血管内皮细胞中缺氧相关基因的可变剪接研究

发布时间：2018-06-30 01:04

本文选题：缺氧损伤 + 低氧诱导因子　；参考：《中国人民解放军军事医学科学院》2008年硕士论文

【摘要】： 可变剪接(Alternative splicing)广泛存在于人类基因组中,是构成蛋白质多样性与基因表达复杂程度的主要原因。虽然目前有报道发现细胞低氧应答通路中的转录因子——低氧诱导因子HIF-1α(hypoxia inducible factor-1α, HIF-1α)的一种可变剪接可对HIF-1α起负性调控作用。但可变剪接机制作为一种重要的基因表达调控手段,在细胞缺氧、动物缺血损伤中发挥的调节作用,目前仍然所知甚少。为了探讨血管内皮细胞缺氧损伤相关基因的可变剪接变化,我们首先通过NCBI GenBank数据库和ECgene数据库分析获得低氧通路相关基因的潜在的可变剪接形式,用RT-PCR鉴定CoCl2诱导的人脐静脉内皮细胞系(ECV304细胞)缺氧损伤后HIFs和EGLNs家族各基因的可变剪接异构体,发现HIF-1α的3个、EGLN1的2个和EGLN3的1个可变剪接异构体,而且各个可变剪接异构体的表达水平随着缺氧时间的延长而具有不同的变化趋势,可能与不同的缺氧损伤状态有关。其中新发现3种可变剪接异构体:HIF1α-3,EGLN1-8和EGLN3-5,提交GenBank数据库,获得的序列接受号分别为:DQ975378,DQ975380和DQ975379。这些结果提示可变剪接在细胞缺氧损伤过程中可能发挥重要作用。为了以全基因组水平研究血管内皮细胞缺氧损伤中的可变剪接,我们进一步利用Affymetrix GeneChip? Human Exon 1.0 ST Array系统检测CoCl2诱导的人脐静脉内皮细胞(HUVEC细胞)缺氧损伤后基因的可变剪接模式。在HUVEC细胞缺氧损伤后,显著差异的基因共1583个,其中表达上调的基因300个,表达下调的基因1283个;显著差异的外显子共342个(涉及到293个基因),其中134个外显子所属基因的mRNA表达水平也显著差异表达。通过GO(基因本体论)和KEGG数据库对差异表达的基因和外显子所属基因的编码产物进行生物学意义注释、聚类,结果显示:“organelle organization and biogenesis”与“nucleobase”属性相关基因产物表达显著下降,具体表现在以下几个表达显著下降的通路:focal adhesion,regulation of actin cytoskeleton,cell cycle,pyrimidine metabolism和TGF-Beta signaling pathway;与此相反,“programmed cell death”属性基因的表达显著上升,表现在表达显著上调的通路:MAPK signaling pathway,proteasome,antigen processing and presentation。同时,大量与细胞低氧反应/缺氧损伤相关的基因表达发生了显著的变化,包括HIF-1α(hypoxia inducible factor-1α, HIF-1α),VEGFC(vascular endothelial growth factor C),ADCY3 ( adenylate cyclase 3), CAD(carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase)基因等。这些结果提示:在HUVEC细胞损伤模型中,细胞外基质减少,细胞间通讯破坏,细胞的生长受到抑制,细胞活性下降;同时,一个新的以热休克蛋白超家族(Hsp27, Hsp70, Hsp105和DnaJ参与缺氧应答)为中心的热休克蛋白通路被激活起应激反应,通过泛素化降解通路使蛋白降解增加,凋亡通路被启动,并发生细胞死亡。 UniProt数据库分析表明,差异表达基因与差异表达外显子所属基因的蛋白功能分类均主要是可变剪接调控(24.1%, 32.1%)、核蛋白(22.4%, 23.5%)、磷酸化(19.7%, 22.9%)等属性,说明与可变剪接密切相关的基因(起调控作用的剪接因子和被可变剪接调控的基因)在CoCl2诱导的HUVEC细胞缺氧损伤过程中发挥重要的调控作用,或通过可变剪接调控对损伤起应答反应。CoCl2诱导的HUVEC细胞缺氧损伤后,重要的组成性剪接因子和特异性剪接因子如SF3A2(splicing factor 3a, subunit 2, 66kDa),SFRS7(splicing factor, arginine/serine-rich 7, 35kDa),SFRS1 (splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate splicing factor) ),PTBP1( polypyrimidine tract binding protein 1),RBM14(RNA binding motif protein 14)等的mRNA表达水平均显著下降,可改变细胞内众多基因的剪接事件,这与250个可变外显子保留的剪接异构体表达上升或出现、92个可变外显子去除的剪接异构体表达水平下降或缺失结果一致。在342个可变剪接事件中,基因可变剪接类型的发生率:47.95%为cassette exon,16.96%为alternative promoter,19.01%为alternative polyadenylation,其它的为内含子保留、互斥外显子等剪接类型。这与文献报道的cassette exon是可变剪接的主要类型一致。可变剪接通过改变蛋白质的结构域而改变基因的功能,改变启动子结构而调控基因的表达。在HUVEC细胞缺氧损伤后,我们通过RT-PCR验证的具有可变剪接异构体的14个基因中,仅有HNRPD, TNFRSF10B, PHF14, ALAS1, BBS9基因的可变剪接异构体具有RefSeq注释。基因的可变剪接调节机制十分复杂。在HUVEC细胞缺氧损伤中,可变剪接事件不仅发生在与缺氧相关的基因上,在剪接因子中也普遍存在。这提示:可变剪接在细胞缺氧损伤中通过剪接因子来发挥重要的调控作用;剪接因子可能通过可变剪接调节自身的表达,然后调控一系列与之相关的基因发生剪接改变以适应环境的改变。然而这些剪接因子在缺氧损伤中究竟怎样调控基因的表达以及调节哪些基因的可变剪接事件,目前还不清楚,尚需深入对剪接因子的调控进行系统的功能研究。目前,我们的研究表明:CoCl2诱导的HUVEC细胞缺氧损伤模型中,基因转录表达水平与基因的可变剪接调控机制共同调节细胞对缺氧损伤起应答反应。另外,我们已成功制备C57BL/6J小鼠大脑中动脉阻塞模型,并利用该模型研究可变剪接在大脑缺血及缺血再灌注损伤中的调控机制,目前已获得初步结果。综上所述,本研究初步揭示细胞缺氧损伤中,可变剪接起到重要的调控作用。为了解细胞缺氧损伤机制提供了重要数据,有助于从基因转录表达水平和可变剪接调控水平理解脑血管病的发病机制。
[Abstract]:Variable splicing (Alternative splicing) is widely found in the human genome, which is the main cause of the complexity of protein diversity and gene expression. Although there are reports of a variable splicing of the transcription factor of hypoxia inducible factor HIF-1 a (hypoxia inducible factor-1 alpha, HIF-1 a) in the cellular hypoxia response pathway However, as an important means of gene expression regulation, variable splicing mechanism, as an important means of gene expression and regulation, is still poorly understood in cell hypoxia and animal ischemic injury.
In order to investigate the variable splicing changes of hypoxia injury related genes in vascular endothelial cells, we first analyzed the potential variable splicing form of hypoxia pathway related genes through NCBI GenBank database and ECgene database, and identified HIFs and EGLNs in CoCl2 induced human umbilical vein endothelial cell line (ECV304 cells) induced by CoCl2 after hypoxia injury. The variable splicing isomers of the family genes found 3 HIF-1 alpha, 2 EGLN1 and 1 variable splicing isomers of EGLN3, and the expression level of the variable splice isomers varied with the prolongation of the anoxic time, which may be related to the different state of hypoxia injury. Among them, 3 alterable splicing isomers were discovered. HIF1 alpha -3, EGLN1-8 and EGLN3-5 were submitted to the GenBank database. The sequence numbers obtained were DQ975378, DQ975380, and DQ975379., which suggested that the variable splicing might play an important role in the process of cell hypoxia injury.
In order to study the variable splicing in the anoxic damage of vascular endothelial cells at the whole genome level, we further used the Affymetrix GeneChip? Human Exon 1 ST Array system to detect the variable splicing pattern after hypoxia injury induced by CoCl2 induced human umbilical vein endothelial cells (HUVEC cells). There were 1583 genes, including 300 up-regulated genes and 1283 down-regulated genes, and 342 exons of significant differences (involving 293 genes), of which 134 exons were also significantly expressed in the mRNA expression level. The genes and exons expressed by the GO (gene ontology) and KEGG database The results showed that the expression of "organelle organization and biogenesis" and "nucleobase" attribute related gene products decreased significantly, which was manifested in the following pathways: focal adhesion, regulation of actin cytoskeleton, cell cell. Abolism and TGF-Beta signaling pathway, on the contrary, the expression of the "programmed cell death" attribute gene is significantly increased, manifested in a significant up-regulated pathway: MAPK signaling pathway, proteasome, antigen, and a large number of gene expressions associated with cellular hypoxia response / hypoxia injury. The changes include HIF-1 alpha (hypoxia inducible factor-1 alpha, HIF-1 alpha), VEGFC (vascular endothelial growth factor C), ADCY3 (2) genes, etc. These results suggest that the extracellular matrix is in the cell damage model. Decreased cell communication was destroyed, cell growth was inhibited and cell activity decreased; at the same time, a new heat shock protein pathway centered on the heat shock protein superfamily (Hsp27, Hsp70, Hsp105 and DnaJ) was activated to activate the stress response, and the protein degradation increased through the ubiquitination degradation pathway and the apoptotic pathway was activated. And cell death occurred.
UniProt database analysis showed that the protein functional classification of genes of differentially expressed and differentially expressed exons were mainly variable splicing regulation (24.1%, 32.1%), nuclear protein (22.4%, 23.5%), phosphorylation (19.7%, 22.9%), indicating genes closely related to variable splicing (the splicing factors regulating the splicing and variable splicing). Regulatory genes play an important regulatory role in the process of CoCl2 induced HUVEC cell hypoxia injury, or through variable splicing to regulate the hypoxia injury induced by.CoCl2 induced HUVEC cells induced by the damage response, important splicing factors and specific splicing factors such as SF3A2 (splicing factor 3a, subunit 2, 66kDa), SFRS7 (splici) (splici) Ng factor, arginine/serine-rich 7, 35kDa), SFRS1 (splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate splicing)), which are significantly reduced, can change the splicing of numerous genes in the cells. Events, the expression of splice isomers retained with 250 variable exons rose or appeared, and the expression level of splicing isomers removed by 92 exons was reduced or the results were consistent. In the 342 variable splicing events, the incidence of variable splicing types was: 47.95% cassette exon, 16.96% alternative promoter, 19.01% alter Native polyadenylation, other splicing types such as introns, exons and exons. This is the same as the main type of variable splicing that was reported by cassette exon. Variable splicing changes the function of the gene by changing the domain of protein, changes the promoter structure and regulates the expression of genes. After the hypoxia injury of HUVEC cells, Of the 14 genes with variable splicing isomers verified by RT-PCR, the variable splicing isomers with only HNRPD, TNFRSF10B, PHF14, ALAS1, and BBS9 genes have RefSeq annotations.
Variable splicing mechanism is very complex. In HUVEC cell hypoxia damage, variable splicing events occur not only in anoxia related genes, but also in splicing factors. This suggests that variable splicing can play a critical role in cell hypoxia injury through splicing factors, and splicing factors may pass by variable shear. It is not clear how these splicing factors regulate the expression of genes and regulate which genes are variable splicing, but it is still unclear how to regulate the regulation of splicing factors. At present, our study shows that in the CoCl2 induced HUVEC cell hypoxia damage model, the transcriptional expression level of the gene and the variable splicing regulation mechanism of the gene regulate the response of the cell to hypoxia injury.
In addition, we have successfully prepared the C57BL/6J mouse model of middle cerebral artery occlusion and used this model to study the mechanism of variable splicing in cerebral ischemia and ischemia-reperfusion injury, and the preliminary results have been obtained.
To sum up, this study has preliminarily revealed that the variable splicing plays an important regulatory role in the cell hypoxia injury. It provides important data for understanding the mechanism of cell hypoxia injury, and is helpful to understand the pathogenesis of cerebrovascular disease from the level of gene transcription and the regulation of variable splicing.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

【引证文献】