Transcriptome Analysis Of Connective Tissue Growth Factor In The "Vascular-fibrotic Switch" Mechanism

Purpose:Based on transcriptomic analysis of RNA-seq technology,we investigate the effect of connective tissue growth factor(CTGF)on vascular endothelial cells in the retina by comparing differentially expressed genes when CTGF is overexpressed,and the differentially expressed genes of retinal vascular endothelial cells were studied in the model of anti-VEGF treatment of diabetic patients.We studied the relationship between the gene and the regulatory process of fibrosis provides a reference for the study of the molecular mechanism of the "vascular fibrotic switch" in cells,and provides a possible reference value for the exploration of the targeted therapy of proliferative diabetic retinopathy.Method: RF6 A cells were divided into 4 groups: 1:Control group;2:CTGF group;3:High glucose anti-VEGF group;4.High glucose anti-VEGF+Metformin group.Control group VS CTGF group(1VS2);high-sugar anti-VEGF group VS high-sugar anti-VEGF + anti-CTGF group(3VS4).RNA transcriptome analysis was used to perform whole-transcriptome sequencing of the cells.Gene ontology,DAVID,and KEGG were used to analyze differentially expressed genes and functional enrichment to analyze the biologically significant roles and effects of some of the differentially expressed genes.Result: The differential gene expression profiles of vascular endothelial cells induced by CTGF were obtained by RNA transcriptional analysis.The sequencing results of two groups were compared: group 1.Control VS CTGF group: 325 differentially expressed genes,including 152 up-regulated genes and 173 down regulated genes,and 2.HG+anti-VEGF group VS HG+ anti-VEGF+anti-CTGF group: 1337 differential expressions Among them,419 genes were up-regulated and 918 genes were down regulated.GO analysis showed that gene functions mainly included the following categories: signal transduction,energy metabolism,protein synthesis,and so on.The results of Pathway significant enrichment show that the pathways involved include: neural ligand receptor interaction pathway,insulin signaling pathway,cell factor receptor interaction pathway,cell adhesion molecule pathway,actincytoskeleton regulation pathway,extracellular matrix receptor interaction pathway,hierarchical signaling pathway,and MAPK signaling pathway.In the differential genes,SLC12A5 is associated with the regulation of insulin secretion based on glucose stimulation;RET and CNTD2 are related to the migration of tumor cells;PFKFB3 is associated with cell proliferation and migration and angiogenesis;KRT14is associated with the formation of keratinocytes and cytoskeleton;the differential expression of RASAL1,ALK,BAG3,KLF10,FBN2 is associated with the fibrosis process UGCG is associated with cell proliferation and apoptosis.Conclusion:Differential expression of CTGF at different expression levels involves a wide range of biological processes.There may be synergistic or antagonistic effects between the differentially expressed genes.RASAL1 and ALK are down-regulated when CTGF is overexpressed;BAG3 and KLF10 are up-regulated and FBN2 is down-regulated when high-sugar anti-VEGF is used to inhibit CTGF expression;all involved in the fibrosis process mediated by TGF-beta signaling pathway,suggesting that we have multiple genes or factors in the TGF-beta signaling pathway to regulate the fibrosis process at the same time and finally reach the dynamic equilibrium stability of fibrosis.We simulated the RF6 A high glucose environment induced neovascularization,anti-VEGF treatment conditions,knock down the expression of CTGF,can inhibit the process of fibrosis,provide theoretical support for dual target treatment of diabetic fibrosis,suggesting that BAG3,KLF10,FBN2 gene may be possible gene targets,but the signaling pathways involved in these genes and their mechanisms of action require further experimental validation.The effect of CTGF on retinal vascular endothelial cells is multi-level and multifaceted.It affects the function of retinal vascular endothelial cells by influencing EMT by differentiating gene expression,regulating blood glucose level and the process of proliferation and apoptosis.