| Objective: To investigate the effects of circ_CHFR on the proliferation,migration,inflammatory and oxidative stress of VSMCs induced by oxidized low density lipoprotein(ox-LDL).To verify the mechanism by which circ_CHFR regulates the biological behavior of VSMCs at the molecular level.To investigate the regulatory mechanism of circ_CHFR/ mi R-214-3p /Wnt3/ β-catenin pathway in ox-LDL-induced VSMCs proliferation,migration,inflammatory and oxidative stress response.Methods: VSMCs were induced by ox-LDL to construct AS cell model.The relative expression level of circ_CHFR was detected by Real time Quantitative PCR(q RT-PCR).The stability and nucleolus localization of circ_CHFR was determined by RNase R digestion experiment and RNA nucleolus separation.The effect of circ_CHFR on cell proliferation activity was determined by MTT assay and plate cloning assay.The effect of circ_CHFR on cell cycle was detected by flow cytometry.Transwell assay was used to detect the effect of circ_CHFR on cell migration.The effect of circ_CHFR on inflammatory factor secretion was detected by enzyme linked immunosorbent assay(ELISA).The level of Reactive oxygen species(ROS)and the activity of superoxide dismutase(SOD)and glutathione peroxidase(Gpx)was detected by the kit method.The effect of circ_CHFR on Wnt3 expression in cells was determined by q RT-PCR and Western blot.The effects of Wnt3 on cell proliferation and migration were determined by MTT assay,plate cloning,flow cytometry and Transwell assay.The effect of Wnt3 on cell secretion of inflammatory factors was determined by ELISA,The effects of Wnt3 on ox-LDL-induced cellular oxidative stress were detected by kit method.Bioinformatics analysis predicting binding sites,double luciferase reporting experiments and q RT-PCR were used to identify circ_CHFR directly targeting mi R-214-3p.Bioinformatics analysis predicting binding sites,double luciferase reporting experiments,q RT-PCR and Western blot were used to identify mi R-214-3p directly targeting Wnt3.The effects of mi R-214-3p and Wnt3 on circ_CHFR were determined by MTT assay,plate cloning,flow cytometry,Transwell assay and ELISA assay,and Western blot was used to detect whether there was any influence on the downstream β-catenin.Results: Compared with the control group,the increase of circ_CHFR expression in VSMCs was induced by ox-LDL.The results of RNase R digestion experiments and RNA nuclear plasma separation experiments show that circ_CHFR is tolerant to RNase R enzymes and was mainly localized in the cytoplasm.Knockdown of circ_CHFR reversed the proliferation,migration,inflammation and oxidative stress response of ox-LDL-induced VSMCs.Wnt3 gene expression was inhibited when circ_CHFR was knocked down in VSMCs.Knockdown of Wnt3 can reverse the proliferation,migration,inflammation and oxidative stress responses of VSMCs induced by ox-LDL.Circ_CHFR was targeted to mir-214-3p,and the dual-luciferase report showed a significant decrease in luciferase activity between the co-transfected pgl3-circ_CHFR and mir-214-3p mimics group.The decreased expression of mir-214-3p in VSMCs was induced by ox-LDL.When circ_CHFR is knocked out in VSMCs,the expression of mi R-214-3p is promoted.mi R-214-3p was targeted to the 3’UTR of Wnt3,and dual-luciferase reports showed a significant decrease in luciferase activity between the co-transfected p GL3-Wnt3 3’UTR and mi R-214-3p mimics group.After transfection of mi R-214-3p mimics with VSMCs,both m RNA and protein expression of Wnt3 were decreased,and after transfection with mi R-214-3p inhibitor,both m RNA and protein expressions of Wnt3 were increased.Compared with the ox-LDL + si-NC group,the cell proliferation and migration ability of the ox-LDL + si-circ_CHFR#1 group decreased,the concentration of inflammatory factors in the cell culture superclear was lower,the levels of ROS was decreased,the activity of SOD and Gpx were enhanced,and the expression of Wnt3 m RNA,Wnt3 and nuclear β-catenin protein in cells decreased,while the expression of p-β-catenin increased.Compared with the ox-LDL + si-circ_CHFR#1 + anti-mi R-NC group,the cells in the ox-LDL + si-circ_CHFR#1+ anti-mi R-214-3p group showed enhanced proliferation and migration capacity,increased inflammatory factor concentration in cell culture superplasmid,increased intracellular ROS levels,reduced SOD and Gpx activity,increased expression of Wnt3 m RNA and Wnt3 and nuclear β-catenin protein,and decreased expression of p-β-catenin.Compared with the ox-LDL + si-circ_CHFR#1+pc DNA group,cells in the ox-LDL + si-circ_CHFR#1+Wnt3 group showed enhanced proliferation and migration ability,increased inflammatory factor concentration in cell culture superplasmid,increased intracellular ROS levels,reduced SOD and Gpx activity,increased expression of Wnt3 m RNA,Wnt3 and nuclear β-catenin protein,and decreased expression of p-β-catenin.Conclusions: Circ_CHFR expression is elevated in ox-LDL-induced VSMCs,and knockdown of circ_CHFR reversed the proliferation,migration,inflammation and oxidative stress response of ox-LDL-induced VSMCs.The regulation of circ_CHFR on VSMCs proliferation,migration,inflammation and oxidative stress response is achieved by adsorption of mi R-214-3p,which further affects the activity of Wnt3/β-catenin signaling pathway. |
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