人和人疱疹病毒6型miRNA与靶标基因的预测及其功能初步研究

发布时间：2018-03-30 10:13

本文选题：miRNA　切入点：miR-941　出处：《东北林业大学》2012年硕士论文

【摘要】：MiRNA介导的基因表达沉默,在以前的研究中已经被报道过在许多生理学和病理学中具有重要的作用。在本研究中,探索了一个真正人类特有的miRNA(miRNA-941),以及在人类特有的功能进化中的作用和基因表达上的变化。为了确定人类基因组中特有的1miRNA,在11个物种中,利用生物信息学方法,找到12种人类特有的miRNA,并且在其他11个物种中没有直向同源物；其中,其他所有的人类特有miRNA表达水平相对于保守的miRNA都极低,甚至根本不表达,只有miR-941的表达量最高,特别是在大脑和小脑的两个区域中,它的表达要比其他人类特有miRNA高,在此基础上进一步研究了miR-941调控的靶标基因及其功能。通过Northern Blot实验,在人的大脑、小脑、肾脏以及视觉皮层组织中验证miR-941的存在；结果显示,miR-941在人的大脑、小脑及肾脏中都有所表达,但在视觉皮层中无表达。通过对人类急性单细胞白血病细胞系(Human acute monocytic leukemia cell line,THP-1)数据的分析显示,miR-941在细胞质中大量存在,其表达量高于功能miRNA表达量的平均水平；并且miR-941与Argonaute(AGO)蛋白结合,与其他与AGO结合的miRNA相比,miR-941的表达水平与保守的miRNA相当,甚至有时高于那些已发现的保守的功能miRNA.综上所述,miR-941具备功能性miRNA所具有的一切特征,包括序列和表达,所以它是一个真正的miRNA. 通过生物信息方法对miR-941调控的靶标基因进行预测,再通过细胞转染实验进行验证。将重组的载体转入3种人细胞系中(293T、HEK及HSF2),转染后24小时,提取细胞RNA,进行基因芯片数据分析；根据数据绘制出累积分布函数曲线结果显示,在3种细胞系中,对于TargetScan预测的靶标基因,其在转染的细胞系中的表达水平要显著低于对照组,说明miR-941在细胞中起到了基因沉默的作用。MiR-941在hedgehog信号通路和insulin信号通路上有大量的结合位点,证明了在细胞分化和寿命长短中的作用。综上所述,这些结果证实了miR-941在人类特有的表型进化中的作用。本研究还对人疱疹病毒6型(HHV-6)的miRNA及其调控基因进行了预测分析。考虑到miRNA在基因表达调控中所起到的重要作用,病毒这种只具有有限基因编码能力的生物同样可能利用miRNA调节宿主或自身某些基因的表达。在本部分研究中,基于HHV-6基因组数据预测了HHV-6编码的前体miRNA,以及这些前体miRNA成熟后序列,通过RNAFOLD软件,绘制出miRNA的二级结构图,在图中可见功能miRNA的颈环结构；在此基础上,进一步预测了这些miRNA可能调控的靶标基因,并分析确定了其中与人体免疫相关的靶标基因。综上所述,这些结果证实了HHV-6编码的miRNA应该与人体免疫相关。
[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.

【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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