Studies On The Effect And Mechanism Of AZIN1 RNA Editing In Promoting Angiogenesis In Colorectal Cancer

Objective:Angiogenesis plays an important role in tumorigenesis and progression.In the early stages of tumorigenesis,tumor must generated new vessels to maintain oxygen and nutrient delivery when the dimeter exceeds than 2-3mm.Tumor blood vessels are characterized by multiple pores,rough surface,loose intercellular connections,which enhances the permeability of tumor vessels,facilitates metastasis of tumor cells and forms a special microenvironment.At present,targeted drugs to inhibit tumor angiogenesis have been successfully developed and widely used in clinical practice,and these drugs have achieved favorable theraputic effect.However,a series of problems appeared,such as side effects,low efficacy and resistance.Therefore,it is of vital importance to explore other potential molecular targets against tumor angiogenesis.RNA editing of antizyme inhibitor 1(AZIN1)is an important post-transcriptional mechanism involved in tumor development.In this study,we investigate the influence and mechanism of RNA editing in tumor angiogenesis by overexpressing edited AZIN1 in tumor cells.Methods:1.Real-time PCR was used to detect the expression of AZIN1 in colorectal cancer cell lines.2.We constructed plasmids,packaged the lentivirus,and used lentivirus to infect HCT 116 and HT-29 cells.The AZIN1edited,wide-type(WT)and control cells were transfected stably,and then puromycin was used to select and maintain.3.We performed Western blot and Real-time PCR to detect the expression of AZIN1 of stably transfected cells at protein and mRNA level respectively.4.Conditional medium was extracted from edited,WT and control cells and was cocultured with human umbilical vein endothelial cells(HUVEC).5.CCK-8 was used to examine the effect of CM from each group on HUVEC proliferation.6.Wound healing and transwell were used to detect the influence of CM from each group on HUVEC migration.7.We used matrigel to compare the effect of CM from each group on tube formation of HUVEC.8.Immunodeficient mice were injected subcutaneously in the right flank with 3×10~6HCT116 cells/100?l and in the left flank with 3×10~6 HCT116 cells mixed with6×105HUVEC/100?l,and tumor volume was measured twice a week.9.Micro-CT scanning was used to test the intratumoral vessel distribution.10.We used the Human angiogenesis Array for qualitative comparison of angiogenic cytokine among CM from edited and WT cells.ELISA was used to quantify the secreted factor PEDF and PDGF-AA in CM from each group.11.Metabolomic was performed to detect the differential metabolite between each group.12.Gradient concentration of NAC was added to HUVEC,and CCK-8 was tested the effect of NAC on proliferation.Transwell and wound healing was detected the influence of NAC on migration,and tube formation experiment was used to examine the role of NAC in tube formation.Results:1.The conditional medium from edited cells significantly promoted HUVEC migration and angiogenesis,but had no significant effect on HUVEC proliferation.2.The volume of transplanted tumor injected with edited cells was significantly higher than that with WT and control group.And the volume of transplanted tumor co-culturing with HUVEC was significantly higher than that of single inoculation group.3.The vascular density in the transplanted tumor injected with edited cells was significantly higher than that with WT and control group.4.The concentration of PEDF in CM from edited cells was significantly lower than in WT and control group.There was no significant difference in PDGF-AA between edited,WT and control group.5.The content of NAC in the CM from edited cells was higher than that of WT and control group,and the effect of exogenous addition of NAC on HUVEC was dose-dependent.High concentration of NAC showed significant inhibition to the biological behavior of HUVEC,while low concentration of NAC promoted the migration and tube formation of HUVEC,and showed no significant effect on proliferation.Conclusion:Edited AZIN1 promoted vascular endothelial cell migration and tube formation,which may up-regulate the expression of NAC,inhibit the expression of PEDF and ultimately promoting angiogenesis.