Study On The Gene Expression Profile Of Human Cord Vein Endothelial Cells Early After Hypoxia

Background:The organ failure at early postburn stage is one of the most common and severe complications of burn patients, in which the hypoxia plays a key role in initiation of this pathological process. Due to the increase of vascular permeability immediately after burns, and the sharp decrease of blood volume and supply, the cells will subjected to hypoxia. This will cause the injury of cell structure and dysfunction of cell metabolism, and finally promote the development of systemic organ dysfunction.As a intima lining of the blood vessels, the vascular endothelial cells is one of the most oxygen sensitive cells and regulate a variety of functions, which has been identified to be pivotal in the onset of early organ dysfunction after burns. In common occasions, the vascular endothelial cells maintain a quiescent condition. But when subjected to extracellular stimulation, it is activated and then participated in various biological and pathological process. The endothelial activation may exhibit protective effects on the body through initiation of a series of cell defensive and adaptive mechanisms early after hypoxia, but it may turn to be distructive to cell function as time prolongs and the degree of hypoxia aggravates. So the research on the adaptive or pathological response of vascular endothelial cells and its mechanism after hypoxia is vital for the management of hypoxic injury after burns. As to now, most of reserches in this field focued on the study of the changes in certain special genes, but not on the regulation of the whole genomics, yet the mechanism of endothelial response is not fully understood.Because the cell function is determined by the changes in the gene expression, the study of genomic changes in vascular endothelial cells after hypoxia is an important and effective way for the research of mechanism of endothelial response after hypoxia, and the hooking of significant endogenous protective factors. Among so many genormic research methods, serial analysis of gene expression (SAGE) has many advantages, including high accuracy and reproducibility, ability to quantitate the differentially expressed genes, and so on. Long SAGE is more powerful for its improvement in the identification of gene specificity. So in this study, human cord vein endothelial cell model of hypoxia (3 h) was established based on the preliminary experiment, and normal EA.hy926 cells were enrolled as controls. Then we used Long SAGE technology to construct normoxic and hypoxic endothelial SAGE libraries, and make wide research on the changes and mechanism of endothelial response to short-term hypoxia through bioinformatic and special gene functional studies.Methods:1. To establish hypoxia model of EA.hy926 cells, and detect the changes in ICAM-1 and HIF-1αprotein expression before hypoxia and at 1,3,6,12 post-hypoxia hours(PHH).2. The cells were divided into normoxia and short-hypoxia(with hypoxic treatment for 3 hrs)groups. Total RNA from the 2 groups were extracted for the construction of Long SAGE libaries. The process is briefly described as follows: poly(A)-RNA was isolated using oligo(dT) magnetic beads and converted to cDNA. The resulting cDNA library was digested with NlaIII (anchoring enzyme), and divided into two populations following extensive washing. Each population was ligated with one of the two annealed linker pairs. The tags besides the NlaIII restriction site (CATG) of each transcript were released from the magnetic beads by cleavage with MmeI (tagging enzyme). The two tag populations were then blunted and ligated to produce ditags. The Ditags were amplified and isolated with PCR, and then self-ligated to produce long concatemers. After the resulting concatemers were ligated into the pZErO-1, the ligated products were transformed into One Shot- TOP10 electrocompetent Escherichia coli, seeded onto a low salt LB plate containing 50ug/ml zeocin, and cultured at 37℃overnight. White colonies were screened by PCR to select long inserts (>600 bp) for automated sequencing. SAGE tags of 17 bp were extracted, filtered, and tabulated using SAGE2000 software.3. Bioinformatics was used to analyze the Long SAGE libraries, including gross analysis of tags in SAGE libraries; Draw the gene expression maps according to the gene data of the 2 libraries; Application of Gominer platform to make classification of gene function categories, as well as gene enrichment analysis for the study of signaling pathway changes after hypoxia. 4. Realtime PCR was used to determine the changes in the expression of 5 selected differentially expressed genes , so as to check the reliability of SAGE libraries; Generation of longer cDNA fragments from SAGE tags for gene identification(GLGI) was used to prolong 3'EST fragment of 3 no-matched tags in SAGE libraries; At the same time, the protein expression of VEGF and PCNA by western blot, growth curve by cell counting, cell cycle by flowcytometry, as well as apoptosis by Annexin V methods were also determined to verify the endothelial functional changes in proliferation and apoptosis compared with the result in SAGE libaries.5. According to the bioinformatic analysis, we examined the changes in protein expression of Integrinβ1 after hypoxia with western blot. Then we determined the changes in the promoter activity of Integrinβ1 under hypoxic conditions 48 hrs after transfection of luciferase reporter plasmid containing full-length of Integrinβ1 promoter. Meanwhile, we constructed fluorescent Integrinβ1 siRNA plasmid, transfected it into EA.hy926 cells, and got stably expressed clones after G418 selection. The growth curves of the normal cells without hypoxia, normal cells with 3 hrs of hypoxia, si- Integrinβ1 cells without hypoxia, and si- Integrinβ1 cells with 3 hrs of hypoxia were drawn by cell counting for the study of the influence of Integrinβ1 on the proliferation of EA.hy926 cells under hypoxic conditions.Results:1. Totally 31213 tags, and 13665 unique tags were got from library of normoxia EA.hy926 cell line, while 30756 tags and 13879 unitags were got from hypoxia (3 h) library. The repeated Ditags in the 2 libraries were 762 and 758, respectively. There were 112 genes with obvious differential expression(P<0.05), among which 62 were evidently up-regulated, and other 50 were markedly down-regulated. In order to avoid the possibility of sequencing errors and to perform a valuable comparison between two libraries, the unique tags with a combination of 1:0, 0:1, or1:1 tags were removed. The remaining tags were designated as meaningful unique tags. These genes were used in our bioinformatic analysis.2. Bioinformatic analysis revealed that short-term hypoxia could promote ECM receptor interaction and lipid metabolism; It could also promote cell proliferation by down regulation of the expression of genes (CAV1,GJA1,HMOX1) which exhibit negative effect in regulation of proliferation. In addition, we found that PI3K-Akt,TGF-β, and Notch signaling pathway participated in the positive regulation of proliferation after hypoxia. We also found that short-term hypoxia promote apoptosis and angiogenesis, affect the cell cycle progress, and decrease the defence ability. Furthermore, we observed that HIF-1 tags was not in our SAGE library, indicating endothelial gene expression after short-term hypoxia was mainly modulated by HIF-independent way.3. Realtime PCR results showed that after 3 hrs of hypoxia, the mRNA expression of ANGPTL4, HNRPDL were up-regulated, while that of HMOX1, FTH1 and CTGF were down-regulated. The results were in concord with that of SAGE libraries.4. GLGI results showed that each selected tags was prolonged to some extent. BLAST showed that all of them were known genes.5. In the study of influence of hypoxia on the proliferation and apoptosis of endothelial cells, left move of the growth curve occurred after hypoxia. The proliferation index obviously increased at 1 PHH, maintained to 6 PHH, and decreased at 12 PHH. The expression of VEGF and PCNA increased at 1 PHH and 3 PHH, respectively. The VEGF expression decreased at 12 PHH, while PCNA still maintained at high level at this time-point. All these results indicated that short-term hypoxia could promote endothelial proliferation. Additionally, the occurance of apoptosis was significantly increased at 3 and 12 PHH, indicating pro-apoptotic effect of short-term hypoxia on endothelium.6. Hyoxia could markedly upregulate the protein expression of Integrinβ1, promote the luciferase reporter gene activity. After hypoxia, the growth curve of both cell lines turned to left, though the proliferation of siIntegrinβ1 cells was enhanced compared with that of normal cells, it was obviously decreased when compared with that of hypoxic Eahy926 cells, indicating that the increase of Integrinβ1 expression is benefitial for the EA.hy926 cell proliferation early after hypoxiaConclusion:1. We successfully constructed two Long SAGE libraries: One is about normoxia EA.hy926 cells, the other is about short-term hypoxic(3 hrs) EA.hy926 cells. Each library contains more than 10,000 transcripts, among which 112 were obvious differentially expressed genes(P<0.05).2. Short-term hypoxia promotes cell proliferation, lipid metabolism and angiogenesis, etc through regulation of various signaling pathways and genes. These effects are benefitial for the initiation of self-adaptive system. Meanwhile, short-term hypoxia also promotes apoptosis, affect cell cycle process and decreases the defence ability, which represents harmful effects to endothelial cells. The endothelial gene expression after hypoxia may be regulated through HIF-1 independent way. The differentially expressed genes aquired from our libraries provide direction and basis for the deep study in the future.3. The verification of SAGE libraries from single gene expression and special gene function show the good quality of our SAGE library and good reliability of our bioinformatic analysis.4. Integrinβ1 protein expression is up-regulated early after hypoxia through activation of its promoter, and which make it partly participate in the pro-proliferative effect early after hypoxia.