Studies On The Splicing Regulation In Hypoxia/Ischemia Injuries By Exon Array

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 arrayHypoxia 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 injuryStroke 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 dataThe 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.