长链非编码RNA PVT1通过竞争性结合miR-152参与肝纤维化的研究

发布时间：2018-05-03 13:18

本文选题：肝星状细胞 + PVT1　；参考：《南方医科大学》2017年博士论文

【摘要】：研究背景和目的:肝纤维化主要特征为肝脏内细胞外基质(extracellular matrix,ECM)的过度沉积。肝星状细胞(Hepatic stellate cell,HSC)是肝主要的ECM产生细胞,其活化过程被视为肝纤维化的关键环节。近来研究显示Hedgehog(Hh)调控的内皮间叶化进程(EMT)参与HSC活化。以往视为“噪音”的长链非编码RNA(Long non-coding RNAs,lncRNAs)现已被证实与多种疾病相关且能调节基因功能。浆细胞瘤变异易位1(Plasmacytoma variant translocation 1,PVT1)是一个肿瘤相关lncRNA,但其在肝纤维化中的作用尚不清楚。本研究目的是探索PVT1在肝纤维化中的表达改变、功能及其相关分子机制。方法:四氯化碳(CC14)诱导的小鼠肝纤维化模型接受重组腺病毒Ad-shPVT1或丹酚酸B(SalvianolicacidB,SalB)的治疗并检测肝脏胶原量的改变。原代HSCs接受PVT1 siRNA或Sal B处理后检测增殖和胶原改变。实时荧光定量PCR和Western blot分别检测EMT、Hedgehog信号及其它相关基因mRNA和蛋白改变。亚硫酸氢盐测序法用于分析PTCH1启动子甲基化。荧光素报告基团及Pull down实验验证PVT1和microRNA-152(miR-152)间是否存在关联。结果:与对照组相比,PVT1在纤维化肝组织和活化的HSCs中表达升高。在HSCs中,沉默PVT1有效抑制HSC活化包括增殖和胶原蛋白的下降;体内PVT1的减少也能抑制CC14诱导的I型胶原。PVT1下降引起EMT进程的阻滞,伴有E-cadherin的上调和desmin及vimentin的下调。PVT1下降也抑制了Hedgehog信号,伴有PTCH1的上调和SMO及GLI2的下调。此外,我们的研究表明Sal B也有良好的体内外抗肝纤维化疗效。在Sal B处理后的HSCs中,我们再次确证了 Hedgehog信号下降会伴有EMT进程阻滞。研究发现作为Hedgehog信号负向调控因子的PTCH1,其启动子在肝纤维化进展时会高度甲基化。Sal B能抑制PTCH1的甲基化并恢复其表达水平。深入研究证实靶向DNA甲基转移酶1的miR-152调控PTCH1甲基化进程。我们发现PVT1下调不仅抑制体内外PCTH1的甲基化还伴有miR-152的升高。值得注意的是,下降的细胞增殖和胶原表达以及增强的PTCH1去甲基化和PTCH1表达水平,这些PVT1下调所带来的效应均被miR-152抑制剂所阻断。荧光素报告基团实验证实PVT1与miR-152间存在靶向关系。Pull down实验也证实PVT1能够直接结合miR-152。此外,在Sal B处理后的HSCs中沉默PVT1能增强Sal B对细胞增殖和胶原的抑制作用。结论:PVT1在CC14诱导后的纤维化肝组织和活化的HSCs中表达均上调,而PVT1下调后则表现出体内外抗纤维化效果。我们证实PVT1通过竞争性结合miR-152继而表观性抑制PTCH1表达,从而促进肝纤维化中Hedgehog信号活化和EMT发展。本研究表明了 PVT1/miR-152/PTCH1这一新颖的信号通路存在于肝纤维化中且Sal B能通过该信号发挥抗纤维化作用。
[Abstract]:Background and purpose: liver fibrosis is mainly characterized by excessive deposition of extracellular matrix (ECM) in the liver. Hepatic stellate cell (HSC) is the major ECM producing cell of the liver, and its activation process is considered as a key link in liver fibrosis. Recent studies have shown that Hedgehog (Hh) Regulation of endothelial foliage has been shown. EMT (Long non-coding RNAs, lncRNAs), formerly known as "noise", has been confirmed to be associated with a variety of diseases and can regulate gene function. The mutation translocation 1 (Plasmacytoma variant translocation 1, PVT1) of plasma cell tumor (Plasmacytoma variant translocation 1, PVT1) is a tumor associated lncRNA, but its role in liver fibrosis is not clear. The aim of this study was to explore the expression changes, functions and related molecular mechanisms of PVT1 in liver fibrosis. Methods: the mice liver fibrosis model induced by carbon tetrachloride (CC14) accepted the treatment of recombinant adenovirus Ad-shPVT1 or salvianolic acid B (SalvianolicacidB, SalB) and the changes in the liver collagen content. The original HSCs accepted PVT1 siRNA or Sal B. After treatment, the proliferation and collagen changes were detected. Real-time fluorescence quantitative PCR and Western blot were used to detect EMT, Hedgehog signal and other related genes mRNA and protein changes. The hydrogen sulfite sequencing method was used to analyze PTCH1 promoter methylation. The fluorescein report group and Pull down experiment verified whether there was any association between PVT1 and microRNA-152 (miR-152). Results: compared with the control group, the expression of PVT1 increased in the fibrosis liver tissue and activated HSCs. In HSCs, the silence of PVT1 effectively inhibited the activation of HSC, including the decrease of proliferation and collagen, and the decrease of PVT1 in the body could inhibit the CC14 induced I type collagen.PVT1 decrease to block the EMT process, accompanied by E-cadherin up and desmin. The downregulation of.PVT1 also inhibited the Hedgehog signal, with the up-regulation of PTCH1 and the downregulation of SMO and GLI2. In addition, our study showed that Sal B also had a good therapeutic effect on liver fibrosis. In HSCs after Sal B, we confirmed that Hedgehog signal decline would be accompanied by EMT process block. To the regulatory factor PTCH1, its promoter highly methylation.Sal B can inhibit the methylation of PTCH1 and restore its expression level when liver fibrosis progresses. Further studies have confirmed that the miR-152 targeting DNA methyltransferase 1 regulates the process of PTCH1 methylation. We found that the down regulation of PVT1 not only inhibits the methylation of PCTH1 in and out of the body, but also is associated with miR-152. It is worth noting that decreased cell proliferation and collagen expression as well as enhanced PTCH1 demethylation and PTCH1 expression levels, the effects of these down-regulation were blocked by miR-152 inhibitors. The fluorescein report group experiment confirmed the existence of the targeting relationship between PVT1 and miR-152,.Pull down experiment also confirmed that PVT1 can be directly combined with miR-15. 2. in addition, the silencing of PVT1 in HSCs treated with Sal B enhanced the inhibitory effect of Sal B on cell proliferation and collagen. Conclusion: the expression of PVT1 in the fibrosis liver tissues and activated HSCs after CC14 induction is up, and the down regulation of PVT1 shows the effect of anti fibrosis in vivo and in vivo. Inhibiting the expression of PTCH1 and promoting the activation of Hedgehog signal and the development of EMT in liver fibrosis. This study shows that the novel signal pathway of PVT1/miR-152/PTCH1 exists in liver fibrosis and Sal B can play anti fibrosis effect through the signal.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575.2

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