参与先天性免疫应答的miRNA的鉴定及其功能的初步研究

发布时间：2018-01-13 23:23

本文关键词：参与先天性免疫应答的miRNA的鉴定及其功能的初步研究　出处：《安徽大学》2012年硕士论文　论文类型：学位论文

【摘要】：microRNA (miRNA)是一类由基因组编码的长度为18-26个核苷酸的单链非编码小RNA,它们广泛存在于真核细胞中,在进化上高度保守。在RNA聚合酶Ⅱ或Ⅲ的作用下,miRNA以前体的形式从基因组中转录出来。它的形成过程涉及一系列蛋白分子对其转录前体的剪切和转运,最终成熟的miRNA在细胞质中与其它相关蛋白分子一起构成沉默复合体(RISC),通过与靶mRNA分子的3’UTR中相应序列的结合,导致靶mRNA的降解或者转录后翻译受到抑制。到目前为止,已在生物体内发现上万种的miRNA,它们构成了一个复杂的调控网络,参与细胞增殖分化、生物体发育、机体免疫应答和肿瘤发生等生理及病理过程。当病原微生物入侵时,机体的先天性免疫应答会被迅速激活,并通过其介导的炎症和抗病毒反应来杀灭和清除病原微生物。NF-κB是先天性免疫应答过程中的一个重要信号通路,当它被激活时,能够启动下游一系列细胞因子的转录,从而促进机体的炎症反应的发生,与此同时先天性免疫应答诱导产生的Ⅰ型干扰素在机体的抗病毒反应中也发挥了重要作用。宿主的抗感染先天性免疫应答一方面要产生足够强烈的炎症反应以彻底消灭入侵的病原微生物,另一方面又要防止这一反应的过度激活而对机体本身带来的损伤。在这一信号转导过程中,存在着许多正负调控因子,以保障机体先天性免疫应答的适度激活。越来越多的文献报道miRNA参与了先天性免疫应答,并发挥重要的调控作用。发现和鉴定参与调控先天性免疫应答的miRNA及对其分子机制的研究,可以加深对宿主先天性免疫应答调控的理解,为研制抗感染药物提供新的靶标。在本课题中,我们初步鉴定出了38种可能调控先天性免疫应答信号通路的miRNA,并对其中的miR-526a、miR-617和miR-623进行了进一步的功能研究。 1高通量筛选参与先天性免疫应答的miRNA: 通过IFNβ和IL-1p(NF-κB调控的细胞因子)的检测平台,对140个miRNA进行筛选,从中鉴定出了38种参与先天性免疫应答的功能miRNA。其中21种能够上调IFNβ的表达水平,22种能够上调IL-1p的表达水平。 2miR-526a在先天性免疫应答中功能的初步研究：通过荧光素酶报告基因和酶联免疫吸附试验发现,miR-526a能够明显上调IFNp和IL-1p的表达水平。因此,我们进一步研究了miR-526a在宿主先天性免疫应答中的作用。通过荧光定量PCR的方法,我们发现在VSV和LPS诱导THP-1细胞的过程中,miR-526a的表达水平随着时间的变化逐渐增强,这说明miR-526a可能参与了病原体感染的宿主免疫应答。接下来,通过生物信息学的方法预测到CYLD可能是miR-526a的靶点。通过荧光定量PCR的方法,我们发现细胞中过表达miR-526a能够在转录和翻译水平上抑制CYLD的表达。CYLD是一个去泛素化酶,能够通过对RIG-I的去泛素化作用抑制RIG-I的信号转导；同时它还能够通过与MyD88的相互作用抑制其对下游分子的磷酸化进而负调控TLR4信号通路,从而下调宿主先天性免疫应答水平。上述结果初步证明miR-526a可能通过负调控CYLD的表达水平促进IFNp和IL-1β的产生。 3参与炎症反应的miR-617和miR-623在肿瘤发生中功能的初步研究：通过酶联免疫吸附试验发现,miR-617和miR-623能够明显上调IL-1β的表达水平,而IL-1p是受NF-κB调控的最重要的细胞因子之一。NF-κB信号通路在先天性免疫促炎症反应中发挥着重要的作用。大量研究表明,在肿瘤的发生过程中,常伴有NF-κB信号通路特征性的表达变化,其中炎症相关分子参与了肿瘤的发生,并在肿瘤形成的不同阶段起着决定性的作用。IL-1p是一个前炎症因子,它介导的炎症反应所产生的微环境能够导致正常细胞突变同时促进细胞的增殖。其它相关课题的研究中发现,miR-617和miR-623在乳腺癌发生过程中表达增高,因此我们探讨了miR-617和miR-623在乳腺癌发生中的作用。通过细胞计数和划痕实验,我们发现在乳腺癌细胞系ZR-75-1中过表达miR-617和miR-623能够导致肿瘤细胞增殖和迁移能力的显著增加。上述结果表明miR-617和miR-623可能通过影响细胞的炎症反应参与肿瘤的发生,为我们进一步研究miR-617和miR-623的功能打下了基础。综上所述,本课题初步鉴定了38种可能参与先天性免疫应答的miRNA,进一步研究表明,miR-526a可能通过抑制负调控因子CYLD从而上调IFNβ和IL-1β的表达水平；此外参与炎症反应的miR-617和miR-623能够促进肿瘤的生长与转移。这些研究为进一步揭示miRNA在先天性免疫应答及炎症介导的肿瘤发生中的分子机制提供了重要线索。
[Abstract]:MicroRNA (miRNA) is a class of single stranded genome by encoding length 18-26 nucleotides encoding non small RNA, which widely exists in eukaryotic cells, highly conserved in evolution. In the RNA polymerase II or III under the action of miRNA before the form from the genome to record its forming process. To cut and transport of a series of proteins of the transcripts, eventually mature miRNA in the cytoplasm and other related protein molecules together constitute the silencing complex (RISC), by combining the corresponding sequence and the target molecule mRNA 3 'UTR, leading to the target mRNA degradation or translation after transcription inhibition to the current. So far, thousands of miRNA have been found in the organism, they constitute a complex regulatory network involved in cell proliferation and differentiation, organism development, immune response and tumorigenesis of physiological and pathological process.
When the invasion of pathogenic microorganisms, the body's innate immune response will be rapidly activated, inflammation and antiviral responses and through its mediated to sterilize and remove pathogenic microorganisms of.NF- kappa B is an important signaling pathway in the innate immune response, when it is activated, can activate the downstream transcription of a series of cells factor, so as to promote the body's inflammatory response occurred at the same time, type I interferon induced innate immune response in antiviral responses also played an important role in anti infection. The host innate immune response to a strong enough to produce inflammation completely destroy invading pathogenic microorganisms, on the other hand to prevent this reaction caused excessive activation of the body itself damage. In the signal transduction process, there are a lot of positive and negative regulatory factors, in order to safeguard the innate Moderate activation of immune response. More and more reports of miRNA involved in the innate immune response, and play an important role. The discovery and study of identification is involved in the regulation of the innate immune response of miRNA and its molecular mechanism, can deepen our understanding of the host innate immune response regulation understanding for anti infection drugs provide a new target.
In this study, we initially identified 38 kinds of miRNA that might regulate the signal transduction pathway of congenital immune response, and further studied the functions of miR-526a, miR-617 and miR-623.
1 high throughput screening of miRNA: involved in congenital immune response
Through the detection platform of IFN beta and IL-1p (NF- kappa B regulated cytokines), 140 miRNA were screened, and 38 kinds of functional miRNA. participating in innate immune response were identified. 21 of them could upregulate the expression level of IFN beta, and 22 of them could upregulate IL-1p expression level.
Preliminary study on the function of 2miR-526a in the innate immune response:
By luciferase assay and enzyme-linked immunosorbent assay showed that miR-526a expression levels were significantly up-regulated the expression of IFNp and IL-1p. Therefore, we further study the role of miR-526a in innate immune response. By fluorescence quantitative PCR method, we found that the process of THP-1 cells induced by VSV and LPS, the expression level of miR-526a enhanced gradually with the change of time, indicating that miR-526a may be involved in the host immune response to pathogen infection. Then, through the method of bioinformatics to predict CYLD may be the target of miR-526a. By fluorescence quantitative PCR method, we found that cells overexpressing miR-526a could inhibit the expression of.CYLD CYLD in the level of transcription and translation is a deubiquitinating enzyme, can go through ubiquitination of RIG-I inhibition of RIG-I signal transduction; at the same time it can also pass and MyD88 Interaction inhibits the phosphorylation of downstream molecules, thereby negatively regulates the TLR4 signaling pathway, thereby reducing the level of host innate immune response. The above results preliminarily prove that miR-526a may promote IFNp and IL-1 beta production by negatively regulating the expression level of CYLD.
3 preliminary studies on the function of miR-617 and miR-623 involved in inflammatory reactions in the carcinogenesis:
By ELISA, miR-617 and miR-623 expression of IL-1 beta, and IL-1p is one of the most important cytokines.NF- B signaling pathway by NF- kappa B regulation plays an important role in the inflammatory reaction in innate immunity and promoting. A large number of studies show that, in the process of tumor, often accompanied by changes in the expression characteristics of NF- kappa B signaling pathway, in which inflammation related molecules involved in tumor development, tumor formation in different stages and plays a decisive role in.IL-1p is a proinflammatory cytokine, microenvironment generated inflammation mediated can cause normal cells to promote cell proliferation and mutation study found. Other relevant subjects, the expression of miR-617 and miR-623 in the process of breast cancer increased, so we investigate the role of miR-617 and miR-623 in breast cancer cells through. Counting and scratch test, we found that overexpression of miR-617 and miR-623 resulted in a significant increase in tumor cell proliferation and migration in breast cancer cell line ZR-75-1. The results showed that miR-617 and miR-623 may occur through the effect of inflammatory cells in the tumor, for us to further study the function of miR-623 and the miR-617 foundation.
To sum up, this paper identified 38 may be involved in the innate immune response of miRNA, further study showed that miR-526a could inhibit the expression by negative regulation factor CYLD and upregulation of IFN beta and IL-1 beta; also involved in inflammatory reaction of miR-617 and miR-623 can promote the growth and metastasis of tumor. In these studies provide important clues in order to further reveal the molecular mechanism of miRNA in innate immune response and inflammation mediated tumorigenesis.

【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

【相似文献】