强直性脊柱炎患者外周血单个核细胞中5种microRNAs表达的检测及miR-495对DVL-2蛋白的调控作用

发布时间：2018-05-02 00:39

本文选题：强直性脊柱炎 + microRNA　；参考：《宁夏医科大学》2015年硕士论文

【摘要】：目的通过对AS患者PBMC中5种微小RNA(micro-RNA,mi RNAs)表达量的检测,筛选出差异性表达的mi RNAs。探讨mi RNAs是否可以作为AS患者潜在的诊断标志物,并对mi R-495在DVL-2蛋白表达中的调控作用进行研究。方法1.利用mi Randa、Star Base等生物信息学软件,筛选与AS致病密切相关的mi RNAs。并利用Real-time PCR方法对临床61例AS患者和31例健康者PBMC中mi R-17、mi R-106、mi R-181、mi R-495和mi R-30的表达情况进行检测,同时利用统计软件对实验结果进行分析,筛选可以作为AS诊断潜在生物标志物的mi RNAs。2.利用mi Randa、Star Base等生物信息学软件,以AS患者PBMC中表达量具有显著性差异的mi R-495为研究对象,对其潜在的靶基因进行预测。通过双荧光素酶报告基因系统及Western-Blot实验验证mi R-495与DVL-2的靶向关系。3.应用ELISA对28例AS患者和14例健康者血浆中的DVL-2蛋白量进行检测,研究AS患者PBMC中mi R-495的表达量与血浆中DVL-2蛋白水平的相关性以及mi R-495通过靶向抑制DVL-2蛋白调控AS发病机制。结果1.在AS患者PBMC中表达上调的有mi R-106与mi R-30;表达下调mi R-181(p0.05)、mi R-495(p0.05)和mi R-17。并分别对mi R-181和mi R-495作为临床诊断进行ROC曲线分析,其中mi R-181的AUG为0.697,敏感性达76.67%,特异性达54.84%,正确性为68.48%。通过对结果的分析,我们认为mi R-181对临床诊断参考价值较低;而mi R-495的AUG为0.729,敏感性达92.23%,特异性达58.06%,正确性为86.9%。结果表明,mi R-495有望成为AS疾病潜在的诊断标志物。2.预测结果表明mi R-495可以靶向作用于DVL-2基因。双荧光素酶报告结果表明:过表达mi R-495可抑制DVL-2的相对荧光素酶活性;相反抑制mi R-495可增加DVL-2的相对荧光活性。Western-Blot结果表明:过表达mi R-495可抑制DVL-2蛋白表达量;而抑制表达mi R-495可促进DVL-2蛋白的表达。由此我们认为mi R-495可以靶向抑制DVL-2。3.进一步通过ELISA方法检测临床样本血浆中DVL-2的表达情况,在AS患者中DVL-2的表达量明显增高。并联合PBMC中mi R-495的表达量与血浆中DVL-2蛋白的表达量进行相关性分析,结果表明mi R-495与DVL-2呈负相关,进一步说明mi R-495可靶向抑制DVL蛋白的表达并参与AS发病机制的调控。结论1.PBMC中mi R-495的表达水平,对AS疾病的临床诊断具有参考意义,有望成为AS疾病潜在的诊断标志物。2.mi R-495可以靶向调节Wnt通路中的关键蛋白DVL-2的表达,在AS疾病中起到调节作用。
[Abstract]:Objective to detect the expression of micro-RNAi RNAs-5 kinds of micro-RNAs in PBMC of patients with as, and to screen out the differentially expressed miRNAs. To explore whether mi RNAs can be used as a potential diagnostic marker in as patients, and to study the regulatory role of mi R-495 in the expression of DVL-2 protein. Method 1. Using the bioinformatics software such as mi Randa Star Base to screen the mi RNAs which are closely related to the pathogenicity of as. The expressions of mi R-17P mi R-106mi R-181mmi R-495 and miR-30 in 61 patients with as and 31 healthy controls were detected by Real-time PCR method. The results of the experiment were analyzed by statistical software. To screen the potential biomarker for as diagnosis, mi RNas. 2. 2. Using the bioinformatics software such as mi Randaanstar Base, the potential target genes of as patients with PBMC were predicted by using mi R-495, which had significant difference in PBMC expression. The targeting relationship between mi R-495 and DVL-2 was confirmed by double luciferase reporter gene system and Western-Blot experiment. The levels of DVL-2 protein in plasma of 28 patients with as and 14 healthy controls were detected by ELISA. The correlation between the expression of mi R-495 in PBMC and the level of DVL-2 protein in plasma was studied. The mechanism of regulation of as by targeting inhibition of DVL-2 protein was studied. Result 1. The up-regulated expression of mi R-106 and mi R-30 was found in PBMC of as patients, and the down-regulation of the expression of mi R-181 p0.05 + mi R-495P 0.05) and mi R-17. The ROC curves of miR-181 and miR-495 were analyzed respectively. The AUG of miR-181 was 0.697, the sensitivity was 76.677.The specificity was 54.844.The accuracy was 68.48. Through the analysis of the results, we think that the reference value of miR-181 for clinical diagnosis is lower, while the AUG of miR-495 is 0.729, the sensitivity is 92.233.The specificity is 58.06, and the correctness is 86.9. The results showed that MMI R-495 might be a potential diagnostic marker for as. The predicted results showed that mi R-495 could target DVL-2 gene. The results of double luciferase report showed that overexpression of mi R-495 could inhibit the relative luciferase activity of DVL-2, whereas inhibition of mi R-495 could increase the relative fluorescence activity of DVL-2. Western-Blot results showed that overexpression of mi R-495 could inhibit the expression of DVL-2 protein. Inhibition of the expression of mi R-495 could promote the expression of DVL-2 protein. Therefore, we think that mi R-495 can be targeted to inhibit DVL 2.3. Furthermore, the expression of DVL-2 in plasma was detected by ELISA method, and the expression of DVL-2 was significantly increased in as patients. The correlation between the expression of miR-495 in PBMC and the expression of DVL-2 protein in plasma was analyzed. The results showed that the expression of miR-495 was negatively correlated with DVL-2, which further indicated that miR-495 could inhibit the expression of DVL protein and participate in the regulation of the pathogenesis of as. Conclusion the expression level of miR-495 in 1.PBMC may be useful for the clinical diagnosis of as, and it may be a potential marker for the diagnosis of as. MiR-495 can regulate the expression of DVL-2, a key protein in Wnt pathway, and play a regulatory role in as disease.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.23

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