循环miR-130b-3p在狼疮性肾炎进展中的作用

发布时间：2018-04-11 17:52

  本文选题：系统性红斑狼疮 + 狼疮性肾炎　； 参考：《上海交通大学》2015年博士论文

【摘要】：【目的】系统性红斑狼疮(SLE)是由于机体免疫耐受缺失而引起的自身免疫性疾病,通常伴随免疫复合物性肾小球肾炎,狼疮性肾炎(LN)是其主要并发症,严重影响患者预后。循环micro RNA(如血清micro RNA)在临床疾病诊断中发挥重要作用,可以作为潜在的诊断标记物。本文拟通过:1.microarray技术分析处于不同进展阶段的LN患者血清mi RNA的变化。2.探讨循环mi RNA对于LN的诊断价值及与LN临床参数相关性。3.从分子生物学机制探讨异常表达的循环mi RNA在LN发生发展中的作用。【方法】1.本研究一共囊括96份血清标本,60例LN患者和36名健康对照组。挑选出其中性别、年龄匹配的12份血清标本用于循环mi RNA表达谱筛选,包括早期LN患者4名(CKD分期1-3期),晚期LN患者4名(CKD分期4-5期)及健康体检者4名。使用mi RNA PCR芯片检测LN患者血清中372个mi RNA变化。2.剩余52名LN患者,包括40名早期LN患者、12名晚期LN患者及32名年龄、性别无差异的健康对照血清样本用于验证mi RNA表达谱筛选结果。200μL血清用于抽提总RNA,最后溶解于12μL无RNA酶水,Nanodrop 2000检测血清RNA抽提浓度;使用tagman茎-环引物探针real-time PCR检测差异循环mi RNA的相对表达量。分析循环mi RNA与LN患者临床参数相关性,受试者工作曲线(ROC)及曲线下面积(AUC)分析循环mi RNA对疾病诊断的价值。3.肾小管上皮细胞(HK-2)分别转染mi R-130b-3p mimics、inhibitor及各自相应的对照,并予以TGF-β1(10ng/ml)刺激。实时定量PCR和westen blot检测转染72h后细胞Ⅰ型胶原、Ⅳ型胶原、E钙连接素(E-cad)、α-平滑肌肌动蛋白(α-SMA)转录水平变化,westen blot检测E-cad、α-SMA蛋白水平变化;检测靶基因ERBB2IP及PPARγ的蛋白在转染mimics后水平变化。构建含有野生型ERBB2IP及PPARγ3’-UTR及突变的PPARγ3’-UTR双荧光素报告酶载体,分别予以对照mimic、突变mimic及正常mimic共转染,48h后检测荧光活性。【结果】1.与对照组相比,早期LN患者5个mi RNA表达下降倍数大于2,33个mi RNA上升倍数大于2。其中7个表达上升的mi RNA差异具有统计学意义;分别是:mi R-1233-3p(P=0.019),mi R-130b-3p(P=0.021),mi R-18a-3p(P=0.021),mi R-628-3p(P=0.023),mi R-1260b(P=0.030),mi R-1539(P=0.041)和mi R-378e(P=0.047),晚期LN组与健康对照组比较,共75个循环mi RNA表达下调大于2倍,其中P值小于0.05的mi RNA有21个;晚期LN组循环与早期LN组比较,共107个循环mi RNA表达下调,其中53个mi RNA其表达下调具有统计学意义(P0.05)。对照组,早期LN患者、晚期LN患者RNA浓度依次为30.8(28.0,35.0)ng/μL、30.5(25.4,33.1)ng/μL、16.4(14.9,19.3)ng/μL,与对照组和早期LN组相比较,晚期LN患者血清总RNA浓度减少,其差异具有统计学意义(P0.01)。2.早期LN组患者与对照组比较,血清中存在mi R-130b-3p升高[IQR 16.2(8.7,42.7)vs 9.6(4.8,17.4),p0.01)],其差异具有统计学意义(P0.01),循环mi R-130b-3p对于区分LN患者与正常对照均具有一定价值价值,ROC曲线下面积(AUC)=0.683±0.062(95%CI=0.561-0.805,P0.01),最优敏感度和特异度分别为35.05%和96.8%;验证组显示早期LN患者血清mi R-1233-3p虽然高于健康对照组患者[IQR 10.4(6.4,16.9)vs 8.7(3.8,16.9)],但其统计学差异未见明显意义(P0.05);相关性分析显示,循环mi R-130b-3p的相对表达量与LN患者疾病活动性各指标,如SLE疾病活动性评分(SLEDAI评分)、血清C3、C4、ds DNA、C反应蛋白(CRP)、红细胞沉降率(ESR)等无明显相关性(P0.05),与24小时尿蛋白、慢性肾脏活动指数(CI)、甘油三酯(TG)呈正相关(P0.05)。3.正常HK-2细胞在转染mi R-130b-3p mimic后,Ⅰ型胶原、Ⅳ型胶原、E钙连接素(E-cad)、α-平滑肌肌动蛋白(α-SMA)表达并没有显著差异;但在TGF-β1刺激时,mi R-130b-3p的转染可以增加小管上皮细胞Ⅰ型胶原、Ⅳ型胶原、α-平滑肌肌动蛋白(α-SMA)的m RNA和蛋白表达水平,并下调E-cad的表达,差异具有统计学意义(P0.05);相反的作用见于转染mi R-130b-3p inhibitor;转染mi R-130b-3p mimics无论伴或者不伴有TGF-β1(10ng/ml)刺激的情况下,与对照组相比,mi R-130b-3p mimics可以显著下调PPAR-γ或者ERBB2IP蛋白表达水平(P0.05);mi R-130b-3p mimics与构建ERBB2IP基因的3'UTR及突变的荧光素酶报告质粒共转染工具细胞293T细胞后,与NC和mut-mi R-130b-3p组相比,wt-mi R-130b-3p mimics与质粒共转染使海肾荧光素酶活性下降,海肾荧光素酶与萤火虫荧光素酶活性之比下降(P0.01),而NC-mimics及mut-mi R-130b-3p对质粒的作用无显著性差异(P0.05);mi R-130b-3p mimics使wt-PPAR-γ-3’-UTR海肾荧光素酶与萤火虫荧光素酶活性之比下降(P0.01),NC mimic及mut-PPAR-γ无此作用。【结论】LN肾炎患者存在血清mi RNA表达异常;对于严重肾功能受损患者,循环mi RNA存在整体下降;早期升高的循环mi R-130b-3p可能通过与PPAR-γ和ERBB2IP 3’-UTR直接结合而调控肾小管上皮细胞EMT作用,参与早期LN肾脏损害。
[Abstract]:[Objective] systemic lupus erythematosus (SLE) is caused by the lack of immune tolerance and autoimmune disease, often accompanied by immune complex glomerulonephritis, lupus nephritis (LN) is the main complication, seriously affect the prognosis of patients with RNA. Micro cycle (such as serum micro RNA) play an important role in the clinical the diagnosis of disease, can be used as a potential diagnostic marker. This paper proposed by: in the analysis of 1.microarray technology.2. LN RNA changes of serum MI in patients with different stages of progress on the role in the occurrence and development of LN in MI RNA mi RNA for cycle cycle the diagnostic value of LN and LN and the clinical parameters between.3. from molecular biology mechanism of abnormal expression. [method] this study included a total of 1. of 96 serum samples, 60 cases of LN patients and 36 healthy controls. The selected one of the gender, age matched 12 serum specimens for circulating mi The expression profile of RNA screening, including 4 patients with early LN (CKD stage 1-3), 4 patients with advanced LN (CKD stage 4-5) and 4 healthy controls. The remaining 52 patients with LN 372 mi RNA.2. to detect changes of serum LN in patients with MI RNA PCR chip, including 40 patients with early LN 12, late stage LN patients and 32 age-matched healthy controls, no gender difference in serum samples used to validate mi RNA expression.200 L screening results for serum total RNA extracted, finally dissolved in 12 L water 2000 Nanodrop without RNA enzyme, serum RNA extraction concentration; the relative expression of circulating mi RNA assay. The use of tagman stem loop primers. Real-time PCR analysis of circulating mi RNA with clinical parameters of patients with LN correlation receiver operating curve (ROC) and area under the curve (AUC) analysis of circulating mi RNA on the diagnosis value of.3. in renal tubular epithelial cells (HK-2) were transfected into mi R-130b-3p mimics, inhib Itor and the corresponding control, and be TGF- beta 1 (10ng/ml) stimulation. Real time quantitative PCR and westen blot detection after transfection of 72h cells of type I collagen, type IV collagen, E calcium ligatin (E-cad), alpha smooth muscle actin (alpha -SMA) transcription level changes, westen blot detection of E-cad, changes of alpha -SMA protein level; protein detection of target gene ERBB2IP and PPAR gamma changes in level mimics after transfection. Construct containing wild type ERBB2IP and PPAR Gamma 3 '-UTR and mutation of PPAR Gamma 3' -UTR dual luciferase reporter vector, respectively, compared with mimic, mimic mutation and normal mimic co transfection, fluorescence detection [48H activity. Results 1.] compared with the control group, 5 patients with early LN mi RNA decreased the expression of multiple mi RNA rose more than 2,33 times greater than 2. of which were statistically significant differences in expression of MI RNA increased 7; respectively is: Mi R-1233-3p (P=0.019), MI R-130b-3p (P=0.021), MI R-18a -3p(P=0.021),mi R-628-3p(P=0.023),mi R-1260b(P=0.030),mi R-1539(P=0.041)鍜宮i R-378e(P=0.047),鏅氭湡LN缁勪笌鍋ュ悍瀵圭収缁勬瘮杈，

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