本文關(guān)鍵詞： Hsa-miR-27b U251細(xì)胞 Engrailed-2 神經(jīng)系統(tǒng)發(fā)育　出處：《中南大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：前期研究顯示HCMV感染U251細(xì)胞后上調(diào)細(xì)胞hsa-miR-27b表達(dá),通過(guò)生物信息學(xué)分析顯示Egrailed-2(EN2)基因?yàn)閔sa-miR-27b調(diào)控的靶基因之一。本研究的目的是通過(guò)實(shí)驗(yàn)證實(shí)hsa-miR-27b靶向調(diào)控EN2,為進(jìn)一步闡明miRNA表達(dá)改變?cè)贖CMV感染導(dǎo)致神經(jīng)系統(tǒng)發(fā)育畸形中的作用提供實(shí)驗(yàn)基礎(chǔ)。 方法：①采用生物信息學(xué)方法分析hsa-miR-27b靶向調(diào)控EN2的靶點(diǎn)位置和EN2的生物學(xué)功能。②構(gòu)建hsa-miR-27b表達(dá)載體LV3-HmiR-27b,將LV3-HmiR-27b表達(dá)載體轉(zhuǎn)染HEK-293T細(xì)胞,轉(zhuǎn)染后24h和48h,用Real-time RT-PCR檢測(cè)其成熟體]HmiR-27b的表達(dá)水平。③構(gòu)建EN2-mRNA-3'UTR正義鏈和反義鏈表達(dá)載體Psi-EN2-UTR-S和Psi-EN2-UTR-AS。將LV3-HmiR-27b和Psi-EN2-UTR-S/AS共轉(zhuǎn)染HEK-293T細(xì)胞,轉(zhuǎn)染后48h檢測(cè)雙熒光素酶活性值,驗(yàn)證靶點(diǎn)的正確性。④通過(guò)嘌呤霉素篩選LV3-HmiR-27b質(zhì)粒穩(wěn)定轉(zhuǎn)染的U251細(xì)胞,Western blot技術(shù)檢測(cè)EN2蛋白表達(dá)水平。 結(jié)果：(DMicroRNA靶點(diǎn)預(yù)測(cè)數(shù)據(jù)庫(kù)包括Targetscan、 miRanda、PicTar和DIANA-microT均能預(yù)測(cè)到hsa-miR-27b與EN2-mRNA-3'UTR有互補(bǔ)結(jié)合區(qū),包含3個(gè)靶點(diǎn)。1niRo數(shù)據(jù)庫(kù)聯(lián)合靶點(diǎn)數(shù)據(jù)庫(kù)分析EN2參與神經(jīng)元、中腦和間腦的發(fā)育。GO數(shù)據(jù)庫(kù)對(duì)EN2基因分析顯示EN2主要分布于細(xì)胞核內(nèi)和細(xì)胞膜,參與神經(jīng)元、中腦、間腦以及多細(xì)胞機(jī)體的發(fā)育,且正調(diào)控RNA聚合酶II啟動(dòng)子的表達(dá)。②成功構(gòu)建了含有雙熒光素酶報(bào)告基因的表達(dá)載體Psi-EN2-UTR-S/AS。③成功構(gòu)建了Hsa-miR-27b前體表達(dá)載體LV3-HmiR-27b, Real-time RT-PCR檢測(cè)顯示,其成熟體在HEK-293T細(xì)胞中能高水平表達(dá),其表達(dá)水平比空白對(duì)照組高20倍。④雙熒光素酶實(shí)驗(yàn)證明EN2是Hsa-miR-27b的靶基因。⑤通過(guò)嘌呤霉素成功篩選出LV3-HmiR-27b質(zhì)粒穩(wěn)定轉(zhuǎn)染的U251細(xì)胞：與空白對(duì)照組和陰性對(duì)照組(shNC)細(xì)胞比較,LV3-HmiR-27b組U251細(xì)胞呈現(xiàn)細(xì)胞形態(tài)學(xué)改變,主要表現(xiàn)為細(xì)胞變細(xì)長(zhǎng),且神經(jīng)細(xì)胞突起數(shù)量增多；Western blot分析顯示EN2蛋白的表達(dá)水平下降。 結(jié)論：EN2為hsa-miR-27b調(diào)控的靶基因,hsa-miR-27b能下調(diào)U251細(xì)胞EN2蛋白的表達(dá)水平。HCMV可能通過(guò)上調(diào)hsa-miR-27b而下調(diào)EN2的蛋白表達(dá)這一機(jī)制,影響神經(jīng)系統(tǒng)發(fā)育,導(dǎo)致畸形的發(fā)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R739.4

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本文編號(hào)：1455430