利用外显子芯片研究缺血/缺氧损伤相关的剪接调控机制
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摘要
对于人类而言,氧气是“天空”,血液是“河流”。如果缺少了氧气和血液,人类的细胞、组织和器官都必将面临损伤和死亡。本文特别关注了缺血/缺氧性损伤相关疾病的关键病理过程及其分子机制,从剪接调控这个全新的视角阐述可变剪接在缺血/缺氧性损伤发生和发展过程中的重要作用。本文利用外显子芯片技术渐进式地研究了人内皮细胞缺氧模型和小鼠脑缺血再灌注损伤模型中基因的剪接表达模式和调控特征,从功能、通路、网络模块等角度进行了系统生物学探讨,并尝试性地研究了剪接和转录的共调控现象。在进行数据分析的同时,我们还发展了用于外显子芯片数据可变剪接预测的带分层惩罚项的线性回归模型。因此,本文是以实验数据为立论依据和验证支持,以生物信息学数据分析为主线的“干湿”结合的系统生物学课题,以下将根据研究内容简要地介绍各部分的主要结果和结论:
1)人脐静脉内皮细胞模拟缺氧状态下基因的转录和剪接调控
缺氧是机体内常见的生理和病理环境之一,在众多疾病发生和发展的过程中都会存在局部或者全局的微循环障碍和组织缺氧。内皮细胞位于血管的内表面,在一定的缺氧条件下会发生细胞凋亡并造成内皮组织损伤。压力诱导的内皮细胞生存对抗凋亡的动态平衡状态是维持血管完整性和血管内稳态非常重要的细胞过程,同时也是众多血管相关疾病发生和发展过程中决定血管发生(angiogenesis)的重要因素之一。然而,内皮细胞生存对抗凋亡的动态平衡状态的分子机制至今研究较少,尽管已经有研究报道转录和剪接是内皮细胞缺氧环境下重要的分子应激机制,但是目前还缺乏全基因组水平的剪接表达研究,同时缺氧环境下的转录和剪接调控关系研究也是空白。
本研究首先使用不同浓度的氯化钴在不同时间点对人脐静脉内皮细胞进行处理,发现300μM氯化钴对细胞干预24hrs会诱导早期的细胞凋亡,此时内皮细胞处于细胞生存对抗凋亡的动态平衡状态。然后,我们利用昂飞公司的外显子芯片系统全基因组地筛选差异表达的基因和外显子,鉴定出1583个差异表达基因和342个可变剪接事件,16个可变剪接事件通过了RT-PCR实验验证。对剪接事件进行剪接模式分类,并和文献报道的外显子芯片基准数据集比较,发现盒式外显子的比例在缺氧状态下增高近一倍。进一步地,差异表达基因的基因本体论分析、通路表达富集分析以及受可变剪接影响的蛋白质功能域分析从功能角度均直接反映出内皮细胞生存对抗凋亡的动态平衡状态。重要地,我们基于文献挖掘的方法发现了一个以热休克蛋白家族成员为核心的新功能模块,它在缺氧状态下被显著激活。最后,我们意外地发现46%的可变剪接基因本身也存在基因表达差异,剪接的外显子包含-缺失模式和基因的上调-下调表达模式之间具有非常显著的相关性,这些均暗示缺氧状态下可能存在转录和剪接的共调控。因此,我们的研究表明转录和剪接及其共调控关系可能在内皮细胞缺氧状态下发挥了重要的调控作用,同时对内皮细胞生存对抗凋亡状态的深入探讨可能为血管相关疾病发生和发展过程中关键病理生理机制的研究提供了新的线索。
2)小鼠脑缺血-再灌注损伤模型中的剪接调控研究
脑中风是现代社会危害人类健康的第三大“杀手”,大约87%的脑中风属于脑血管阻塞引起的缺血性脑中风,可导致不同脑区的损伤并造成机体功能丧失,因此及时的血液再通是缺血性脑中风必需和必要的治疗手段。众多文献报道可变剪接是脑缺血再灌注损伤过程中重要的分子调控机制之一,脑缺血再灌注损伤的剪接调控研究将会对脑中风的诊断和治疗具有非常重要的借鉴意义。另外,皮层和海马作为大脑的重要功能区域,对缺血损伤的耐受性和敏感度具有明显差异,因此研究皮层和海马的剪接调控的组织差异将会有助于更加深入地理解脑中风的分子机制。我们首先制备了右侧损伤的小鼠大脑中动脉阻塞-再灌注模型模拟脑中风的缺血再灌注损伤过程,然后使用昂飞公司的外显子芯片系统分别分析小鼠脑双侧半球皮层和海马的外显子表达谱,从左侧海马、右侧海马、左侧皮层和右侧皮层的缺血再灌注损伤进程中鉴定出614个可变剪接事件。我们发现右侧海马的可变剪接事件的数量要远远高于其它脑区,并随着损伤进程数量逐渐增加。同时,我们发现各比较组之间可变剪接基因的交集非常少,而且同一比较组内差异表达基因和可变剪接基因的交集也非常少,这很可能是剪接表达时空特异性的直接体现。经过样本探针集的DABG(Detection Above BackGround)值筛选后的外显子表达值聚类结果和主成分分析结果均显示皮层和海马在缺血再灌注过程中有明显的外显子表达差异。紧接着,以损伤过程为主线的GO(Gene Ontology)和exon-GSEA(Gene Set Enrichment Analysis)分析得出了不同脑区缺血再灌注损伤相关的重要的可变剪接基因功能及其所参与的通路,这些结果和脑缺血再灌注损伤有着非常紧密的联系。在此基础上根据外显子在损伤过程中的表达模式的变化,我们定义和分析了缺血再灌注损伤过程中最显著的时序性剪接“转换”标签,这些标签能非常显著地区分不同组织间的外显子表达特征。最后,受到可变剪接影响的蛋白质功能域分析和SRRM1剪接因子发生可变剪接前后的三维结构分子动力学模拟均证明缺血再灌注损伤条件下可变剪接导致蛋白质产物的结构和功能发生改变。综上所述,本文系统地研究了皮层和海马在缺血再灌注损伤条件下产生的可变剪接事件及其影响的蛋白质结构、功能和通路,发现了皮层和海马由不同的缺血耐受性导致的剪接调控差异,为更加深入细致地研究局灶性脑中风的诊断和治疗提供了非常有力的科学依据。
3)利用惩罚分层回归模型REMAS从外显子芯片数据预测可变剪接
在以上的数据分析过程中我们发现,外显子芯片的数据分析涉及到基因和外显子两个层次,而探针集的绝对表达值并不能反映真实的外显子表达水平,因此基于外显子芯片数据预测可变剪接事件是极具挑战性的课题。目前,绝大部分外显子芯片分析工具均采用剪接指数算法推断可变剪接事件,但是这种算法对于小样本实验准确度将受到影响。我们基于基因和外显子的关系开发了新的惩罚分层回归模型来挑选可变剪接基因和外显子。首先用合适的变量来定义发生可变剪接的外显子,然后设计了带分层惩罚项的LASSO线性回归模型表征基因结构,定义和细分表示基因水平和外显子水平的作用因素的参数。对于小样本量大变量空间的数据采用分步选择的策略逐次优先选择剪接趋势最显著的基因和外显子。最后,基于基因的剪接趋势参数对所有可变剪接基因进行排序,保证了可变剪接预测的可靠性。
为了评估新方法的敏感度和特异性,我们有针对地设计了模拟数据集对其进行性能测试。模拟数据的控制因素包括样本量大小、剪接事件的种类、基因的表达模式和基因中可变外显子的数量等,模拟数据集中设置的剪接事件均能够以高比率被挑选出来,且比率远远高于剪接指数算法的效果。在利用已经发表的真实外显子芯片数据集对REMAS进行测试时,挑选出的前500个具有可变剪接趋势的基因中有57个和Gardina等利用剪接指数算法推断的可变剪接基因相同,REMAS挑选的前10个有可变剪接趋势的基因中有4个通过了RT-PCR实验验证,而57个交集可变剪接基因中也已有20个通过了RT-PCR实验验证或者得到文献支持。由此可见,REMAS作为新的带分层惩罚项的线性回归模型,能够有效可靠地从外显子芯片数据中预测可变剪接事件。
To human, oxygen is the sky and blood is the river. Human cells, tissues and organisms will die without sufficient supply of oxygen and blood. We focus on the important pathological cell processes and molecular mechanisms of hypoxic and ischemic diseases. As a new point of view, alternative splicing and its regulations response to hypoxic and ischemic injuries were studied in this paper. We used Affymetrix exon array to profile exon expression and detetc splicing patterns in hypoxic human umbilical vein endothelial cells (HUVECs) and mouse middle cerebral artery occlusion (MCAO) model. Systematic function analysis including GO analysis, GSEA analysis and network-module analysis were carried out to investigate the underlying regulations in injuries. We also attempted to study the combinational regulation between transcription and splicing under the stress. Finally, a linear regression model named REMAS with hierarchical penalties was developed to select alternatively spliced genes and exons. Briefly, this is a bioinformatics study based on the supports and validation of experiment results. In other word, this is an integrative systems biology study with both“dry”and“wet”works. Then results and conclusions of each part will be introduced below.
1) Transcription and splicing regulation in human umbilical vein endothelial cells under hypoxic stress conditions by exon array
Hypoxia is one of the most popular physiological and pathological stresses in human bodies. Focal or global vessel hypoxia is primary to the occurrence and development of many vasular diseases. As the first layer of the vessels, endothelial cells are easily insulted by hypoxia, which will result in cell apoptosis and endothelium dysfunction. The balance between endothelial cell survival and apoptosis during stress is an important cellular process for vessel integrity and vascular homeostasis, and it is also pivotal in angiogenesis during the development of many vascular diseases. However, the underlying molecular mechanisms remain largely unknown. Although both transcription and alternative splicing are important in regulating gene expression in hypoxic endothelial cells, the underlying regulatory mechanisms and their interactions have not been studied in genome-wide. HUVECs were treated with cobalt chloride (CoCl2) to mimic hypoxia and induce cell apoptosis and alternative splicing responses. Cell apoptosis rate analysis indicated that HUVECs exposed to 300μM CoCl2 for 24 hrs were initially counterbalancing apoptosis with cell survival. We therefore used the Affymetrix exon array system to determine genome-wide transcript- and exon-level differential expression. Other than 1583 differentially expressed transcripts, 342 alternatively spliced exons were detected and classified by different splicing types. Sixteen alternatively spliced exons were validated by RT-PCR. Furthermore, direct evidence for the ongoing balance between HUVEC survival and apoptosis was provided by Gene Ontology (GO) and protein function, as well as protein domain and pathway enrichment analyses of the differentially expressed transcripts. Importantly, a novel molecular module, in which the heat shock protein (HSP) families play a significant role, was found to be activated under mimicked hypoxia conditions. In addition, 46% of the transcripts containing stress modulated exons were differentially expressed, indicating the possibility of combinatorial regulation of transcription and splicing. The exon array system effectively profiles gene expression and splicing on the genome-wide scale. Based on this approach, our data suggest that transcription and splicing not only regulate gene expression, but also carry out combinational regulation of the balance between survival and apoptosis of HUVECs upon mimicked hypoxia conditions. Since cell survival following the apoptotic challenge is pivotal in angiogenesis during the development of many vascular diseases, our results may advance the knowledge of multilevel gene regulations in endothelial cells under physiological and pathological conditions.
2) Alternative splicing in mouse cerebral cortex and hippocampus response to focal ischemia-reperfusion injury
Stroke is the third leading“killer”of modern society, and approximately 87% of them are focal ischemic stroke caused by cerebral artery occlusion. Ischemic stroke will damage different brain regions and cause serious, long-term disability. Therefore, timely reperfusion is important and necessary to therapy for ischemic stroke patients. Previous studies had reported that alternative splicing was one of the important molecular mechanisms under ischemia and ischemia-reperfusion conditions, on which the splicing regulation researches are helpful to the ischemic stroke diagnosis and therapy. In addition, cortex and hippocampus are two important regions with different tolerance and sensitivity to ischemia. The investigation of differential splicing regulation between cortex and hippocampus will grow on the understanding of stroke pathology.
First, we successfully prepared the C57BL/6J mouse MCAO model, which is the well-known animal model for ischemic stroke. Then the Affymetrix exon array system was used to profile exon expression of cortex and hippocampus of two hemispheres. We identified 614 alternative splicing events in sample comparison along the ischemia-reperfusion process in left/right part of cortex and hippocampus. We found that the alternative splicing frequency in right hippocampus was much higher than other cerebral regions, and it increased with the progress of ischemic injuries. Few overlapping splicing events were detected in intra-samples comparison and inner-sample comparison between differentially expressed genes and alternatively spliced genes. Hierarchical clustering analysis and principle component analysis based on exon expression filtered by probeset DABG values along all samples demonstrated that cortex and hippocampus have different exon expression patterns. Subsequently, GO and exon-GSEA analysis along the ischemia-reperfusion injury told us which functions and pathways were closely correlated with the pathological process. After that, we defined the splicing switch signatures to reveal the continuous splicing patterns, which could distinguish the exon expression among different cerebral regions. Finally, protein domains influenced by alternative splicing events and the molecular dynamic simulation of two isoforms of splicing factor SRRM1 demonstrated that ischemia-reperfusion injury induced alternative splicing events which affected important structure and function domains of proteins. Together these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for ischemia-reperfusion injury.
3) REMAS: a new regression model to identify alternative splicing events from exon array data
The pipeline of exon array data analysis has two individual levels for gene and exon expression. Since the absolute value after normalization and summarization does not directly figure the real exon expression, it is challenging to predict alternative splicing events based on exon array data. Currently, most of tools for exon array data analysis utilize the“splicing index”algorithm to infer the alternative splicing events, but they have low accuracy for small-sample studies. We developed a new linear regression model named REMAS with hierarchical penalties arisen from the relationship between gene and exon to select alternatively spliced genes and exons. Firstly, features of alternatively spliced exons were scaled by reasonably defined variables. Secondly, we designed a hierarchical model which can represent gene structure and transcriptional influence to exons, and the lasso type penalties were introduced in calculation because of huge variable size. Thirdly, an iterative two-step strategy was developed to select alternatively spliced genes and exons. To avoid negative effects introduced by small sample size, we ranked genes by parameters indicating their AS capabilities in an iterative manner.
In order to evaluate the sensitivity and specificity of REMAS, we designed comprehensive simulation data by tuning several important biological parameters which might influence the performance of REMAS, e.g. sample size, splicing patterns, gene expression patterns and number of alternative spliced exons in a gene. Simulated alternatively spliced genes and exons could be successfully selected out with a high frequency near to 100% within 1000 times of selections. The selection rates of REMAS were much higher than that of the“splicing index”algorithm. In real data evaluation, there were 57 overlapping alternatively spliced genes between the results of REMAS and“splicing index”algorithm. Four of the top ten alternative splicing events selected by REMAS had been validated by RT-PCR. Among those 57 overlaps, 20 events had been validated by RT-PCR or supported by literatures. Conclusively, as a new linear regression model with hierarchical penalties, REMAS has been demonstrated to be a reliable and effective method to identify AS events from exon array data.
1)人脐静脉内皮细胞模拟缺氧状态下基因的转录和剪接调控
缺氧是机体内常见的生理和病理环境之一,在众多疾病发生和发展的过程中都会存在局部或者全局的微循环障碍和组织缺氧。内皮细胞位于血管的内表面,在一定的缺氧条件下会发生细胞凋亡并造成内皮组织损伤。压力诱导的内皮细胞生存对抗凋亡的动态平衡状态是维持血管完整性和血管内稳态非常重要的细胞过程,同时也是众多血管相关疾病发生和发展过程中决定血管发生(angiogenesis)的重要因素之一。然而,内皮细胞生存对抗凋亡的动态平衡状态的分子机制至今研究较少,尽管已经有研究报道转录和剪接是内皮细胞缺氧环境下重要的分子应激机制,但是目前还缺乏全基因组水平的剪接表达研究,同时缺氧环境下的转录和剪接调控关系研究也是空白。
本研究首先使用不同浓度的氯化钴在不同时间点对人脐静脉内皮细胞进行处理,发现300μM氯化钴对细胞干预24hrs会诱导早期的细胞凋亡,此时内皮细胞处于细胞生存对抗凋亡的动态平衡状态。然后,我们利用昂飞公司的外显子芯片系统全基因组地筛选差异表达的基因和外显子,鉴定出1583个差异表达基因和342个可变剪接事件,16个可变剪接事件通过了RT-PCR实验验证。对剪接事件进行剪接模式分类,并和文献报道的外显子芯片基准数据集比较,发现盒式外显子的比例在缺氧状态下增高近一倍。进一步地,差异表达基因的基因本体论分析、通路表达富集分析以及受可变剪接影响的蛋白质功能域分析从功能角度均直接反映出内皮细胞生存对抗凋亡的动态平衡状态。重要地,我们基于文献挖掘的方法发现了一个以热休克蛋白家族成员为核心的新功能模块,它在缺氧状态下被显著激活。最后,我们意外地发现46%的可变剪接基因本身也存在基因表达差异,剪接的外显子包含-缺失模式和基因的上调-下调表达模式之间具有非常显著的相关性,这些均暗示缺氧状态下可能存在转录和剪接的共调控。因此,我们的研究表明转录和剪接及其共调控关系可能在内皮细胞缺氧状态下发挥了重要的调控作用,同时对内皮细胞生存对抗凋亡状态的深入探讨可能为血管相关疾病发生和发展过程中关键病理生理机制的研究提供了新的线索。
2)小鼠脑缺血-再灌注损伤模型中的剪接调控研究
脑中风是现代社会危害人类健康的第三大“杀手”,大约87%的脑中风属于脑血管阻塞引起的缺血性脑中风,可导致不同脑区的损伤并造成机体功能丧失,因此及时的血液再通是缺血性脑中风必需和必要的治疗手段。众多文献报道可变剪接是脑缺血再灌注损伤过程中重要的分子调控机制之一,脑缺血再灌注损伤的剪接调控研究将会对脑中风的诊断和治疗具有非常重要的借鉴意义。另外,皮层和海马作为大脑的重要功能区域,对缺血损伤的耐受性和敏感度具有明显差异,因此研究皮层和海马的剪接调控的组织差异将会有助于更加深入地理解脑中风的分子机制。我们首先制备了右侧损伤的小鼠大脑中动脉阻塞-再灌注模型模拟脑中风的缺血再灌注损伤过程,然后使用昂飞公司的外显子芯片系统分别分析小鼠脑双侧半球皮层和海马的外显子表达谱,从左侧海马、右侧海马、左侧皮层和右侧皮层的缺血再灌注损伤进程中鉴定出614个可变剪接事件。我们发现右侧海马的可变剪接事件的数量要远远高于其它脑区,并随着损伤进程数量逐渐增加。同时,我们发现各比较组之间可变剪接基因的交集非常少,而且同一比较组内差异表达基因和可变剪接基因的交集也非常少,这很可能是剪接表达时空特异性的直接体现。经过样本探针集的DABG(Detection Above BackGround)值筛选后的外显子表达值聚类结果和主成分分析结果均显示皮层和海马在缺血再灌注过程中有明显的外显子表达差异。紧接着,以损伤过程为主线的GO(Gene Ontology)和exon-GSEA(Gene Set Enrichment Analysis)分析得出了不同脑区缺血再灌注损伤相关的重要的可变剪接基因功能及其所参与的通路,这些结果和脑缺血再灌注损伤有着非常紧密的联系。在此基础上根据外显子在损伤过程中的表达模式的变化,我们定义和分析了缺血再灌注损伤过程中最显著的时序性剪接“转换”标签,这些标签能非常显著地区分不同组织间的外显子表达特征。最后,受到可变剪接影响的蛋白质功能域分析和SRRM1剪接因子发生可变剪接前后的三维结构分子动力学模拟均证明缺血再灌注损伤条件下可变剪接导致蛋白质产物的结构和功能发生改变。综上所述,本文系统地研究了皮层和海马在缺血再灌注损伤条件下产生的可变剪接事件及其影响的蛋白质结构、功能和通路,发现了皮层和海马由不同的缺血耐受性导致的剪接调控差异,为更加深入细致地研究局灶性脑中风的诊断和治疗提供了非常有力的科学依据。
3)利用惩罚分层回归模型REMAS从外显子芯片数据预测可变剪接
在以上的数据分析过程中我们发现,外显子芯片的数据分析涉及到基因和外显子两个层次,而探针集的绝对表达值并不能反映真实的外显子表达水平,因此基于外显子芯片数据预测可变剪接事件是极具挑战性的课题。目前,绝大部分外显子芯片分析工具均采用剪接指数算法推断可变剪接事件,但是这种算法对于小样本实验准确度将受到影响。我们基于基因和外显子的关系开发了新的惩罚分层回归模型来挑选可变剪接基因和外显子。首先用合适的变量来定义发生可变剪接的外显子,然后设计了带分层惩罚项的LASSO线性回归模型表征基因结构,定义和细分表示基因水平和外显子水平的作用因素的参数。对于小样本量大变量空间的数据采用分步选择的策略逐次优先选择剪接趋势最显著的基因和外显子。最后,基于基因的剪接趋势参数对所有可变剪接基因进行排序,保证了可变剪接预测的可靠性。
为了评估新方法的敏感度和特异性,我们有针对地设计了模拟数据集对其进行性能测试。模拟数据的控制因素包括样本量大小、剪接事件的种类、基因的表达模式和基因中可变外显子的数量等,模拟数据集中设置的剪接事件均能够以高比率被挑选出来,且比率远远高于剪接指数算法的效果。在利用已经发表的真实外显子芯片数据集对REMAS进行测试时,挑选出的前500个具有可变剪接趋势的基因中有57个和Gardina等利用剪接指数算法推断的可变剪接基因相同,REMAS挑选的前10个有可变剪接趋势的基因中有4个通过了RT-PCR实验验证,而57个交集可变剪接基因中也已有20个通过了RT-PCR实验验证或者得到文献支持。由此可见,REMAS作为新的带分层惩罚项的线性回归模型,能够有效可靠地从外显子芯片数据中预测可变剪接事件。
To human, oxygen is the sky and blood is the river. Human cells, tissues and organisms will die without sufficient supply of oxygen and blood. We focus on the important pathological cell processes and molecular mechanisms of hypoxic and ischemic diseases. As a new point of view, alternative splicing and its regulations response to hypoxic and ischemic injuries were studied in this paper. We used Affymetrix exon array to profile exon expression and detetc splicing patterns in hypoxic human umbilical vein endothelial cells (HUVECs) and mouse middle cerebral artery occlusion (MCAO) model. Systematic function analysis including GO analysis, GSEA analysis and network-module analysis were carried out to investigate the underlying regulations in injuries. We also attempted to study the combinational regulation between transcription and splicing under the stress. Finally, a linear regression model named REMAS with hierarchical penalties was developed to select alternatively spliced genes and exons. Briefly, this is a bioinformatics study based on the supports and validation of experiment results. In other word, this is an integrative systems biology study with both“dry”and“wet”works. Then results and conclusions of each part will be introduced below.
1) Transcription and splicing regulation in human umbilical vein endothelial cells under hypoxic stress conditions by exon array
Hypoxia is one of the most popular physiological and pathological stresses in human bodies. Focal or global vessel hypoxia is primary to the occurrence and development of many vasular diseases. As the first layer of the vessels, endothelial cells are easily insulted by hypoxia, which will result in cell apoptosis and endothelium dysfunction. The balance between endothelial cell survival and apoptosis during stress is an important cellular process for vessel integrity and vascular homeostasis, and it is also pivotal in angiogenesis during the development of many vascular diseases. However, the underlying molecular mechanisms remain largely unknown. Although both transcription and alternative splicing are important in regulating gene expression in hypoxic endothelial cells, the underlying regulatory mechanisms and their interactions have not been studied in genome-wide. HUVECs were treated with cobalt chloride (CoCl2) to mimic hypoxia and induce cell apoptosis and alternative splicing responses. Cell apoptosis rate analysis indicated that HUVECs exposed to 300μM CoCl2 for 24 hrs were initially counterbalancing apoptosis with cell survival. We therefore used the Affymetrix exon array system to determine genome-wide transcript- and exon-level differential expression. Other than 1583 differentially expressed transcripts, 342 alternatively spliced exons were detected and classified by different splicing types. Sixteen alternatively spliced exons were validated by RT-PCR. Furthermore, direct evidence for the ongoing balance between HUVEC survival and apoptosis was provided by Gene Ontology (GO) and protein function, as well as protein domain and pathway enrichment analyses of the differentially expressed transcripts. Importantly, a novel molecular module, in which the heat shock protein (HSP) families play a significant role, was found to be activated under mimicked hypoxia conditions. In addition, 46% of the transcripts containing stress modulated exons were differentially expressed, indicating the possibility of combinatorial regulation of transcription and splicing. The exon array system effectively profiles gene expression and splicing on the genome-wide scale. Based on this approach, our data suggest that transcription and splicing not only regulate gene expression, but also carry out combinational regulation of the balance between survival and apoptosis of HUVECs upon mimicked hypoxia conditions. Since cell survival following the apoptotic challenge is pivotal in angiogenesis during the development of many vascular diseases, our results may advance the knowledge of multilevel gene regulations in endothelial cells under physiological and pathological conditions.
2) Alternative splicing in mouse cerebral cortex and hippocampus response to focal ischemia-reperfusion injury
Stroke is the third leading“killer”of modern society, and approximately 87% of them are focal ischemic stroke caused by cerebral artery occlusion. Ischemic stroke will damage different brain regions and cause serious, long-term disability. Therefore, timely reperfusion is important and necessary to therapy for ischemic stroke patients. Previous studies had reported that alternative splicing was one of the important molecular mechanisms under ischemia and ischemia-reperfusion conditions, on which the splicing regulation researches are helpful to the ischemic stroke diagnosis and therapy. In addition, cortex and hippocampus are two important regions with different tolerance and sensitivity to ischemia. The investigation of differential splicing regulation between cortex and hippocampus will grow on the understanding of stroke pathology.
First, we successfully prepared the C57BL/6J mouse MCAO model, which is the well-known animal model for ischemic stroke. Then the Affymetrix exon array system was used to profile exon expression of cortex and hippocampus of two hemispheres. We identified 614 alternative splicing events in sample comparison along the ischemia-reperfusion process in left/right part of cortex and hippocampus. We found that the alternative splicing frequency in right hippocampus was much higher than other cerebral regions, and it increased with the progress of ischemic injuries. Few overlapping splicing events were detected in intra-samples comparison and inner-sample comparison between differentially expressed genes and alternatively spliced genes. Hierarchical clustering analysis and principle component analysis based on exon expression filtered by probeset DABG values along all samples demonstrated that cortex and hippocampus have different exon expression patterns. Subsequently, GO and exon-GSEA analysis along the ischemia-reperfusion injury told us which functions and pathways were closely correlated with the pathological process. After that, we defined the splicing switch signatures to reveal the continuous splicing patterns, which could distinguish the exon expression among different cerebral regions. Finally, protein domains influenced by alternative splicing events and the molecular dynamic simulation of two isoforms of splicing factor SRRM1 demonstrated that ischemia-reperfusion injury induced alternative splicing events which affected important structure and function domains of proteins. Together these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for ischemia-reperfusion injury.
3) REMAS: a new regression model to identify alternative splicing events from exon array data
The pipeline of exon array data analysis has two individual levels for gene and exon expression. Since the absolute value after normalization and summarization does not directly figure the real exon expression, it is challenging to predict alternative splicing events based on exon array data. Currently, most of tools for exon array data analysis utilize the“splicing index”algorithm to infer the alternative splicing events, but they have low accuracy for small-sample studies. We developed a new linear regression model named REMAS with hierarchical penalties arisen from the relationship between gene and exon to select alternatively spliced genes and exons. Firstly, features of alternatively spliced exons were scaled by reasonably defined variables. Secondly, we designed a hierarchical model which can represent gene structure and transcriptional influence to exons, and the lasso type penalties were introduced in calculation because of huge variable size. Thirdly, an iterative two-step strategy was developed to select alternatively spliced genes and exons. To avoid negative effects introduced by small sample size, we ranked genes by parameters indicating their AS capabilities in an iterative manner.
In order to evaluate the sensitivity and specificity of REMAS, we designed comprehensive simulation data by tuning several important biological parameters which might influence the performance of REMAS, e.g. sample size, splicing patterns, gene expression patterns and number of alternative spliced exons in a gene. Simulated alternatively spliced genes and exons could be successfully selected out with a high frequency near to 100% within 1000 times of selections. The selection rates of REMAS were much higher than that of the“splicing index”algorithm. In real data evaluation, there were 57 overlapping alternatively spliced genes between the results of REMAS and“splicing index”algorithm. Four of the top ten alternative splicing events selected by REMAS had been validated by RT-PCR. Among those 57 overlaps, 20 events had been validated by RT-PCR or supported by literatures. Conclusively, as a new linear regression model with hierarchical penalties, REMAS has been demonstrated to be a reliable and effective method to identify AS events from exon array data.
引文
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