hsa-miR-27b调控人神经胶质瘤U251细胞Engrailed-2基因表达的初步研究

发布时间：2018-01-22 18:25

本文关键词： Hsa-miR-27b U251细胞 Engrailed-2 神经系统发育　出处：《中南大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：前期研究显示HCMV感染U251细胞后上调细胞hsa-miR-27b表达,通过生物信息学分析显示Egrailed-2(EN2)基因为hsa-miR-27b调控的靶基因之一。本研究的目的是通过实验证实hsa-miR-27b靶向调控EN2,为进一步阐明miRNA表达改变在HCMV感染导致神经系统发育畸形中的作用提供实验基础。方法：①采用生物信息学方法分析hsa-miR-27b靶向调控EN2的靶点位置和EN2的生物学功能。②构建hsa-miR-27b表达载体LV3-HmiR-27b,将LV3-HmiR-27b表达载体转染HEK-293T细胞,转染后24h和48h,用Real-time RT-PCR检测其成熟体]HmiR-27b的表达水平。③构建EN2-mRNA-3'UTR正义链和反义链表达载体Psi-EN2-UTR-S和Psi-EN2-UTR-AS。将LV3-HmiR-27b和Psi-EN2-UTR-S/AS共转染HEK-293T细胞,转染后48h检测双荧光素酶活性值,验证靶点的正确性。④通过嘌呤霉素筛选LV3-HmiR-27b质粒稳定转染的U251细胞,Western blot技术检测EN2蛋白表达水平。结果：(DMicroRNA靶点预测数据库包括Targetscan、 miRanda、PicTar和DIANA-microT均能预测到hsa-miR-27b与EN2-mRNA-3'UTR有互补结合区,包含3个靶点。1niRo数据库联合靶点数据库分析EN2参与神经元、中脑和间脑的发育。GO数据库对EN2基因分析显示EN2主要分布于细胞核内和细胞膜,参与神经元、中脑、间脑以及多细胞机体的发育,且正调控RNA聚合酶II启动子的表达。②成功构建了含有双荧光素酶报告基因的表达载体Psi-EN2-UTR-S/AS。③成功构建了Hsa-miR-27b前体表达载体LV3-HmiR-27b, Real-time RT-PCR检测显示,其成熟体在HEK-293T细胞中能高水平表达,其表达水平比空白对照组高20倍。④双荧光素酶实验证明EN2是Hsa-miR-27b的靶基因。⑤通过嘌呤霉素成功筛选出LV3-HmiR-27b质粒稳定转染的U251细胞：与空白对照组和阴性对照组(shNC)细胞比较,LV3-HmiR-27b组U251细胞呈现细胞形态学改变,主要表现为细胞变细长,且神经细胞突起数量增多；Western blot分析显示EN2蛋白的表达水平下降。结论：EN2为hsa-miR-27b调控的靶基因,hsa-miR-27b能下调U251细胞EN2蛋白的表达水平。HCMV可能通过上调hsa-miR-27b而下调EN2的蛋白表达这一机制,影响神经系统发育,导致畸形的发生。
[Abstract]:Objective: previous studies showed that HCMV upregulated the expression of hsa-miR-27b in U251 cells. Bioinformatics analysis showed that Egrailed-2en _ 2). The aim of this study is to confirm the target regulation of EN2 by hsa-miR-27b through experiments. To further elucidate the role of miRNA expression changes in HCMV infection resulting in nervous system deformities. Methods:. 1 using bioinformatics method to construct hsa-miR-27b expression vector LV3-Hm by analyzing the target position of EN2 regulated by hsa-miR-27b and the biological function of EN2. 2. IR-27b. LV3-HmiR-27b expression vector was transfected into HEK-293T cells at 24 h and 48 h after transfection. Using Real-time. Construction of EN2-mRNA-3'UTR sense chain and antisense strand expression vectors Psi-EN2-UTR-S and P. LV3-HmiR-27b and Psi-EN2-UTR-S/AS were co-transfected into HEK-293T cells. 48 h after transfection, double luciferase activity was detected to verify the correctness of target. 4. The LV3-HmiR-27b plasmid was screened by purine mycin for stable transfection of U251 cells. Western blot technique was used to detect the expression of EN2 protein. Results\\\. Both PicTar and DIANA-microT could predict the complementary binding region between hsa-miR-27b and EN2-mRNA-3'UTR. Three targets. 1niRo database combined with target database were used to analyze the neurons involved in EN2. The analysis of EN2 gene in mesencephalon and diencephalon database showed that EN2 mainly distributed in nucleus and cell membrane and was involved in the development of neurons, mesencephalon, diencephalon and multicellular organism. The expression of RNA polymerase II promoter was regulated. 2. The expression vector Psi-EN2-UTR-S/AS.3 containing double luciferase reporter gene was successfully constructed and Hsa-mi was constructed successfully. R-27b precursor expression vector LV3-HmiR-27b. Real-time RT-PCR assay showed that its mature bodies were highly expressed in HEK-293T cells. Its expression level was 20 times higher than that in the blank control group. The double luciferase assay proved that EN2 was the target gene of Hsa-miR-27b. 5. 5 LV3-HmiR-27b was successfully screened by purine mycin. Plasmid stably transfected U251 cells:. Compared with blank control group and negative control group (. ShNC-) cells were compared. In LV3-HmiR-27b group, U251 cells showed morphological changes, mainly as the cells became slender and the number of neuronal processes increased. Western blot analysis showed that the expression of EN2 protein decreased. Conclusion: 1. EN2 is a target gene regulated by hsa-miR-27b. Hsa-miR-27b can down-regulate the expression of EN2 protein in U251 cells. HCMV may down-regulate the expression of EN2 protein by up-regulating hsa-miR-27b. Affect the development of the nervous system, leading to the occurrence of malformation.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.4

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