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一文了解lncRNA、DNA以及蛋白作用关系

Update:2017-11-15From:小张聊科研 Hot:
  

 A Positive Regulatory Loop between a Wnt-Regulated Non-coding RNA and ASCL2 Controls Intestinal Stem Cell Fate Cell Reports, June 21, 2016)

下载链接:http://pan.baidu.com/s/1nuOeoxr 密码:e5j4

在开始本文前,我们再熟悉一下Wnt/beta-catenin经典通路:

“当Wnt蛋白与细胞表面Frizzled受体家族结合后的一系列反应,包括Dishevelled受体家族蛋白质的激活及最终细胞核内β-catenin水平的变化。 Dishevelled (DSH) 是细胞膜相关Wnt受体复合物的关键成分,它与Wnt结合后被激活,并抑制下游蛋白质复合物,包括axin、GSK-3、与APC蛋白。axin/GSK-3/APC 复合体可促进细胞内信号分子β-catenin的降解。当该复合物被抑制后,胞浆内的β-catenin得以稳定存在,部分 β-catenin进入细胞核与TCF/LEF转录因子家族作用并促进特定基因的表达。”

简单概括:Wnt+DSH下调axin/GSK-3/APC,然后beta-catenin上调(抑制降解),beta-catenin核转位,作用TCF/LEF,促进下游基因表达。

文中涉及到了:Wnt-beta-catenin-TCF当然这不是主要的

先上机制图:

在肠上皮细胞中,经典Wnt路通对干细胞的稳态维持、分化、增殖起到主要作用。在结直肠癌中,活性异常的TCF4/b-catenin转录复合物,是首要的转化因子。作者发现了一个lncRNA,能直接结合TCF4/b-catenin。并且,该分子能调控它基因组附近的一个基因ASCL2(ASCL2作为一个转录因子能控制小肠干细胞的命运),增加ASCL2转录。WiNTRLINC1结合TCF4/b-catenin能介导WiNTRLINC1的启动子与ASCL2调控域形成一个环。反过来,ASCL2蛋白能结合到WiNTRLINC1启动子区,正向调控WiNTRLINC1转录。这个调控回路,在结直肠癌中被显著放大,增加癌细胞转移,降低病人存活率。

  • 作者发现了一个wnt通路调控的lncRNA-WiNTRLINC1

  • 该分子在结直肠癌细胞中是下调的,导致细胞凋亡

  • 该分子通过长途回路及染色质改变来正向调控ASCL2

  • WiNTRLINC1-ASCL2的调控作用在结直肠癌中很显著

点击这里可温习前文lncRNA作用机制的三种模式

开始看主要内容:

1、WiNTRLINC1对细胞水平的影响

WiNTRLINC1能促进细胞增殖,减低细胞凋亡水平。

2、WiNTRLINC1能正向调控ASCL2表达,以及机制相关

作者敲除WiNTRLINC1来寻找有差异表达的基因。其中有一个很显著下调的分子ASCL2,它在染色质上的位置与WiNTRLINC1很近。而且ASCL2是Wnt通路的应答基因,并且在肠道中其蛋白对干细胞的维持起到很大的作用。

而在不同的细胞株中发现,ASCL2的表达与WiNTRLINC1的表达紧密相关。结合第一部分内容,所以作者认为WiNTRLINC1通过调控ASCL2的表达来调控细胞增殖的。

WiNTRLINC1、ASCL2表达具有相关性。并且ASCL2敲除后,其调控水平依然与WiNTRLINC1、ASCL2分别敲除保持一致。

作者认为WiNTRLINC1、ASCL2存在一个拓扑环形式。通过染色体构象捕获技术(3C技术),发现WiNTRLINC1的转录起始位点区域与ASCL2下游基因区域相互作用(该区域为ASCL2的增强子),并且该作用对WiNTRLINC1存在依赖性。

ChIRP技术发现WiNTRLINC1的转录本存在与WiNTRLINC1基因转录区,并且当beta-catenin敲除后WiNTRLINC1下调能引起转录本定位消除。(拓扑排序,验证它们存在一个反馈调节)并且,lncRNA-WiNTRLINC1能富集到其基因的启动子区以及ASCL2的增强子区。

这部分内容较多

  • beta-catenin敲除对假说的影响

  • WiNTRLINC1敲除产生的影响

  • 通过3C技术验证DNA间的作用位置

  • 其中ODD、EVEN、LacZ为连接生物素多肽片段

其中3C技术是通过一对分别与选定的2段DNA配对的引物进行PCR扩增,通过PCR产物的有无、产量的高低等,就可以对是否存在相互作用进行判断。下面有图解。

这部分内容主要描述了TCF4/beta-catenin转录复合物结合并激活到WiNTRLINC1基因的启动子区,以及ASCL2的3'端区,接着,并与转录后的lncRNA-WiNTRLINC1结合,稳定住上述两个DNA形成的环,随后促进ASCL2高度表达

3、WiNTRLINC1-ASCL2调控环在组织水平的放大

该部分内容主要验证了组织水平中情况,以及临床相关系。

这篇文章主要从细胞水平出发,并通过数据库在组织中进行了验证,并非在动物体内做验证。其中作者使用了RIP、ChIRP、3C技术。大家对RIP不陌生,对于后两个实验可以参看下图。

ChIRP

Workflow of ChIRP. Chromatin is crosslinked to lincRNA:protein adducts in vivo. Biotinylated tiling probes are hybridized to target lncRNA, and chromatin complexes are purified using magnetic streptavidin beads, followed by stringent washes. We elute lncRNA bound DNA or proteins with a cocktail of Rnase A and H. A putative lincRNA binding sequence is schematized in orange.

3C(染色体构象捕获)

A) An illustration of the 3C method. Genomic DNA is crosslinked (1), capturing three-dimensional interactions inside the cell. After cell lysis and removal of cell membranes, the captured chromatin is solubilized and digested (2), isolating protein-DNA complexes from the chromatin network. The free DNA ends are then ligated together (3) in dilute conditions favoring intra-molecular ligation, creating new DNA junctions representing the proximity of restriction fragments in the fixed sample. After ligation, the crosslinks are reversed (4) and 3C template is purified to eliminate cellular debris. Finally, the ligation products are detected (5) using PCR-based methods. After quantification (6) the results are plotted as a 3C profile (7), revealing interactions between anchor (labeled “A”) and all other fragments in the genomic regions, which mirrors 3D spacing in the nucleus.

B) Possible outcome of ligation reaction between two restriction fragments. As seen in part A, there are many restriction fragments contained within one complex. To further understand the ligation step, we have simplified the reality and show a view where one complex contains only two restriction fragments -red and blue. The 5′ and 3′ ends are indicated for each strand. Each digested end has been numbered 1–4. Also indicated are the locations where 3C primers have been designed. Note that all the primers are on the “forward” strand, located near the restriction site. There are six possible ligation products that result from this molecule. Two of these produce self circles, which are not of interest. Only one of the remaining four ligation products results in a detectable product - that is when end 2 and end 4 are ligated to each other. This ligation event will bring the primers into the proper orientation to produce a PCR product. None of the other ligation products will be detected

DNA 关系 lncRNA 蛋白作用
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