本文選題：miRNA　切入點：miR-941　出處：《東北林業(yè)大學(xué)》2012年碩士論文

【摘要】：MiRNA介導(dǎo)的基因表達沉默,在以前的研究中已經(jīng)被報道過在許多生理學(xué)和病理學(xué)中具有重要的作用。在本研究中,探索了一個真正人類特有的miRNA(miRNA-941),以及在人類特有的功能進化中的作用和基因表達上的變化。為了確定人類基因組中特有的1miRNA,在11個物種中,利用生物信息學(xué)方法,找到12種人類特有的miRNA,并且在其他11個物種中沒有直向同源物；其中,其他所有的人類特有miRNA表達水平相對于保守的miRNA都極低,甚至根本不表達,只有miR-941的表達量最高,特別是在大腦和小腦的兩個區(qū)域中,它的表達要比其他人類特有miRNA高,在此基礎(chǔ)上進一步研究了miR-941調(diào)控的靶標(biāo)基因及其功能。 通過Northern Blot實驗,在人的大腦、小腦、腎臟以及視覺皮層組織中驗證miR-941的存在；結(jié)果顯示,miR-941在人的大腦、小腦及腎臟中都有所表達,但在視覺皮層中無表達。通過對人類急性單細胞白血病細胞系(Human acute monocytic leukemia cell line,THP-1)數(shù)據(jù)的分析顯示,miR-941在細胞質(zhì)中大量存在,其表達量高于功能miRNA表達量的平均水平；并且miR-941與Argonaute(AGO)蛋白結(jié)合,與其他與AGO結(jié)合的miRNA相比,miR-941的表達水平與保守的miRNA相當(dāng),甚至有時高于那些已發(fā)現(xiàn)的保守的功能miRNA.綜上所述,miR-941具備功能性miRNA所具有的一切特征,包括序列和表達,所以它是一個真正的miRNA. 通過生物信息方法對miR-941調(diào)控的靶標(biāo)基因進行預(yù)測,再通過細胞轉(zhuǎn)染實驗進行驗證。將重組的載體轉(zhuǎn)入3種人細胞系中(293T、HEK及HSF2),轉(zhuǎn)染后24小時,提取細胞RNA,進行基因芯片數(shù)據(jù)分析；根據(jù)數(shù)據(jù)繪制出累積分布函數(shù)曲線結(jié)果顯示,在3種細胞系中,對于TargetScan預(yù)測的靶標(biāo)基因,其在轉(zhuǎn)染的細胞系中的表達水平要顯著低于對照組,說明miR-941在細胞中起到了基因沉默的作用。MiR-941在hedgehog信號通路和insulin信號通路上有大量的結(jié)合位點,證明了在細胞分化和壽命長短中的作用。綜上所述,這些結(jié)果證實了miR-941在人類特有的表型進化中的作用。 本研究還對人皰疹病毒6型(HHV-6)的miRNA及其調(diào)控基因進行了預(yù)測分析�？紤]到miRNA在基因表達調(diào)控中所起到的重要作用,病毒這種只具有有限基因編碼能力的生物同樣可能利用miRNA調(diào)節(jié)宿主或自身某些基因的表達。在本部分研究中,基于HHV-6基因組數(shù)據(jù)預(yù)測了HHV-6編碼的前體miRNA,以及這些前體miRNA成熟后序列,通過RNAFOLD軟件,繪制出miRNA的二級結(jié)構(gòu)圖,在圖中可見功能miRNA的頸環(huán)結(jié)構(gòu)；在此基礎(chǔ)上,進一步預(yù)測了這些miRNA可能調(diào)控的靶標(biāo)基因,并分析確定了其中與人體免疫相關(guān)的靶標(biāo)基因。綜上所述,這些結(jié)果證實了HHV-6編碼的miRNA應(yīng)該與人體免疫相關(guān)。
[Abstract]:MiRNA mediated gene silencing, in previous studies has been reported in many plays an important role in the physiology and pathology. In this study, to explore a real human specific miRNA (miRNA-941), and the changes in expression of human specific function evolution and the role of the gene in order to determine the unique human genome in 1miRNA, in the 11 species, using bioinformatics methods, found 12 human specific miRNA, and in the other 11 species have orthologs; among them, all the other human specific miRNA expression levels relative to the conservative miRNA are very low, not even the expression, expression of only miR-941 is the highest, especially in the two regions of the brain and cerebellum, its expression than other human specific miRNA target genes, on the basis of further research on the regulation of miR-941 and its function Can.
Through the Northern Blot experiment, the cerebellum in the human brain, and verify the existence of miR-941 kidney and visual cortex tissues; results showed that miR-941 in the human brain, cerebellum and kidney have expressed, but no expression in the visual cortex. The human acute leukemia cell line cells (Human acute monocytic leukemia cell line, THP-1) data analysis shows that a large number of miR-941 in the cytoplasm, the expression level was higher than the average level of the expression function of miRNA; and miR-941 and Argonaute (AGO) protein binding, compared with other miRNA and AGO, the expression level of miRNA miR-941 and conservative, and sometimes even higher than those who have the conserved function of miRNA. in miR-941 has all the functional features of miRNA, including sequence and expression, so it is a real miRNA..
Predicted by the Bioinformatics Method of target genes regulated by miR-941, and then verified by cell transfection experiments. Vector transfected into 3 human cell lines (293T, HEK and HSF2), 24 hours after transfection, RNA cells were extracted, microarray data analysis; according to the data to draw the cumulative distribution curve the results showed that in the 3 cell lines, for the prediction of TargetScan target genes, their expression levels in the transfected cells was significantly lower than the control group, indicating that miR-941 cells play a role in silencing of the.MiR-941 gene in the hedgehog pathway and the insulin pathway has a large number of binding sites, proved in cell differentiation and lifespan in vitro. In summary, these results confirm the role of miR-941 in phenotypic evolution in human beings.
The study of human herpesvirus 6 (HHV-6) miRNA and its regulatory genes were predicted and analyzed. Considering the expression of miRNA played an important role in the regulation of gene expression in, this virus has only limited gene encoding ability of biological may also modulate the host itself or some genes by miRNA in this part. In the study, HHV-6 genomic data to predict the precursor of miRNA based on HHV-6 encoding, and these precursor miRNA mature sequences by RNAFOLD software, draw the two level structure of miRNA, visible in figure miRNA neck ring structure; on this basis, further predicted the target genes of these miRNA may control. And the analysis of identified target genes associated with human immunity. In summary, these results demonstrate that HHV-6 encoding miRNA should be associated with the human immune system.

【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R373

【參考文獻】

相關(guān)期刊論文 前2條

1 秦一雨;全志偉;李濟宇;;微小RNA生物功能的研究進展[J];實用醫(yī)學(xué)雜志;2007年23期

2 張樂;周乙華;;microRNA與病毒感染[J];現(xiàn)代生物醫(yī)學(xué)進展;2009年17期

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本文編號：1685472