5’tiRNA-Cys-GCA Regulates The Proliferation And Phenotypic Transition Of Vascular Smooth Muscle Cells By Targeting STAT4

Objective This study aims to explore the regulation and mechanism of tRNA-derived small RNA(ts RNA)on vascular smooth muscle cell function in aortic dissection(AD),and provide new precision targets for the prevention and treatment of aortic dissection.Method(1)After reviewing the literature,we screened a stress-induced RNA(tRNA-derived stress-induced RNA,tiRNA),namely 5’tiRNA-Cys-GCA,and there is no research about cardiovascular disease and tiRNA.Samples of aortic dissection and normal aorta were collected clinically,and the cells were localized by Fluorescence in situ hybridization(FISH)and the expression of 5’tiRNA-Cys-GCA was quantified.Mice model of aortic dissection were constructed and RT-qPCR(Real-time quantitative polymerase chain reaction)was used to detect the expression of 5’tiRNA-Cys-GCA.(2)Construct 5’tiRNA-Cys-GCA mimics and inhibitors to transfect human aortic vascular smooth muscle cells(HASMCs),and verify their transfection efficiency by RT-qPCR.CCK8 and Ed U were used to detect the proliferation ability of smooth muscle cells,scratch and transwell were used to detect cell migration ability,and Western blot was used to detect the expression of contractile marker proteins(α-SMA,CNN1 and MHC).Used TUNEL and Western blot to detect cell apoptosis condition and the expression level of apoptotic protein.(3)Predicted the potential targets of 5’tiRNA-Cys-GCA through biological software,and then used pull down and literature data to screen out STAT4 as the best downstream target.RT-qPCR and Western blot were used to verify the regulation of STAT4 by 5’tiRNA-Cys-GCA.The binding sites of 5’tiRNA-Cys-GCA and STAT4 were predicted by biological software and verified using dual luciferase reporter genes.(4)Constructed small interfering RNA targeting STAT4 and verify its knockdown efficiency by Western blot.The effects of knockdown of STAT4 on the function of vascular smooth muscle cells treated with ox-LDL were detected by CCK8 and Ed U,scratch and transwell,and Western blot.(5)Constructed 5’tiRNA-Cys-GCA agomir to treat mice with aortic dissection,and calculate the survival rate of each group.RT-qPCR detected the expression of 5’tiRNA-Cys-GCA in each group.The HE staining method was used to stain mouse aortic tissue and detect the changes in media thickness.Immunohistochemistry(Immunohistochemistry,IHC)was used to detect the expression of STAT4 protein in the aortic tissues of mice in each group.Results(1)5’tiRNA-Cys-GCA was highly expressed in vascular smooth muscle cells and was down-regulated in human aortic dissection tissue and ox-LDL-treated vascular smooth muscle cells.(2)RT-qPCR verified that 5’tiRNA-Cys-GCA mimics and inhibitor have good transfection effects.Compared with the control group,the proliferation,migration and phenotypic conversion ability of vascular smooth muscle cells overexpressing 5’tiRNA-Cys-GCA was significantly inhibited.The vascular smooth muscle that knocked down 5’tiRNA-Cys-GCA had higher cell proliferation,migration and phenotypic switching capabilities.Moreover,the overexpression of 5’tiRNA-Cys-GCA significantly inhibited the proliferation,migration and phenotypic transition of vascular smooth muscle cells induced by ox-LDL.(3)The results of in situ hybridization showed that 5’tiRNA-Cys-GCA was localized in the cytoplasm,and the downstream target was predicted by bioinformatics.Using pull down,RT-qPCR and dual luciferase reporter gene verification,STAT4 was screened and identified as the best downstream target of5’tiRNA-Cys-GCA.(4)Knockdown of STAT4 inhibited ox-LDL-induced vascular smooth muscle cell proliferation,migration and phenotypic transition,and was reversed by 5’tiRNA-Cys-GCA inhibitors.Conclusion Our study revealed for the first time that 5’tiRNA-Cys-GCA may affect the proliferation,migration and phenotype transition of vascular smooth muscle cells by targeting STAT4,and potentially inhibit the occurrence and development of aortic dissection disease.This research lays a theoretical foundation for 5’tiRNA-Cys-GCA to become a potential diagnostic marker and therapeutic target for aortic dissection,and may provide a new therapeutic strategy for clinical diagnosis and treatment.