Study On The Variation Of Apelin-affected Human Umbilical Vein Endothelial Cells With Detection Of Affimetrimex Expression Microarray

BackgroundCurrently we know Apelin-13 is involved in the pathological angiogenesis formation of many diseases,such as cancer,chronic inflammation,gynecological diseases,cardiovascular diseases,cerebral ischemia,Alzheimer’s disease,neuropathy and the like.Recently researches mainly focus on Apelin/APJ signalling system and the relationships between Apelin-13 and signalling factors like TGF-beta,FGF,VEGF,HIF and PDGF.However,the occurrence of cardiovascular diseases and its development is a multi-gene regulatory system.So,besides the Apelin/APJ signalling pathway,whether Apelin-13 regulates other physiological processes,angiogenesis-related factors and signalling pathways still needs further study.ObjectiveIn this study,we used human umbilical vein endothelial cells(HUVECs)as the research object,and detected the curves of migration,proliferation,and apoptosis.To explore its genome mechanism,cells were subjected to Affimetrimex expression microarray to analyze the variation of gene expression after being treated with Apelin-13.These experiments could provide a more intuitive and accurate research method for further study of the molecular mechanism of Apelin-13.Methods1.HUVECs were cultured in DMEM culture medium containing 10%FBS at incubator.In the process of cell culture,the growth state of cells need often be observed under inverted microscope.After morphological test,normal cells can be used for the following detections and varing concentrations of Apelin-13 were added to intervene the growth state according to the experimental settings.2.The proliferation and migration state of Apelin-13 treated cells were detected by real time cells analysis(RTCA)system.The HUVECs were inoculated into proliferation plate with 3.5×104/well and migration plate with 4.0×l04/well.After 3 hours of starvation,the wells were divided into different groups(Control groups,0.01nM Apelin-13 groups,1nM Apelin-13 groups,100M Apelin-13 groups)according to the concentration gradients of Apelin-13.Then cell proliferation and migration curves were recorded.3.The effect of Apelin-13 on apoptotic cells was detected by flow cytometry.The 3h starved endothelial cells were divided into experimental groups:Apelin-13(100nM)and H2O2(200μM)co-stimulated group and H2O2(200μM)stimulated alone.The stimulating times both kept for 12h.After being stained with Annexin V-FITC apoptosis detection kit,the samples were subject to flow cytometry to detect.4.Affimetrix expression microarray was used to detect the differences of HUVECs gene expression profiles between 100nM Apelin-13 stimulated and unstimulated groups.Using the same growth state of HUVECs in both groups,then total RNA of both groups was extracted and reverse transcribed into double strands cDNA after 12h culture.Following staining and elution,samples were subjected to Affymetrix scanning 3000 to get the original images.Differently expressed mRNA whose LogFC>3 or≤-1 were classified and clustered for further analysis.5.Reverse transcription-polymerase chain reaction(RT-PCR)and Western blot were used to confirm the results of microarray did not have flase positive.Cells in the same growth state as the chip used were also treated with varing concentrations of Apelin-13(0.01nM,1nM,100nM)for 12h.The mRNA of both groups were extracted and then reverse transcribed into cDNA for PCR and beta-actin was used as internal standard.The ratio of optical density between each product and the standard was identified as the relative content of mRNA.The extraction of the target protein was used to test the different expressions levels of genes in protein by Western blot analysis.Cells in the same growth state were also treated with the concentration gradient of Apelin-13 for 12h,and then the protein bands were transferred to the PVDF membrane followed by being extracted and separated by 10%SDS-PAGE electrophoresis.Being incubated overnight with mouse monoclonal antibody at 4°C the membrane were then incubated with goat anti-mouse IgG conjugated horseradish peroxidase at room temperature.The ratio of grey value between each product and reference beta-actin was identified as the relative level of protein OSBPL8 and BMPR2.Finally the statistical analysis was carried on.Results1.Normal cells were polygonal,presented like paving stone,larger than other kinds of cells and grew without overlapping.2.The RTCA experimental results showed that Apelin-13 could stimulate the proliferation and migration process of HUVECs,and its effect had dose-dependent manner.The growth and migration of HUVECs could reach maximum rate when concentration of Apelin-13 was 100nM.3.Flow cytometry and Annexin V-FITC apoptosis detection kits were used to detect the effect of Apelin-13 on early and late apoptosis cells.Results showed that compared with the groups H2O2 stimulated alone,Apelin-13 and H2O2 combined treated cells were scarce in early and late apoptotic stages,and living cells’ percentage increased remarkably.4.GO gene enrichment analysis and KEGG pathways enrichment analysis,and statistical test methods were used to analyze the results of microarray to find out the genes and their pathways which were significantly different in two gene expression libraries.The results of GO gene enrichment analysis showed that cell cycle and apoptosis-related genes were in high concentration and of significantly difference.KEGG pathways enrichment analysis showed that pathways associated with cell cycle and degradation pathway mediated by ubiquitin’s protein were both in the top ten differently expressed pathways with the ratio of 7.4%and 8.2%respectively.Thus we chose genes whose LogFC was large,and closely related to the critical pathways in proliferation,migration and apoptosis processes of HUVECs after Apelin-13 was added.5.RT-PCR and Western blot were used to identified the results of Affimetrix did not have false positive.The results showed that Apelin-13 greatly enhanced mRNA expressions of STAG2,ITGAV,RICTOR,NEDD4,BMPR2 and protein expression of BMPR2,meanwhile reduced the expression of OSBPL8 mRNA and protein.These results were coincided with Affimetrix microarray’s results.ConclusionA large amount of differently expressed genes could be got in Affimetrimex expression microarray of HUVECs after cells were treated with Apelin-13.GO gene enrichment analysis and KEGG pathways enrichment analysis showed that differently expressed genes were closely related to cell cycle regulation,cell apoptosis,the formation and development of blood vessels,the development and spread of tumors and so forth.Genes related to these highly enriched biochemical processes,cell components and signalling pathways were selected as research objects such as STAG2,ITGAV,BMPR2 and OSBPL8.Then RT-PCR and Western blot test were used to detect the results of the gene chip.The results showed that Apelin-13 did have great influences to the expressions of these genes.Further evidences showed that Apelin-13 may involve in the regulation of angiogenesis process in HUVECs by regulating cell cycle,increasing their proliferation and migration rates,and inhibiting cell apoptosis process through these signal factors.This study provided a large amount of data to explore possible mechanisms of Apelin-13 action and offered a new way of thinking to continue the study of the pathology in vascular treatment furthermore,it was useful and showed great significance.