Effect Of Circ-ZNF609 Mediated Cell Communication On Vascular Cell Function And Its Molecular Mechanism

Vascular network is the structural basis for oxygen and nutrients delivery and transporting metabolic waste.Angiogenesis,as a normal physiological process,spreads through various organs of the human body and permeates all stages of embryonic and ontogeny.It is also associated with cardiovascular diseases,tumor diseases,rheumatoid arthritis,diabetic retinopathy,wound healing and other diseases.Therefore,it has been paid much attention to reveal the mechanism of angiogenesis and the influencing factors of angiogenesis.Circular RNA(circRNA)is a unique class of endogenous noncoding RNA,which exists in mammalian cells and has the function of regulating gene expression.It is one of the research hotspots in recent years.There are complex and detailed information links between cells.Vascular smooth muscle cell(VSMC),located in the media of aorta,can secrete growth factors to activate a variety of signal transduction pathways,thus making the vascular proliferation,apoptosis,migration,inflammatory response and many other biological effects.However,it is unclear whether VSMC can influence the biological behavior of endothelial cells(EC)through circRNA-mediated intercellular communication.This study aimed to elucidate the mechanism of intercellular communication between smooth muscle cells and endothelial cells and its significance in angiogenesis.Part one VSMC derived circ-ZNF609 inhibits angiogenesis by decreasing VEGFA expressionObjective: In this part,VSMC specific Sirt1-Tg mice were selected as the study subjects.Total RNA of Sirt1-Tg and wild type(WT)VSMCs was extracted and screened for differentially expressed circRNAs.The effect of SIRT1 and hypoxia on circRNA and VEGFA was detected by real-time quantitative PCR and Western blot respectively.Methods:1.In this part,VSMC specific Sirt1-Tg mice were selected as the study subjects.The matrigel plugs with or without VEGFA was injected into opposite iliac regions of mice.Endothelial cell-specific CD31 antibodies was used for immunofluorescence staining of frozen sections of matrigel plugs.2.Total RNA of Sirt1-Tg and WT VSMCs was extracted and screened for differentially expressed circRNAs.The effect of SIRT1 and hypoxia on circRNA and VEGFA was detected by real-time quantitative PCR and Western blot respectively.Results:1.Angiogenesis was impaired in Sirt1-Tg mice.2.circ-ZNF609 had a rich expression in VSMCs and ECs,and it was significantly higher in Sirt1-Tg-VSMCs than that in WT-VSMCs.Hypoxia induced circ-ZNF609 expression in Sirt1-Tg-VSMCs and circ-ZNF609 entered into ECs.3.VSMC-derived circ-ZNF609 inhibited VEGFA expression in ECs.Part two circ-ZNF609 blocks angiogenesis by inhibiting nuclear trans-location of HIF1αObjective: Based on the part one research and previous reports,circ-ZNF609 and HIF1 alpha were used as the research objects.We aimed to find out the specific regulation mechanism of circ-ZNF609 on VEGFA in angiogenesis from the relationship between circ-ZNF609 and HIF1α.Methods:1.After knocking down and overexpressing circ-ZNF609,the expression level of HIF1α and nuclear translocation were detected by Western blot and immunofluorescence.2.RNA-binding protein immunoprecipitation(RIP),RNA pull-down and fluorescence in situ hybridization(FISH)were used to study RNA and protein interactions.After overexpression of circ-ZNF609,chromatin immunopre cipitation(CHIP)was used to confirm the effect of HIF1α on transcriptional regulation of VEGFA.Results:1.Knockdown of circ-ZNF609 in endothelial cells promoted nuclear translocation of HIF1α.Conversely,overexpression of circ-ZNF609 in endothelial cells inhibited nuclear translocation of HIF1α.2.Results of RIP,RNA pull-down and FISH showed that circ-ZNF609 interacts with and co-localizes with HIF1α in the cytoplasm of ECs.3.The results of CHIP showed that overexpression of circ-ZNF609 decreased the enrichment of HIF1α in the VEGFA promoter region.Part three Expression characteristics of circ-ZNF609 derived from colorectal cancer and its relationship with vascular cell behaviorObjective: Colorectal cancer is one of the most common digestive tract malignancies in the world.Tumor growth and metastasis are inseparable from angiogenesis.In this part,we aimed to explore the expression characteristics of circ-ZNF609 derived from colorectal cancer and its relationship with vascular cell behavior.Methods:1.The changes of circ-ZNF609 in HT-29 and medium were detected by real-time quantitative PCR.2.The correlation of tumor conditioned medium to proliferation of VSMCs was verified by Western blot,MTT and cell count.3.Annexin V-FITC/PI,TUNEL and JC-1 were used to detect the effect of tumor-conditioned medium on the apoptosis of VSMCs.4.The changes of microvessel and VSMCs in colorectal cancer tissue were verified by immunohistochemical staining.The change of circ-ZNF609 in colorectal cancer tissues was detected by real-time quantitative PCR.Results:1.Circ-ZNF609 expression decreased in HT-29 cell and medium.2.TCM inhibited the proliferation of VSMCs,but there was no difference of the PCNA expression between control and TCM groups.3.TCM promoted VSMC apoptosis by inducing Caspase-3 activation and increasing Bax/ bcl-2 ratio.4.The blood vessels of colorectal cancer tissues showed heterogeneity,and circ-ZNF609 decreased in colorectal cancer tissues.Conclusions:1.Hypoxia-induced sirt1-Tg-VSMCs express and secret circ-ZNF609.2.Smooth muscle derived circ-ZNF609 mediates communication between smooth muscle cells and endothelial cells,and inhibits endothelial VEGFA expression and angiogenesis.3.Circ-ZNF609 interacts with HIF1α in cytoplasm,and inhibits HIF1α nuclear translocation and VEGFA transcriptional activity.4.Circ-ZNF609 expression and secretion decreased in colorectal cancer;TCM induces VSMCs apoptosis.