The Study Of Alternative Splicing Of Hypoxia Insult-induced Genes In Vascular Endothelial Cells

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.