急性中长波紫外线辐射“旁观者效应”相关分泌型miRNA的筛选及其功能预测

发布时间：2018-01-03 14:10

本文关键词：急性中长波紫外线辐射“旁观者效应”相关分泌型miRNA的筛选及其功能预测　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景及目的辐射包括电离辐射和非电离辐射。紫外线(ultraviolet,UV)是波长为100-400 nm的非电离辐射。根据紫外线波长的不同,可将其分为短波紫外线UVC(200-280 nm)、中波紫外线 UVB(280-320 nm)和长波紫外线 UVA(320-400 nm)。而在透过大气层时波长小于290 nm的紫外线会被大气层中的臭氧吸收掉,能够到达地面的主要是UVA和少量UVB。辐射诱导的旁观者效应(Bystander effects,BE)是指旁观者细胞出现诸如细胞死亡、基因突变、染色体不稳定等类似于细胞直接受到辐射作用后出现的反应。旁观者细胞指与受辐射细胞邻近的细胞,也指用受辐射细胞的上清液培养的细胞。紫外线除了引起受辐射细胞的直接损伤外,还可以通过紫外线辐射旁效应(Ultraviolet irradiation induced bystander effects,UV-BE)引起临近细胞的间接损伤,这些旁观者效应包括氧化应激反应,基因突变,凋亡,炎症反应,免疫抑制,甚至肿瘤病变等。为了消除紫外线辐射所带来的这些影响,维持染色体健全,细胞产生了一系列保护机制,包括DNA修复,细胞周期阻滞以及凋亡。在旁观者效应中,受辐射细胞的DNA损伤反应通过细胞间缝隙连接、细胞外可溶性因子与未受辐射细胞进行细胞间通信。最近的研究表明小分子核糖核酸(microRNA,miRNA)在受辐射细胞和旁观者细胞的信号通路中扮演重要的角色。以往人们都认为miRNA只在细胞内发挥作用,然而随着研究的深入,研究者发现miRNA也会被分泌到细胞间隙中并发挥功能。大量的数据表明其实细胞外的miRNA十分稳定,miRNA之所以能在细胞间隙中稳定存在,是因其包裹于细胞外囊泡中,细胞外囊泡(Extracellular vesicles,EVs)是由磷脂双分子层封闭组成不同大小的微粒(20～2000 nm),在体内和体外几乎所有类型的细胞都会释放EVs到细胞外介质中。到目前为止,根据不同的形成机制和生理特征分成三不同的EVs形式:外泌体、细胞膜微粒、调亡小体。外泌体产生于核内体携带的多泡体,细胞膜微粒和调亡小体来源于细胞质膜释放的微囊泡(Microvesicles,MVs)。被包裹在EVs中的miRNA称为"分泌型miRNA",EVs保护"分泌型miRNA",使其免受各种酶类的降解,从而被运输到相应的受体细胞并调节受体细胞的生物功能。那么,在紫外线辐射"旁观者效应"中细胞的损伤是否由其中的miRNA介导或者由何种miRNA介导呢?本研究的目的在于筛选出有可能在急性中长波紫外线辐射"旁观者效应"中发挥重要作用的分泌型miRNA,并预测其可能的机制。方法1、紫外线辐射"旁观者效应"模型的建立及验证分别采用不同剂量中长波紫外线(Ultraviolet A/Ultraviolet B,UVA/UVB)UVA 20J/cm2、UVB60mJ/cm2辐射人皮肤成纤维细胞(human skin fibroblasts,HSF),用特别配置的无胞外囊泡培养基孵育HSF,于照射后24h提取细胞上清液,与正常HSF共育,分为对照组(Ctr组)、UVA辐射组(UVA组)、UVB辐射组(UVB组)、旁观者细胞对照组(BE-Ctr组)、旁观者细胞UVA组(BE-UVA组)、旁观者细胞UVB组(BE-UVB组),比较直接受辐射细胞和旁观者细胞的变化,倒置显微镜下观察细胞形态,细胞计数CCK-8法检测细胞增殖率,流式细胞仪检测细胞凋亡率,DCFH-DA试剂盒检测细胞内的ROS量。2、miRNA芯片筛选差异表达的分泌型miRNA及生物信息学分析采用以上剂量中长波紫外线辐射人皮肤成纤维细胞,用无胞外囊泡培养基孵育24h,收集直接受辐射细胞各组上清液,进行miRNA芯片分析,以归一化强度的|log2(Ratio)|≥5为条件筛选上述模型中差异表达的上调miRNA、下调miRNA,利用qRT-PCR实验法验证UVA vs Ctr组、UVB vs Ctr组差异倍数均大于5倍的上调miRNA。通过TargetScan、microRNAorg、PITA数据库对差异表达的miRNA进行靶基因预测,并对所得差异miRNA的靶基因进行GO分析以及pathway富集分析。3、qRT-PCR法深入验证差异表达的分泌型miRNA利用qRT-PCR(Quantitative real time PCR,实时定量PCR)实验法对上述差异表达的miRNA进行深入验证,分别检验直接受辐射细胞、直接受辐射细胞上清液和旁观者细胞中miRNA的表达量,筛选出可能与紫外线"旁观者效应"相关的分泌型miRNA;绘制差异miRNA时效曲线图;检验其前体RNA(pre-miRNA)在旁观者细胞中的表达量。结果1、紫外线辐射人皮肤成纤维细胞后,可诱导紫外线"旁观者效应"的发生经中长波紫外线UVA20J/cm2、UVB 60mJ/cm2辐射诱导生成的上清液与旁观者细胞共孵育后24h,倒置显微镜下观察直接受辐射细胞和旁观者细胞,直接受辐射细胞和旁观者细胞均出现较多的细胞碎片,细胞体积变大,过度伸展;CCK-8结果提示直接受辐射细胞和旁观者细胞增殖率均减缓;流式细胞法结果提示直接受辐射细胞和旁观者细胞表现出凋亡率升高;ROS结果提示,直接受辐射细胞和旁观者细胞活性氧荧光强度均高于未辐射组。2、miRNA芯片筛选出差异表达miRNA紫外线辐射诱导生成的上清液中提取分泌型miRNA进行质量验证并予芯片筛选,结果发现,利用差异倍数Fold change值进行筛选,标准为Fold change值=5.0,UVA vs Ctr组中有54个miRNA表达上调、11个miRNA表达下调;UVBvsCtr组中有50个miRNA表达上调,4个miRNA表达下调。其中,上调的 miRNA 中,UVA vs Ctr 组 Fold change 值=5.0 同时 UVB vs Ctr 组 Fold change值=5.0 的 miRNA 有 19 个,分别为 hsa-miR-8071、hsa-miR-769-5p、hsa-miR-758-3p、hsa-miR-7515、hsa-miR-6856-5p、hsa-miR-6837-5p、hsa-miR-6769a-5p、hsa-miR-6743-5p、hsa-miR-564、hsa-miR-4694-3p、hsa-miR-4655-3p、hsa-miR-4514、hsa-miR-4513、hsa-miR-4422、hsa-miR-432-5p、hsa-miR-3659、hsa-miR-3163、hsa-miR-22-5p、hsa-miR-1299。对差异表达的分泌型miRNA进行生物信息学分析,预测靶基因功能,UVA组与UVB组显著富集代谢通路大部分重叠,基因主要富集在能量代谢、转录调节、细胞增殖、跨膜信号转导等生物过程中,并且这些基因主要参与Rap1信号通路、粘着斑激酶(FAK)相关信号通路、MAPK信号通路等重要代谢通路。3、差异表达的分泌型miRNA的进一步验证在直接受辐射细胞和旁观者细胞中hsa-miR-4655-3p、hsa-miR-769-5p均呈现高表达;hsa-miR-4655-3p、hsa-miR-769-5p在24h时间点表达量最高,上清液中miRNA变化趋势与直接受辐射细胞中类似,但变化幅度更大;在旁观者细胞中 pre-miR-4655-3p、pre-miR-769-5p 均呈现低表达,结论急性中长波紫外线辐射人皮肤成纤维细胞可诱导出旁观者效应;急性中长波紫外线辐射可介导分泌型miRNA的表达,其中hsa-miR-4655-3p、hsa-miR-769-5p显著上调,极有可能参与调节紫外线"旁观者效应",值得进一步研究。
[Abstract]:Background and objective: radiation including ionizing and non ionizing radiation. Ultraviolet (ultraviolet, UV) is a non ionizing radiation 100-400 nm wavelength. According to the different wavelengths of ultraviolet light, which can be divided into short wave ultraviolet UVC (200-280 nm), ultraviolet UVB (280-320 nm) and ultraviolet (320-400 UVA nm). And through the atmosphere below the wavelength of 290 nm ultraviolet ozone in the atmosphere will be absorbed, can reach the ground is mainly the bystander effect induced by UVA radiation and a small amount of UVB. (Bystander effects BE) refers to the bystander cells such as cell death, gene mutation, chromosome instability and other similar to the cells directly by the role of radiation after the reaction. The cells and bystander cells irradiated cells adjacent to the culture supernatant, also refers to the irradiated cells. In addition to causing ultraviolet irradiated cells directly Damage, but also through the ultraviolet radiation side effect (Ultraviolet irradiation induced bystander effects, UV-BE) caused by indirect injury to adjacent cells, the bystander effect including oxidative stress, gene mutation, apoptosis, inflammation, immunosuppression, and even tumors. In order to eliminate ultraviolet radiation caused by these effects, the maintenance of chromosomal sound the cell, resulting in a series of protection mechanisms, including DNA repair, cell cycle arrest and apoptosis in bystander effect in response to DNA damage by radiation cells connected by intercellular gap, extracellular soluble factor and non irradiated cells were intercellular communication. Recent studies show that microRNAs (microRNA, miRNA) in the play an important role in radiation cells and bystander cells. The signaling pathway is considered to play a role in intracellular miRNA However, with the deepening of the study, the researchers found that miRNA can be secreted into the intercellular space and function. A large number of data indicate that extracellular miRNA is very stable, miRNA can exist stably in the intercellular space, because it is wrapped in extracellular vesicles, extracellular vesicles (Extracellular vesicles, EVs) is a phospholipid bilayer enclosed particles of different sizes (20 ~ 2000 nm), in vitro and in vivo in almost all cell types will release EVs into the extracellular medium. So far, according to the different formation mechanism and physiological characteristics of EVs are divided into three different forms: exosomes. The cell membrane particles, apoptotic bodies. Exosomes originate in endosomes carrying multivesicular bodies, Microcystis cell membrane particles and apoptotic bodies comes from the release of cell membrane vesicles (Microvesicles, MVs). The EVs is wrapped in a miRNA called "secretory miRNA", EVs "Protection of secretory miRNA, degradation from the various enzymes, which are transported to the corresponding receptor cells and regulate receptor cell biological function. Then, in the ultraviolet radiation" bystander effect "in the cell injury by the miRNA mediated by miRNA mediated or what? The purpose of this study is to screening out possible in acute ultraviolet radiation" bystander effect "play an important role in the secretion of type miRNA, and forecast its possible mechanism. Methods 1, establishment and validation of ultraviolet radiation" bystander effect "model respectively with different doses of UVA (Ultraviolet A/Ultraviolet B, UVA 20J/cm2, UVA/UVB) UVB60mJ/cm2 radiation human skin fibroblasts (human skin, fibroblasts, HSF), with a special configuration of the no extracellular vesicles with HSF medium incubated in 24h cells after irradiation, extraction supernatant were incubated with normal HSF points For the control group (Ctr group), UVA radiation group (UVA group), UVB group (group UVB), radiation bystander cells in the control group (BE-Ctr group), UVA group of bystander cells (BE-UVA group), UVB group of bystander cells (BE-UVB group), compared with directly irradiated cells and bystander cell changes were observed morphology under inverted microscope, cell proliferation was detected by cell counting CCK-8 assay, cell apoptosis was detected by flow cytometry, ROS.2 DCFH-DA kit to detect the intracellular and secreted miRNA and bioinformatics analysis of the dose of UVA irradiation of human skin fibroblasts miRNA chip to screen differentially expressed, with no extracellular vesicles with 24h medium were collected, directly irradiated cells supernatant, miRNA microarray analysis, using normalized intensity |log2 (Ratio) | more than 5 conditions are differentially expressed in the above model on miRNA, downregulation of miRNA, using qRT-PCR test method Validation of the UVA vs Ctr miRNA. UVB vs Ctr group, raised the group differences multiples are greater than 5 times by TargetScan, microRNAorg, PITA database of differentially expressed miRNA target gene prediction and target genes for income differences were analyzed by GO and miRNA pathway.3 qRT-PCR enrichment analysis method, in-depth verification of secretory miRNA expression by qRT-PCR (Quantitative real time PCR, real-time quantitative PCR) experimental method on the expression of the differences in the miRNA for further inspection, were directly irradiated cells, directly irradiated cells supernatant and the expression of miRNA in bystander cells, secretory miRNA may be screened with ultraviolet "bystander effect" related to the difference of miRNA time curve drawing; check the map; precursor RNA (pre-miRNA) expression in the bystander cells. Results 1, ultraviolet radiation in human fibroblasts can be induced by ultraviolet radiation, bystander "The occurrence of the effect of ultraviolet radiation UVA20J/cm2, UVB 60mJ/cm2 and the supernatant induced bystander cells were incubated with 24h, directly irradiated cells and bystander cells were observed under inverted microscope, directly irradiated cells and bystander cells showed more cell debris, cell size, excessive stretch; CCK-8 showed directly the slow rate of radiation and cell proliferation of bystander cells; flow cytometry showed that directly irradiated cells and bystander cells showed apoptosis rate increased; ROS results suggest that radiation directly affected by the activity of cells and the bystander cell oxygen fluorescence intensity were higher than the radiation group.2, miRNA microarray selected153differentially expressed supernatant miRNA ultraviolet radiation induced the extraction of secretory miRNA quality verification and to the chip screening results showed that using the difference of multiple Fold change value selection Fold change, the standard value of =5.0, expression of 54 miRNA UVA vs in the Ctr group, 11 miRNA expression; expression of 50 miRNA in the UVBvsCtr group, 4 miRNA expression. The upregulation of miRNA, UVA vs Ctr Fold change =5.0 and the UVB value of group vs group Ctr Fold change the value of =5.0 19 in miRNA, respectively hsa-miR-8071, hsa-miR-769-5p, hsa-miR-758-3p, hsa-miR-7515, hsa-miR-6856-5p, hsa-miR-6837-5p, hsa-miR-6769a-5p, hsa-miR-6743-5p, hsa-miR-564, hsa-miR-4694-3p, hsa-miR-4655-3p, hsa-miR-4514, hsa-miR-4513, hsa-miR-4422, hsa-miR-432-5p, hsa-miR-3659, hsa-miR-3163, hsa-miR-22-5p, hsa-miR-1299. on the differential expression of secreted miRNA by bioinformatic analysis, the predicted target gene function, UVA group and UVB group significantly enriched the metabolic pathways of most overlapping genes mainly enriched in energy metabolism, transcription regulation, fine The process of cell proliferation, transmembrane signal transduction in organisms, and these genes mainly involved in Rap1 signaling pathway, focal adhesion kinase (FAK) signaling pathway, MAPK signaling pathway and other important metabolic pathways of.3, differential expression of secretory miRNA to further validate the directly irradiated hsa-miR-4655-3p cells and bystander cells, showed a high hsa-miR-769-5p the expression of hsa-miR-4655-3p, hsa-miR-769-5p in 24h; time expression was the highest, and directly affected by the trend of similar changes in miRNA in the supernatant of cell radiation, but larger change; pre-miR-4655-3p in bystander cells, pre-miR-769-5p showed low expression, long wave ultraviolet radiation in acute human skin fibroblasts can induce bystander effect in the long wave; acute ultraviolet radiation can mediate the expression of secretory miRNA in hsa-miR-4655-3p, hsa-miR-769-5p was down regulated, is likely to. It is worth further study to adjust the "bystander effect" of ultraviolet light.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R758.1

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