miR-30和lincRNA-p21调控肝纤维化的作用机制研究

发布时间：2019-05-28 04:54

【摘要】：肝纤维化在组织水平上的直接表现是细胞外基质(extracellular matrix, ECM)过度沉积,肝脏正常结构被破坏。从细胞水平来看,肝脏内过度活化的肝星状细胞大量分泌ECM被普遍认为是导致肝纤维化发生的直接原因。然而,肝细胞的持续坏死和凋亡可以引发肝组织内早期炎症反应,直接激活肝星状细胞。因此,肝细胞凋亡在肝纤维化发生过程中起关键作用。尽管大量的实验证据表明肝星状细胞活化和肝细胞凋亡与TGF-β信号通路密切相关,但是对于肝纤维化过程中TGF-β信号参与调控这些细胞生命活动的具体分子机制,目前的认识依然非常局限。因此,更加全面、深入地阐明肝星状细胞过度活化和肝细胞持续凋亡与TGF-β信号通路深层次的调控关系仍然是当前亟待解决的关键科学问题。充分的研究证据表明,微小RNA (microRNA, miRNA)和长链非编码RNA (long non-coding RNAs, lncRNAs)作为非编码RNA (non-coding RNA)家族中最重要的两种类型参与了诸多人类疾病的发生发展过程,它们作为重要的功能分子参与调控各种信号通路以及一系列细胞生物学过程也被广泛地揭示。然而,目前关于microRNA和lncRNA参与肝纤维化过程中TGF-β信号通路的调控机制以及它们之间存在的表达调控关系尚不清楚。因此,基于以上科学问题,我们对microRNA和lncRNA与TGF-β信号通路之间的关系以及它们参与调节肝星状细胞活化和肝细胞凋亡的过程进行了深入地分析和探讨。在本文中,我们首先探究了肝纤维化过程中microRNA对活化肝星状细胞内TGF-β信号通路的调控机制。利用CCl4诱导的肝纤维化小鼠模型,我们首先观察到纤维化过程中星状细胞内miR-30的表达显著下调。同时,TGF-β诱导的转录调节因子KLF11的表达显著上调。接下来,我们分别在肝星状细胞和肝纤维化动物模型中研究了miR-30和其潜在的作用靶点KLF11的相互作用及其对肝星状细胞活化的影响。我们在肝纤维化小鼠模型中利用腺病毒介导肝星状细胞特异性表达miR-30的方法以研究miR-30的生物学功能。结果发现在肝星状细胞中过表达miR-30显著抑制了CCl4诱导的肝纤维化；同时,有效抑制了小鼠肝星状细胞中KLF11表达并上调Smad7的水平。进一步对miR-30作用机制的探索表明,KLF11是miR-30的直接作用靶点,并且miR-30可以通过抑制肝星状细胞内KLF11的表达而削弱TGF-β信号通路传导,而KLF11本身又通过抑制负反馈调节物Smad7的表达而加强TGF-β信号通路发生。随后的调查发现,利用KLF11过表达载体或siRNA来上调或下调肝星状细胞中KLF11的水平可以促进或抑制肝星状细胞的活化;同时,利用miR-30的前体或者抑制剂上调或下调它在肝星状细胞内的水平证明miR-30可以通过抑制肝星状细胞的增殖和迁移达到抑制细胞活化的口的。因此,这部分研究揭示了miR-30作为肝纤维化发生的重要阻遏物通过抑制TGF-β诱导的早期转录因子KLF11的表达来削弱肝星状细胞活化过程中TGF-β信号通路的传导,并且为肝纤维化的机制探索提供了重要的研究视野。尽管lncRNAs具有重要的生物学功能,但它是否以及如何参与肝纤维化的调控仍然未知。在本部分研究中,我们首先利用高通量测序方法对CCl4诱导的肝纤维化小鼠肝细胞中lncRNAs的表达情况进行了检测。结果发现纤维化小鼠肝细胞相对于正常肝细胞,63个lncRNAs发生了显著上调,同时48个lncRNAs发生了显著下调。进一步地筛查发现,IincRNA-p21的表达明显上调约4倍且关于它在肝纤维化中的具体作用机制研究尚属空白。于是,我们分别在肝细胞和肝纤维化小鼠模型中研究了lincRNA-p21参与肝细胞凋亡及其对肝纤维化发生的影响。我们结合Northern blot、qRT-PCR以及RT-PCR等方法对肝纤维化过程肝组织和不同类型细胞内的lincRNA-p21表达情况进行了分析,发现在CCl4诱导的小鼠肝纤维化过程中,肝细胞内的lincRNA-p21表达特异性升高。接下来,我们利用腺病毒介导肝细胞特异性干扰lincRNA-p21的方法以研究lincRNA-p21的生物学功能,结果发现下调小鼠肝细胞内lincRNA-p21水平对肝纤维化具有显著治疗作用,且下调此过程中lincRNA-p21的表达可以有效抑制肝细胞的凋亡并促进肝细胞增殖。我们又对lincRNA-p21影响肝细胞凋亡的作用机制进行了探究,利用生物信息学分析结合荧光素酶报告实验发现lincRNA-p21序列中存在与miR-30家族种子序列保守结合的位点。与此同时,在体外分别对miR-30和lincRNA-p21进行生物素标记并对肝细胞进行RNA pulldown和RNA沉淀实验发现lincRNA-p21可与miR-30特异性地结合。最后,通过干扰小鼠肝细胞miR-30的表达抵消了lincRNA-p21干扰腺病毒在肝纤维化模型中的疗效。因此,我们的研究从肝细胞入手,揭示了lincRNA-p21在肝纤维化过程中被诱导表达并通过与肝细胞内的miR-30竞争性结合而参与肝纤维化的发生发展过程,并确定了lincRNA-p21可作为一种新型的竞争性内源非编码RNA参与肝细胞凋亡和增殖的调控,这也为肝纤维化的治疗提供了新的靶点和研究思路。
[Abstract]:The direct expression of hepatic fibrosis at the tissue level is the excessive deposition of the extracellular matrix (ECM) and the normal structure of the liver is destroyed. From the cell level, a large number of secretory ECMs of the hyperactivated hepatic stellate cells in the liver are generally considered to be a direct cause of the occurrence of hepatic fibrosis. However, the continuous necrosis and apoptosis of the liver cells can initiate the early inflammatory reaction in the liver tissue and directly activate the hepatic stellate cells. Therefore, hepatocyte apoptosis plays a key role in the pathogenesis of hepatic fibrosis. Although a large number of experimental evidence suggests that the activation of the hepatic stellate cells and the apoptosis of the hepatocytes are closely related to the TGF-signaling pathway, the present recognition is still very limited to the specific molecular mechanism involved in the control of these cellular life activities during the liver fibrosis. Therefore, it is still a key scientific problem to be solved urgently. The sufficient study evidence indicates that microRNA (microRNA, miRNA) and long-chain non-coding RNAs (lncRNAs) are the most important two types of non-coding RNA (non-coding RNAs) in the development of many human diseases, They are also widely disclosed as important functional molecules involved in the regulation of various signal pathways and a range of cell biological processes. However, the regulation and regulation of TGF-signaling pathway in the process of liver fibrosis with microRNA and lncRNA are not clear. Therefore, based on the above scientific problems, we deeply analyze and discuss the relationship between microRNA and lncRNA and TGF-I signal pathway and their participation in the regulation of hepatic stellate cell activation and hepatocyte apoptosis. In this paper, we first study the regulation and control mechanism of microRNA in the activation of TGF-1 signaling pathway in hepatic stellate cells. In the model of liver fibrosis induced by CCl4, we first observed that the expression of miR-30 in the star-like cells was significantly reduced in the course of fibrosis. At the same time, the expression of the transcription regulatory factor KLF11 induced by TGF-1 was up-regulated. Next, we studied the interaction of miR-30 and its potential target KLF11 and its effect on the activation of hepatic stellate cells in the model of hepatic stellate and hepatic fibrosis, respectively. We use adenovirus-mediated hepatic stellate cell specific expression of miR-30 in the liver fibrosis mouse model to study the biological function of miR-30. The results showed that the expression of miR-30 in the hepatic stellate cells significantly inhibited the liver fibrosis induced by CCl4, and the expression of KLF11 in the hepatic stellate cells of the mice was effectively inhibited and the level of Smad7 was increased. Further exploration of the action mechanism of miR-30 shows that KLF11 is a direct target of miR-30, and miR-30 can weaken the conduction of the TGF-1 signal by inhibiting the expression of KLF11 in the hepatic stellate cell, and the KLF11 itself enhances the TGF-1 signal path by suppressing the expression of the negative feedback regulator Smad7. Subsequent investigations have found that the level of the KLF11 in the hepatic stellate cells can be raised or reduced by the use of the KLF11 overexpression vector or siRNA to promote or inhibit the activation of the hepatic stellate cells, Up-regulation or down-regulation of its level in the hepatic stellate cell using a precursor or inhibitor of miR-30 demonstrates that miR-30 can inhibit cell activation by inhibiting the proliferation and migration of hepatic stellate cells. Therefore, this part of the study revealed that the important repressor of miR-30 as a hepatic fibrosis, by inhibiting the expression of the early transcription factor KLF11 induced by TGF-1, weakened the conduction of the TGF-1 signal pathway in the activation of the hepatic stellate cells, And provides an important field of research for the mechanism of liver fibrosis. Although lncRNAs have important biological functions, it is not known whether or not it is involved in the regulation of hepatic fibrosis. In this part of the study, we first used the high-throughput sequencing method to detect the expression of lncRNAs in the liver-fibrosis mouse liver cells induced by CCl4. The results showed that the liver cells of the fibrotic mice were significantly up-regulated with respect to the normal hepatocytes and 63 lncRNAs, and the 48 lncRNAs were down-regulated. Further screening found that the expression of IincRNA-p21 was significantly up-regulated by about 4 times and a study on its specific mechanism of action in hepatic fibrosis was still blank. So, we studied the effect of lincRNA-p21 in the hepatocyte and the liver fibrosis mouse model, and the effect on the hepatic fibrosis. The expression of lincRNA-p21 in the liver and different types of cells in the liver fibrosis was analyzed by using the methods of Northern blot, qRT-PCR and RT-PCR. Next, we studied the biological function of lincRNA-p21 by using adenovirus-mediated hepatocyte-specific interference line cRNA-p21 to study the biological function of lincRNA-p21, and found that the down-regulation of the level of lincRNA-p21 in the mouse hepatocytes has a significant therapeutic effect on the liver fibrosis, And the expression of the lincRNA-p21 in the process can effectively inhibit the apoptosis of the liver cells and promote the proliferation of the liver cells. We also explored the mechanism of the effect of linkcRNA-p21 on the apoptosis of hepatocytes, and by using the bioinformatics analysis combined with the luciferase reporter, it was found that there was a conserved binding site of the seed sequence of the miR-30 family in the linkcRNA-p21 sequence. At the same time, in vitro, the miR-30 and the lincRNA-p21 were labeled with biotin and the hepatocytes were subjected to RNA pulpit and RNA precipitation experiments, and the linkcRNA-p21 can be specifically combined with the miR-30. Finally, by interfering with the expression of miR-30 in the mouse liver cells, the effect of the lincRNA-p21 interference adenovirus in the liver fibrosis model was cancelled. Therefore, our research starts with the liver cells, and reveals that the linkcRNA-p21 is induced to express in the liver fibrosis process and is involved in the development of the liver fibrosis through the competitive binding of the miR-30 in the liver cells, And it is determined that the linkcRNA-p21 can be used as a novel competitive endogenous non-coding RNA to participate in the regulation of the apoptosis and the proliferation of the liver cells, which also provides a new target point and a research thinking for the treatment of the liver fibrosis.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R575.2

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