MiRNAs在白癜风外周血单个核细胞中的表达及miR-3940-5p对T细胞作用机制的研究

发布时间：2018-07-28 14:29

【摘要】：目的应用miRCURY LNATM microRNA Array微阵列芯片技术检测非节段型白癜风(Non-Segmental Vitiligo, NSV)患者外周血单个核细胞(Peripheral blood mononuclear cells, PBMCs)中microRNAs的表达模式,并筛选其和健康人相比差异性表达的miRNAs。运用实时荧光定量PCR技术对差异性表达的miRNAs进行验证。体外培养白癜风患者PBMCs,提取miRNA检测胸腺肽Tα1对筛选的miRNA表达变化的直接效应。应用靶基因预测数据库选择白介素2受体作为miR-3940-5p调控的靶基因,构建慢病毒载体抑制miR-3940-5p表达并感染人皮肤T细胞株HuT78细胞,进一步研究miR-3940-5p对白癜风免疫相关基因的靶向调节,探讨差异表达的miRNAs和Tα,在非节段型白癜风中的作用机制。方法第一部分：收集5例进展期非节段型白癜风患者和5例年龄性别相匹配的健康人外周静脉血,分离单个核细胞,提取总RNA。应用第七代miRCURYTMLNA阵列(v.18.0) (Exiqon)芯片杂交技术对NSV患者和健康人PBMCs总RNA进行miRNAs的表达谱系差异分析,运用MEV软件(v4.6, TIGR)进行聚类分析；收集32例进展期NSV患者和18例健康人外周血并提取单个核细胞总RNA,茎环引物法逆转录合成cDNA,通过实时荧光定量PCR技术进行miRNA差异表达验证;体外培养白癜风患者外周血单个核细胞,将不同浓度胸腺肽α.加入细胞悬液,72h后离心收集细胞,提取miRNA进行RT-qPCR分析,检测差异性miRNAs的相对表达量。分析胸腺肽α1对白癜风患者PBMCs中差异性miRNAs表达水平的直接效应。第二部分：应用生物信息数据库miRDB和Targetscan预测筛选的miRNA-3940-5p可能调控的与白癜风自身免疫相关的靶基因IL-2RG。构建低表达miR-3940-5p慢病毒载体,转染至人T淋巴细胞株HuT78细胞,设立慢病毒阴性对照组；miR-3940-5p表达下调是通过应用预先设计的反义RNA序列竞争结合到miR-3940-5p前体并克隆到GV159载体Hl-MCS-CMV-EGFP来实现的。3-4天后观察绿色荧光蛋白表达阳性的细胞转染率,收获细胞沉淀；通过ELISA和Western Blot技术检测培养的淋巴细胞上清液和细胞中IL-2R Gamma蛋白含量及表达水平,分析miRNA-3940-5p抑制表达对靶基因IL-2RG的调控作用。结果一、通过高通量miRNA微阵列芯片检测技术,对NSV患者和健康人外周血免疫细胞中的miRNA表达谱进行全面地分析,结果显示与年龄性别相匹配的健康人相比,NSV患者PBMCs miRNA表达谱中有4个miRNAs呈显著差异性表达,其中miR-224-3p, miR-2682-3p和miR-4712-3p表达明显上调(p0.05), miR-3940-5p表达明显下调(p0.05)。二、应用茎环引物逆转录SYBR Green PCR技术在较大的患者人群中分析了这四个miRNAs的表达水平,结果发现NSV患者PBMCs中miR-224-3p、 miR-4712-3p表达均上调,miR-3940-5p表达下调,差异均有统计学意义(p0.05),这与miRNA微阵列数据分析结果相一致。微阵列芯片检测技术和实时荧光定量PCR技术这两种检测方法呈高度正相关(相关系数r=1.000,p=0.005)。但是和健康人相比,miR-2682-3p的表达水平升高但差异没有统计学意义(p0.05)。三、倒置相差显微镜下观察到体外培养的白癜风患者外周血单个核细胞形态圆而透亮,悬浮分布。和空白对照组相比,加入不同浓度胸腺肽a1(50、100ug/ml)处理后实验组细胞数量和聚集性明显增加,与处理前相比差异有统计学意义(p0.05)。胸腺肽α1处理72h后对4个差异性miRNAs表达水平进行PCR检测,发现与未经处理的对照组相比,处理组PBMC中miR-224-3p, miR-2682-3p和miR-4712-3p表达下降,而miR-3940-5p表达升高,但两个浓度之间比较差异无统计学意义(p0.05)。四、体外实验结果显示和慢病毒阴性对照组HuT78细胞相比,目的基因miRNA-3940-5p低表达转染实验组可促进T细胞增殖,淋巴细胞上清液中的IL-2RG含量明显升高,实验组细胞中的IL-2RG蛋白表达上调(p0.05)。结论1、非节段型白癜风患者外周血单个核细胞miRNA表达谱中4个miRNAs发生了差异性表达,其中3个miRNAs表达上调,1个miRNA表达下调。2、实时荧光定量PCR技术检测证实了这四种miRNAs的异常表达与微阵列芯片分析结果一致。3、免疫调节剂胸腺肽α1干预对白癜风患者外周血单个核细胞miRNAs的表达谱产生直接影响。4、miRNA-3940-5p在人皮肤T淋巴细胞系中存在一定表达。LV-hsa-miR-3940-5p-inhibit on可转染至人T淋巴细胞株HuT78细胞。5、miR-3940-5p通过对细胞因子受体IL-2R Gamma基因进行负调控发挥作用。
[Abstract]:Objective to detect the expression patterns of peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMCs) in patients with non segmental vitiligo (Non-Segmental Vitiligo, NSV) using miRCURY LNATM microRNA Array microarray technology, and to screen their differential expression by real-time quantitative fluorescence spectrometry. R technique was used to verify the differential expression of miRNAs. In vitro culture of vitiligo patients PBMCs, miRNA was extracted to detect the direct effect of thymosin T alpha 1 on the changes of miRNA expression. The target gene prediction database was used to select the interleukins 2 receptor as the target gene regulated by miR-3940-5p, and the lentivirus vector was constructed to inhibit miR-3940-5p expression and infected people. Skin T cell line HuT78 cells, further study the target regulation of miR-3940-5p on the immune related genes of vitiligo, explore the mechanism of differential expression of miRNAs and T alpha in non segmental vitiligo. Methods first part: collect 5 patients with progressive non segmental vitiligo and 5 healthy people with matched age and sex in peripheral venous blood. To isolate mononuclear cells and extract total RNA. with seventh generation miRCURYTMLNA array (v.18.0) (Exiqon) chip hybridization technique, the difference analysis of the expression lineage of miRNAs in NSV patients and healthy people PBMCs RNA was analyzed. The cluster analysis was carried out by MEV software (v4.6, TIGR), and 32 cases were collected and 18 healthy human peripheral blood were collected and isolated. Nuclear cell total RNA, stem ring primer reverse transcriptase synthesis of cDNA, miRNA differential expression verification by real-time fluorescence quantitative PCR; cultured peripheral blood mononuclear cells from patients with vitiligo in vitro, different concentrations of thymosin alpha into cell suspension, 72h after 72h centrifugation to collect cells, and miRNA for RT-qPCR analysis to detect the relative miRNAs The direct effect of thymosin alpha 1 on the differential miRNAs expression in PBMCs of patients with vitiligo. The second part: the application of the biological information database miRDB and Targetscan predicted that miRNA-3940-5p may regulate the target gene IL-2RG. related to the autoimmune disease of vitiligo to construct a low expression of miR-3940-5p lentivirus vector, and transfection To the human T lymphocyte strain HuT78 cells, the lentivirus negative control group was set up. The downregulation of miR-3940-5p expression was to observe the cell transfection rate of green fluorescent protein expression positive by using pre designed antisense RNA sequence combined with miR-3940-5p precursor and cloned to GV159 carrier Hl-MCS-CMV-EGFP to observe the transfection rate of green fluorescent protein expression positive. The content and expression level of IL-2R Gamma protein in the cultured lymphocyte supernatant and cells were detected by ELISA and Western Blot techniques. The effect of miRNA-3940-5p inhibition expression on the target gene IL-2RG was analyzed. The miRNA expression profile was analyzed comprehensively. The results showed that 4 miRNAs expressions were significantly different in the PBMCs miRNA expression profiles of NSV patients, in which miR-224-3p, miR-2682-3p and miR-4712-3p expressions were significantly up-regulated (P0.05) and miR-3940-5p expression decreased significantly (P0.05). Two, using stem ring primers for reverse transcription. SYBR Green PCR technology analyzed the expression level of these four miRNAs in a large number of patients. The results showed that miR-224-3p, miR-4712-3p expression was up and miR-3940-5p expression was down regulated in NSV patients' PBMCs, and the difference was statistically significant (P0.05), which was in accordance with the results of miRNA microarray data analysis. Microarray detection technology and real time The two methods of fluorescence quantitative PCR showed a highly positive correlation (correlation coefficient r=1.000, p=0.005). But compared with healthy people, the expression level of miR-2682-3p increased but the difference was not statistically significant (P0.05). Three, the morphology of peripheral blood mononuclear cells in the patients cultured in vitro under the inverted phase contrast microscope was round and bright. Floating distribution. Compared with the blank control group, the number and aggregation of the cells in the experimental group were significantly increased after adding different concentrations of thymosin A1 (50100ug/ml). The difference was statistically significant compared with that before treatment (P0.05). After 72h thymosin alpha 1 treatment, 4 differential miRNAs expression levels were detected by PCR, and compared with that of the untreated control group. The expression of miR-224-3p, miR-2682-3p and miR-4712-3p decreased in PBMC, but the expression of miR-3940-5p increased, but there was no significant difference between the two concentrations (P0.05). Four. In vitro, the experimental results showed that the low expression of the target gene miRNA-3940-5p in the experimental group could promote the proliferation of T cells compared with the HuT78 cells in the negative control group of the slow virus. The content of IL-2RG in the cell supernatant increased significantly, and the expression of IL-2RG protein in the experimental group was up regulated (P0.05). Conclusion 1, 4 miRNAs in the miRNA expression profiles of peripheral blood mononuclear cells in patients with non segmental vitiligo were expressed differently, of which 3 miRNAs expressions were up, 1 miRNA expressed down regulated.2, and real-time fluorescent quantitative PCR technique detection It was confirmed that the abnormal expression of these four kinds of miRNAs was consistent with the results of microarray microarray analysis. The interference of thymosin alpha 1 on the expression profiles of miRNAs in peripheral blood mononuclear cells of patients with vitiligo was directly affected by.4. MiRNA-3940-5p could be transfected into human T lymphocyte lines in human skin T lymphocyte lines and could be transfected to humans by.LV-hsa-miR-3940-5p-inhibit on. T lymphocyte HuT78 cell.5 and miR-3940-5p play a negative role in regulating the cytokine receptor IL-2R Gamma gene.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R758.41

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