發(fā)布時(shí)間：2018-09-04 14:39

【摘要】：子癇前期（pre-eclampsia，PE）是一組妊娠與高血壓共存的疾病，這種疾病嚴(yán)重影響著母嬰健康，是孕產(chǎn)婦及圍產(chǎn)兒死亡率升高的主要原因之一。然而，子癇前期的發(fā)病機(jī)制迄今為止尚未完全明確，該疾病的預(yù)防和治療的進(jìn)展受到嚴(yán)重阻礙，因此，深入研究子癇前期的發(fā)生發(fā)展機(jī)制具有重要的臨床指導(dǎo)意義。微小RNA(microRNA，miRNA)是一種能夠調(diào)節(jié)蛋白表達(dá)但不能編碼蛋白的小RNA，能夠降解靶基因mRNA或者阻礙其翻譯，在基因表達(dá)調(diào)控中具有重要的作用。有研究表明miRNAs在調(diào)控細(xì)胞周期和生命體的生長(zhǎng)發(fā)育中起了重要的作用，子癇前期以及其它妊娠相關(guān)疾病的發(fā)生與胎盤(pán)中miRNAs的表達(dá)失衡密切相關(guān)[1]。我們課題組在前期研究中利用基因芯片技術(shù)及熒光實(shí)時(shí)定量PCR技術(shù)證實(shí)了miR-30a-3p在子癇前期患者胎盤(pán)中的表達(dá)特異性升高，并開(kāi)展體外實(shí)驗(yàn)，研究miR-30a-3p表達(dá)水平對(duì)人正常滋養(yǎng)細(xì)胞系HTR-8/SVneo細(xì)胞功能的影響，結(jié)果發(fā)現(xiàn)，miR-30a-3p表達(dá)水平與HTR-8/SVneo細(xì)胞的凋亡率呈正相關(guān)，我們推測(cè)在胎盤(pán)中miR-30a-3p表達(dá)的升高與子癇前期發(fā)病相關(guān)。然而，miR-30a-3p具體是通過(guò)調(diào)控哪個(gè)或者哪些靶基因而起作用？相關(guān)靶基因又在子癇前期的發(fā)病中扮演了怎樣的角色？為解決這些問(wèn)題,我們進(jìn)一步開(kāi)展了對(duì)miR-30a-3p子癇前期相關(guān)靶基因的預(yù)測(cè)和驗(yàn)證的研究。 目的 1.預(yù)測(cè)miR-30a-3p的靶基因，并從中篩查與子癇前期有關(guān)的基因?yàn)槟康幕颉?2.明確miR-30a-3p對(duì)靶基因的調(diào)控關(guān)系和作用位點(diǎn)。 方法 1.利用生物信息軟件預(yù)測(cè)miR-30a-3p的靶基因，根據(jù)候選靶基因在生物信息軟件中的分值及其與子癇前期相關(guān)性進(jìn)行篩選。 2．利用脂質(zhì)體轉(zhuǎn)染技術(shù)在人正常滋養(yǎng)細(xì)胞系HTR-8/Svneo細(xì)胞中建立miR-30a-3p過(guò)表達(dá)及抑制表達(dá)模型，利用熒光實(shí)時(shí)定量PCR技術(shù)和Western-blot技術(shù)檢測(cè)細(xì)胞模型中的miR-30a-3p與靶基因的表達(dá)水平的相關(guān)性。 3．構(gòu)建目的基因的熒光素酶報(bào)告載體，利用雙熒光素酶載體報(bào)告系統(tǒng)驗(yàn)證miR-30a-3p與靶基因的靶向關(guān)系和結(jié)合位點(diǎn)。 結(jié)果 1．生物信息預(yù)測(cè)軟件預(yù)測(cè)結(jié)果表明：IGF-1為miR-30a-3p的理論靶基因之一，其mRNA序列的3′UTR區(qū)含有五個(gè)理論上可以與miR-30a-3p相結(jié)合的位點(diǎn)。 2．熒光實(shí)時(shí)定量PCR及Western-blot結(jié)果顯示，過(guò)表達(dá)組中miR-30a-3p的表達(dá)量顯著上升（P＜0.05），而IGF-1在mRNA和蛋白水平上的表達(dá)顯著下降（P＜0.05）；抑制表達(dá)組中miR-30a-3p的表達(dá)量顯著下降（P＜0.05），IGF-1在mRNA水平及蛋白水平的表達(dá)上顯著升高（P＜0.05)。 3．雙熒光素酶載體報(bào)告實(shí)驗(yàn)結(jié)果表明: miR-30a-3p和IGF-1有確切的靶向關(guān)系；其中IGF-1mRNA序列的3′UTR區(qū)有四個(gè)可能與miR-30a-3p相結(jié)合的位點(diǎn)。 結(jié)論 IGF-1為miR-30a-3p的靶基因之一，miR-30a-3p對(duì)IGF-1在其mRNA水平和蛋白水平上均有明顯的負(fù)性調(diào)控作用。胎盤(pán)中表達(dá)升高的miR-30a-3p通過(guò)負(fù)性調(diào)控IGF-1，，使其表達(dá)水平下降，滋養(yǎng)細(xì)胞功能發(fā)生障礙，從而導(dǎo)致了子癇前期的發(fā)生。
[Abstract]:Preeclampsia (PE) is a group of pregnant and hypertension coexisting diseases, which seriously affects maternal and infant health and is one of the main causes of maternal and perinatal mortality. MicroRNA (microRNA) is a small RNA that can regulate protein expression but can not encode protein. It can degrade the target gene mRNA or hinder its translation. It plays an important role in the regulation of gene expression. Preeclampsia and other pregnancy-related diseases are closely related to the unbalanced expression of microRNAs in the placenta [1].In our previous study, microarray and real-time fluorescent quantitative PCR were used to confirm the presence of microRNAs-30a-3p in the placenta of preeclampsia patients. In vitro experiments were carried out to investigate the effect of the expression of microRNAs-30a-3p on the function of human normal trophoblast HTR-8/SVneo cells. The results showed that the expression of microRNAs-30a-3p was positively correlated with the apoptosis rate of HTR-8/SVneo cells. We speculated that the increased expression of microRNAs-30a-3p in placenta was associated with the pathogenesis of preeclampsia. Mi-30a-3p plays a role in the pathogenesis of pre-eclampsia by regulating which or which target genes? To solve these problems, we have further carried out the prediction and validation of the target genes related to pre-eclampsia of Mi-30a-3p.
objective
1. predict the target genes of miR-30a-3p and screen the genes associated with preeclampsia as the target genes.
2. clarify the regulatory relationship between miR-30a-3p and target genes.
Method
1. Bioinformatics software was used to predict the target genes of microRNAs-30a-3p. The candidate genes were screened according to their scores in bioinformatics software and their correlation with preeclampsia.
2. Liposome transfection technique was used to establish an overexpression and inhibition model of microRNAs-30a-3p in human normal trophoblastic cell line HTR-8/Svneo. The correlation between microRNAs-30a-3p and target gene expression was detected by real-time fluorescence quantitative PCR and Western-blot.
3. To construct luciferase reporter vectors for target genes and validate the targeting relationship and binding sites between microRNAs-30a-3p and target genes by double luciferase reporter system.
Result
1. Prediction results of bioinformatics software showed that IGF-1 was one of the theoretical target genes of microarray-30a-3p. The 3'-UTR region of its mRNA sequence contained five sites that could theoretically bind to microarray-30a-3p.
2. The results of real-time PCR and Western-blot showed that the expression of miR-30a-3p increased significantly in the overexpression group (P < 0.05), whereas the expression of IGF-1 decreased significantly in the mRNA and protein levels (P < 0.05); the expression of miR-30a-3p decreased significantly in the inhibition group (P < 0.05), and the expression of IGF-1 increased significantly in the mRNA and protein levels. High (P < 0.05).
3. The results of double luciferase vector report showed that there was a definite targeting relationship between microRNAs-30a-3p and IGF-1, and there were four sites in the 3'UTR region of the IGF-1 mRNA sequence that might bind to microRNAs-30a-3p.
conclusion
IGF-1 is one of the target genes of microRNAs-30a-3p. MicroRNAs-30a-3p can negatively regulate the expression of IGF-1 at both mRNA and protein levels.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R714.244

【參考文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 欒麗霞;子癇前期相關(guān)miR-30a-3p的功能研究[D];第四軍醫(yī)大學(xué);2012年

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