TGFβ诱导miRNA及DNA甲基转移酶异常抑制Klotho促进肾脏纤维化表观遗传机制

发布时间：2018-06-17 15:14

本文选题：肾脏纤维化 + 转录生长因子β　；参考：《南京大学》2017年博士论文

【摘要】：慢性肾脏病(Chronic kidney disease,CKD)患病率和病死率居高不下己成为全球范围内的重大公共健康问题,造成了巨大的经济损失。肾脏纤维化是包括糖尿病肾病、高血压肾病、IgA肾病等在内的多种CKD的共同病理特征。肾脏纤维化过程中,αα-平滑肌肌动蛋白(Alpha smooth musle actin,α-SMA)阳性表达的肌成纤维细胞增殖能力增强,细胞外基质(Extracellular matrix,ECM)累积,形成永久的疤痕组织,肾脏功能下降。深入研究肾脏纤维化的分子机制,从根源出发寻找积极、安全、有效缓解肾脏纤维化的干预手段是我们急需面对的问题。Klotho是一个肾脏中特异性高表达的新型抗衰老蛋白,近年来研究发现Klotho有肾脏保护和抗纤维化的作用。Klotho蛋白分为膜型和分泌型,膜型Klotho主要作为FGF23等的共受体发挥作用;分泌型Klotho分布在循环系统中起激素调节作用。在肾脏纤维化过程中,氧化应激和炎症通路、TGFβ信号通路和Wnt/β-catenin信号通路共同调控促纤维化过程。近年来研究表明Klotho蛋白能够抑制炎症反应、氧化应激、TGFβ信号通路和Wnt/β-catenin信号通路。在肾脏纤维化相关疾病患者和动物模型中,Klotho在疾病进程早期即出现一致的表达下调并与疾病进程密切相关,外源性补充Klotho能够有效抵抗肾脏纤维化。综上,Klotho不仅是肾脏疾病早期的诊断标志物还是治疗纤维化相关肾脏疾病的有效靶点。在肾脏纤维化过程中Klotho下调的机制还未完全明确,Klotho基因启动子上含有密集的CpG位点,易受甲基化调控。己证明DNA甲基化调控Klotho的组织特异性表达,表观遗传调控在肾脏纤维化进程也发挥重要作用。种种线索提示在肾脏纤维化过程中Klotho表达可能受表观遗传DNA甲基化调控。我们构建了两个肾脏纤维化小鼠模型:单侧输尿管结扎(UUO)和腺嘌呤(Adenine)模型,检测肾脏组织中Klotho的表达和甲基化情况,结果证明在肾脏纤维化过程中Klotho表达下降且受甲基化调控。DNA甲基化过程主要由DNA甲基转移酶(DNAmethyltransferase,DNMT)将S-腺苷-甲硫氨酸(S-adenosyl methionine,SAM)上的甲基基团转移到胞嘧陡上,引起基因表达抑制。在哺乳动物中DNMT主要有DNMT1、DNMT3a和DNMT3b三种亚型。接下来我们进一步探究造成Klotho启动子甲基化的上游事件,我们进一步检测了肾脏组织中DNMT表达,结果发现纤维化过程中DNMT1和DNMT3a表达增加。研究发现TGFβ有抑制Klotho表达和调控甲基化的双效作用,可能是调控肾脏纤维化过程中Klotho启动子甲基化抑制的关键因子。我们的实验结果证明纤维化肾脏组织中TGFβ表达急剧增加,且TGFβ单独处理能抑制肾脏上皮细胞中Klotho的表达,促进Klotho启动子甲基化,并能促进DNMT1和DNMT3a的表达,引起和纤维化肾脏中一致的变化。通过网站分析预测与DNMT1和DNMT3a评分较高的两种miRNA,miR-152和miR-30a。在纤维化的肾脏组织和TGFβ诱导的肾脏上皮细胞中miR-152和miR-30a表达减少,抑制TGFβ Ⅰ型受体后,miR-152和miR-30a的表达抑制作用减弱。过表达miR-152和miR-30a能够分别抑制DNMT1和DNMT3a的表达。证明在肾脏纤维化过程中TGFβ通过抑制miR-152和miR-30a增加DNMT1和DNMT3a的表达量。采用小发卡RNA分别干扰DNMT1和DNMT3a表达的研究结果证明Klotho的启动子甲基化过程由DNMT1和DNMT3a共同调控。为了验证TGFβ和DNA甲基化的作用,我们用TGFβ Ⅰ型受体抑制剂SB431542和DNMT抑制剂地西他滨(5-氮杂-2'-脱氧胞嘧啶核苷,5-Aza-2'-deoxycytidine,5Aza)进行体内实验。SB431542能够有效缓解肾脏纤维化并抑制DNMT1和DNMT3a的表达上调,还能够减弱Klotho启动子甲基化水平并恢复其表达,证明了 TGFβ的关键诱导作用。5Aza能够有效恢复Klotho的表达抑制其甲基化并有效地缓解肾脏纤维化。在小鼠体内干扰Klotho表达后,发现5Aza缓解肾脏纤维化的作用受到抑制,证明Klotho是5Aza抗纤维化作用的关键靶点。综上所述,本研究证实了在肾脏纤维化过程中Klotho的表达抑制受启动子甲基化调控。并证实了在肾脏纤维化过程中Klotho甲基化的上游事件。我们通过体内实验和体外实验证明TGFβ是诱导肾脏纤维化中表观调控的关键病理因素,TGFβ能够抑制miR-152和miR-30a分别上调DNMT1和DNMT3a进而促进Klotho启动子的异常高甲基化和Klotho蛋白的表达抑制。这些变化使Klotho的肾脏保护作用减弱加剧肾脏纤维化。我们的研究证明干预miRNA-DNMT-Klotho通路可能是治疗纤维化相关肾脏疾病的积极有效的新方向。
[Abstract]:The prevalence of chronic kidney disease (Chronic kidney disease, CKD) and high mortality have become a major public health problem worldwide, causing huge economic losses. Renal fibrosis is a common pathological feature of various CKD, including diabetic nephropathy, hypertensive nephropathy, IgA nephropathy, and so on. In the process of renal fibrosis, alpha alpha - alpha - The proliferation ability of myofibroblast (Alpha smooth musle actin, alpha -SMA) positive expression of myofibroblast increased, the extracellular matrix (Extracellular matrix, ECM) accumulated, formed permanent scar tissue, and the renal function decreased. The molecular mechanism of renal fibrosis was studied in depth, to find active, safe and effective kidneys from the origin. The intervention of fibrosis is an urgent problem we need to face..Klotho is a novel antiaging protein with high specific expression in the kidney. In recent years, the study found that Klotho has renal protection and anti fibrosis effect,.Klotho protein is divided into membrane type and secretory type. Membrane type Klotho is mainly used as FGF23 and other common receptors; secretory Klotho During the process of renal fibrosis, oxidative stress and inflammatory pathways, TGF beta signaling pathway and Wnt/ beta -catenin signaling pathway co regulate the process of fibrosis during renal fibrosis. In recent years, studies have shown that Klotho protein can inhibit inflammatory response, oxidative stress, TGF beta signaling pathway and Wnt/ beta -catenin signaling pathway. In patients with renal fibrosis related diseases and animal models, Klotho has a consistent down-regulation in the early stages of the disease and is closely related to the process of disease. Exogenous Klotho can effectively resist renal fibrosis. To sum up, Klotho is not only a diagnostic marker for early renal disease, but also for the treatment of fibrosis related renal diseases. Effective targets. The mechanism of Klotho downregulation in the process of renal fibrosis is not completely clear. The Klotho gene promoter contains dense CpG loci, which is easily regulated by methylation. It has been proved that DNA methylation regulates the specific expression of Klotho, and epigenetic regulation plays an important role in the progression of renal fibrosis. Various clues suggest that the kidney is in the kidney. The expression of Klotho may be regulated by epigenetic DNA methylation during the process of filthy fibrosis. We constructed two mice models of renal fibrosis: unilateral ureteral ligation (UUO) and adenine (Adenine) model to detect the expression and methylation of Klotho in renal tissue. The results showed that the expression of Klotho in renal fibrosis was decreased and methylated in the process of renal fibrosis. The process of.DNA methylation is mainly controlled by DNA methyltransferase (DNAmethyltransferase, DNMT) to transfer the methyl group of S- adenosine methionine (S-adenosyl methionine, SAM) to cytosimon steepness, causing inhibition of gene expression. In mammals DNMT mainly consists of DNMT1, DNMT3a and DNMT3b three subtypes. In the upstream event of Klotho promoter methylation, we further detected the expression of DNMT in the renal tissue, and found that the expression of DNMT1 and DNMT3a increased during the process of fibrosis. The study found that TGF beta has the double effect of inhibiting the expression of Klotho and regulating methylation, which may be the regulation of the inhibition of the methylation of Klotho promoter in the process of renal fibrosis. Key factors. Our experimental results show that the expression of TGF beta in the renal fibrotic tissue increases sharply, and the TGF beta alone can inhibit the expression of Klotho in the renal epithelial cells, promote the methylation of the Klotho promoter, and promote the expression of DNMT1 and DNMT3a, and cause the same changes in the renal fibrosis. The two kinds of miRNA with higher T3a score, miR-152 and miR-30a. were reduced in the renal tissue of fibrosis and the expression of miR-152 and miR-30a in the renal epithelial cells induced by TGF beta. The inhibition of the expression of miR-152 and miR-30a was weakened after the inhibition of TGF beta type I receptor. In the process of fibrosis, TGF beta increases the expression of DNMT1 and DNMT3a by inhibiting miR-152 and miR-30a. The results of DNMT1 and DNMT3a expression using small hairpin RNA show that the promoter methylation process of Klotho is regulated by DNMT1 and DNMT3a. The experimental.SB431542 in 431542 and DNMT inhibitors (5- -2'- deoxycytidine nucleoside, 5-Aza-2'-deoxycytidine, 5Aza) can effectively alleviate renal fibrosis and inhibit the up-regulated expression of DNMT1 and DNMT3a, and also reduce the level of Klotho promoter methylation and restore its expression, which proves that the key inducement of TGF beta is.5A. Za can effectively restore the expression of Klotho to inhibit its methylation and effectively alleviate renal fibrosis. After interfering with Klotho expression in mice, it is found that the role of 5Aza in alleviating renal fibrosis is inhibited, which proves that Klotho is the key target of the anti fibrosis effect of 5Aza. To sum up, this research confirms Klotho in the process of renal fibrosis. The expression inhibits the regulation of promoter methylation and confirms the upstream event of Klotho methylation during renal fibrosis. Through in vivo and in vitro experiments, we have demonstrated that TGF beta is a key pathological factor in the apparent regulation of renal fibrosis. TGF beta can inhibit miR-152 and miR-30a up to DNMT1 and DNMT3a and then promote Klotho, respectively. The abnormal hypermethylation of the promoter and the inhibition of the expression of Klotho protein. These changes weaken the renal protective effect of Klotho and aggravate the renal fibrosis. Our study has shown that intervention in the miRNA-DNMT-Klotho pathway may be a positive and effective new direction for the treatment of fibrosis related renal diseases.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R692

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