| OBJECTIVE The purpose of this study is to establish Cas9 and SV40 LT co-expressed endothelial cell lines that can easliy proliferate in large number and excise SV40 LT or any other gene by CRISPR system.At the same time,it is expected to apply the new endothelial cell model to molecular mechanism research of cardiovascular diseases and cancerMETHODS First,the immortalized human umbilical vein endothelial cell line which co-expressing Cas9 and SV40 LT proteins were constructed.Two technical routes:HUVEC2-CTG was obtained by transfection of lentiviral vector expressing SV40 LT and Cas9 respectively in two-step method.For the One-step method lentiviral vector Lv-EF1α-Cas9-IRES-SV40 LT was constructed by overlap PCR and homologous recombination,and then it is applied to infect primary HUVEC2 to obtain HUVEC2-CT.Then we designed and screened the CRISPR system for SV40 LT,and transfected into the HUVEC-CTG cells to obtain HUVEC2-CΔTG.The excision of SV40 LT was verified at the protein level by Western Blotting,and the CCK8 method was used to detect the proliferation of HUVEC2-CΔTG cells in vitro.CD31,CD34 and VWF were detected by immunocytochemistry.Matrigel microvascule formation of endothelial cells before and after SV40 LT resection were examined,and RNA-seq was employed to compare the transcriptome of endothelial cells before and after SV40 LT resection.The retrospective analysis excavated the CAVD-related gene expression dataset and miRNA dataset on the GEO database,and used bio-informatics methods to screen differential genes and miRNAs.Cluster analysis of differentially expressed genes were proceeded to get biological processes and molecular pathways of CAVD,and then we analyzed of miRNA-gene regulatory network.RESULTS First,we isolated and cultured the primary PUMC-HUVEC2,whose morphology showed a typical cobblestone-like arrangement.After infected with SV40 LT-GFP,the expression of SV40 LT in HUVEC2-TG were detected at the DNA/protein levels by genomic PCR/Western Blotting.HUVEC2-TG has the typical endothelial morphology,and showed lectin binding ability.We transfected HUVEC2-TG with Cas9 and obtained the stable expressed cell strains HUVEC2-CTG.Western Blotting showed that Cas9 were expressed in 23 of the 25 selected colonies,and genomic DNA PCRpositive.HUVEC2-CTG also has a similar endothelial morphology,and proved lectin binding ability.The constructed lentiviral vector LV-EF1α-Cas9-IRES-SV40 LT has been proved correct by sequencing.The HUVEC2-CT with stable expression of Cas9 and SV40 LT wes establishted by one-step infectionwith the new vector.The expression of Cas9 and SV40 LT were detected at the DNA level.The constructed CRISPR system for SV40 LT was transfected into HUVEC2-CTG cells,and HUVEC2-CΔTG was obtained.CCK8 method was used to detect the proliferation of HUVEC2-CΔTG cells in vitro.The OD values of HUVEC-CTGwere(0.45±0.017),(0.64±0.067)and(0.93±0.110)at d3,d5 and d7 respectively while that of HUVEC2-CΔTG cells was significantly decreased at 3 days(0.24±0.005),5 days(0.30±0.011)and 7 days(all P values were less than 0.001).The results showed that the growth rate of the cells after SVLT knockout were significantly lower than that of the control cells.With the best excision effect gRNAs,indicated by the CCK8 detection,the expression of CD31,CD34 and VWF of HUVEC2-CΔTG cells were positive,and there was no significant difference immunocytochemically.Matrigel microvascule formation of endothelial cells showed that these endothelial cells could form typical tube-like structures on matrigel without significant differences.According to the analysis of the transcriptome expression profile,HUVEC2-CTG-8,22,and 27 differentially expressed in proliferation-related genes before SV40 LT resection,which was related to the different insertion positions of SV40 LT and Cas9 in the genome.Then we analyzed the differentially expressed genes after SV40 L resection,and found 246,141 and 506 genes respectively.We further analyzed molecular pathway of the differential genes.It was found that the immune response related pathways were all up-regulated in the three cell lines,while the down-regulated pathways were not consistent.Among them,down-regulation genes of HUVEC2-CΔTG-8 are mainly clustered in cytoskeleton remodeling,cell adhesion and cell cycle related pathways,down-regulation genes of HUVEC2-CΔTG-22 are mainly clustered in fibrogenesis,and down-regulation genes of HUVEC2-CΔTG-27 are mainly clustered in metabolism-related pathways.We focused on the gene changes related to cell proliferation,and found that part of the differential genes after SV40 LT resection showed the same changes in different cell lines.We further analyzed the molecular pathways of proliferation-related differential genes in these cells,and found that proliferation-related differential genes were clustered into insulin-like growth factor(IGF)-related pathways in all three monoclonal cell lines,in which IGF binding proteins(IBP)4-7 were mainly altered.Through bioinformatic analysis of the differential genes of CAVD patients,it is found that the up-regulated genes of CAVD patients are mainly clustered in response to external stimuli,biological macromolecule metabolism,and immune cell activation,while down-regulated genes are concentrated in response to external stimuli,myocardial cell development,and ion homeostasis.In addition,a variety of genes related to the causes of CAVD are partially differentially expressed.At the same time,110 miRNA’s expressions were significantly different.In the down-regulated miRNA-up-regulated gene pattern,three miRNAs that can regulate HLA-DRB1 and HLA-DRB5 and five miRNAs that can regulate VCAM1 were found.Among the up-regulated miRNA-down-regulated genes pattern,HAND2 interacts with hsa-miR-6081,ACTC1 and hsa-miR-4793-5p.CONCLUSION In this study,endothelial cell lines HUVEC2-CTG and HUVEC2-CT co-expressing Cas9 and SV40LT were established by two-step method and one-step method.After the excision of the immortalizing gene SV40 LT using the CRISPR system,the slow-down growth and gene expression profile changproved that vascular endothelial cells could be an optimized model.The potential molecular mechanism of CAVD disease was analyzed by bioinformatics,which will be verified by the established endothelial cell model. |
