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。
Alternative splicing is an important mechanism for increasing the diversity of protein and regulating gene expression in human. Several reports have indicated that an alternative splicing isoform of the transcription factor HIF-1α(hypoxia inducible factor-1α) functioned as a potential dominant negative regulation for fine-tuning the cellular response to hypoxia. However, not a great deal is known about whether alternative splicing is involved in the regulation of genes in the cellular response to hypoxia and anoxic damage.
In order to study the differentially expressed alternative splicing isoforms of hypoxia-responding genes in the endothelial cells exposed to hypoxic condition, we used the RT-PCR technique to detect the alternative splicing events of HIF-αfamily and EGL-Nine homologs (EGLNs) family genes in the human umbilical vein endothelial cell line ECV304 following the bioinformatic analysis based on the annotation of NCBI GenBank and ECgene databases analysis. Three isoforms of human HIF-1α, two isoforms of EGLN1 and one isoform of EGLN3 were detected in the ECV304 cells in hypoxic insults, after which we observed different expression patterns of all isoforms, which maybe have different functions in hypoxic insults induced by CoCl2. In this case, we have successfully identified three novel alternatively spliced variants of the hypoxia-responding genes: HIF1α-3, EGLN1-8 and EGLN3-5, which have been submitted to the GenBank database under accession number DQ975378 for HIF1α-3, DQ975380 for EGLN1-8 and DQ975379 for EGLN3-5.
To evaluate the differentially expressed alternative splicing isoforms of whole genomic genes in the endothelial cells exposed to hypoxic condition, we used the GeneChip? Human Exon 1.0 ST Array to identify the different expression of whole genomic genes and exons in the human umbilical vein endothelial cells HUVEC cells in hypoxic insults. The results showed that there were 1583 genes, in which 300 genes were significantly up-regulated while 1283 down-regulated, and 342 exons (corresponding to 293 genes) strongly differentially expressed in hypoxia-treaded HUVECs. The intersection data, 134 out of 293 represented alternatively spliced genes that were differentially expressed in transcription level simultaneously. The results of GO-based functional analysis and network analysis of the differentially expressed genes and exons based on the KEGG database revealed that the categories“organelle organization and biogenesis”and“nucleobase”mostly distributed in down-regulated group, including focal adhesion, regulation of actin cytoskeleton, cell cycle, pyrimidine metabolism and TGF-Beta signaling pathway. On the contrary, the categories“programmed cell death”distributed in up-regulated group, including MAPK signaling pathway,proteasome,antigen processing and presentation. Some hypoxia insults-induced genes, such as 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), were strongly differentially expressed in hypoxia insulted HUVECs. The results indicated that extracellular matrix (ECM) of cell was reduced, several important signal pathways in cell were destroyed, growth of cell was inhibited and the activity of cell decreased. At the same time, a newly defined heat shock proteins circuit composing subfamilies of Hsp27, Hsp70, Hsp105 and DnaJ was discovered to response to hypoxia insult, protein degraded by ubiquitin/proteasome pathways and more cells were death by acting apoptosis pathway.
According to protein annotation in UniProt knowledge database, function categories of differentially expressed genes and exons were“alternative splicing”(24.1%, 32.1% respectively),“nuclear protein”(22.4%, 23.5% respectively)“phoshporylation”(19.7%, 22.9% respectively) and so on. This indicated that there are numbers of genes (some are splicing factors and some are spliced genes) related with alternative splicing playing important functions in hypoxia treated HUVECs by splicing or being spliced. The splice sites choice are determined by the concentration and localization of splicing factors and the interactions between splicing factors and pre-mRNA in pre-mRNA splicing process. The important consistent splicing factors and alternative splicing factors, such as 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), which were down-regulated, maybe regulated the splicing events(250 exons inclusion and 92 exons skipping) of 293 genes in hypoxia treated HUVECs. Classification of 342 alternative splicing events indicated that cassette exon accounts for 47.95%, consistent with previous reports that cassette exon was the most dominant splicing pattern. Alternative promoter occupied 16.96%, alternative polyadenylation occupied 19.01%, others were intron retention, mutually exclusive exons, et al. The diversity of production of one gene is different from protein domains or promoter caused by alternative splicing regulation. Only five alternative splicing events (happed in HNRPD, TNFRSF10B, PHF14, ALAS1, BBS9 genes) in fourteen alternative splicing events validated by RT-PCR have the RefSeq annotation.
It is believed that regulation of pre-mRNA alternative splicing is very complex. Not only the hypoxia related genes but many important splicing factors regulated by alternative splicing indicated that alternative splicing regulates the expression of genes by splicing factors self-splicing regulation, and then leads the down-stream genes spliced in responding the hypoxia insults. However, we still know little about how the splicing factors regulate pre-mRNA alternative splicing, which need more detail researches. We have attempted to provide insight into the mechanisms and the significance of pre-mRNA splicing alterations in hypoxia treated HUVECs, and we concluded that the transcription and alternative splicing cooperate to regulate the response to hypoxia insults in HUVECs.
In addition, we have successfully prepared the C57BL/6J mouse middle cerebral artery occlusion model, which be used to study the regulation mechanism of alternative splicing in ischemic brain injury.
In conclusion, our results suggest that alternative splicing play important function in cellular response to hypoxic insults. Our conclusions are important for understanding the molecular mechanism of cellular response to hypoxia as well as for ischemic/hypoxia insults in the brain.
引文
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