Study On The Regulatory Mechanism Of MiR-1909-5p/GPX4 On Vascular Endothelial Cell Ferroptosis And Aortic Dissection

Background and ObjectiveAortic dissection（AD）is a kind of aortic emergency with sudden onset,rapid progress,and potentially fatal.It can lead to malignant events such as multi-organ malperfusion,aortic valve insufficiency and even pericardial tamponade.Early diagnosis and treatment are very crucial.50%of AD patients without emergency surgery in the acute stage of AD will die within 24 hours of onset,and the mortality rate can reach 1%-2%per hour.At present,the diagnosis of AD mainly includes echocardiography（ECG）,computerized tomography angiography（CTA）and magnetic resonance imaging（MRI）.The treatment of AD mainly includes surgery and endovascular intervention.However,none of the above diagnosis and treatment methods can prevent the occurrence and progression of AD.On the other hand,current research on molecular targets and mechanisms can provide new strategies for the prevention and early intervention of AD.Therefore,in-depth and extensive mechanism exploration is particularly important for the non-invasive diagnosis and treatment of AD.It has been established that smoking is an important risk factor for AD,and nicotine can exacerbate the formation of AD.Ferroptosis is a regulatory cell death caused by lipid peroxidation.Smoking can also participate in the progression of AD through ferroptosis pathway,but the specific mechanism is still unclear.As a kind of non-coding RNA,microRNAs（miRNAs）can exist stably in body fluids such as blood and urine.It has been reported that miRNAs are widely involved in AD and have high diagnostic sensitivity and specificity.Therefore,the aim of this study is to clarify the regulatory role of miR-1909-5p in long-term smoke-induced AD by exploring the mechanism of ferroptosis in the damaged aorta of AD patients with long-term smoking,and to provide a new target for alleviating aortic injury in long-term smoking AD patients.Methods（1）Aortic tissues from AD patients with long-term smoking history were collected as the disease group,and normal arteries adjacent to the tumor were collected from patients without long-term smoking history and without AD who underwent gastrointestinal tumor resection as the control group.AD mouse model was established by subcutaneous injection of nicotine combined with intraperitoneal injection of AngiotensinⅡ（AngⅡ）andβ-aminopropionitrile（BAPN）.Western blot（WB）was used to detect the expression of glutathione peroxidase 4（GPX4）and prostaglandin endoperoxide synthase 2（ptgs2）in the aorta of long-term smoking AD patients,and the expression of GPX4 in the aorta of nicotine-treated AD mice.Immunohistochemistry（IHC）and immunofluorescence（IHF）were used to detect the expression and localization of GPX4 in the aortae of long-term smoking AD patients and nicotine-treated AD mice.Quantitative real-time polymerase chain reaction（qRT-PCR）was used to detect the expression of GPX4 in three cell lines（VSMCs;HUVECs and RAW 264.7）to identify the study cells.（2）WB was used to detect the expression of GPX4 in HUVECs treated with nicotine at a concentration gradient(10^-6-10^-2M)and a time gradient（0-72h）.After HUVECs were co-treated with nicotine with or without Ferrostatin-1（Fer-1）,GPX4 was detected by WB.（3）Three online bioinformatics databases（Targetscan,miRDB and miRWalk）were used to predict the upstream miRNAs of GPX4 and take the intersection,and miR-1909-5p targeting GPX4 was screened by combining the species conservation,cardiovascular event correlation and oxidative stress correlation of miRNAs in the intersection.The transfection efficiency of miR-1909-5p mimics and miR-1909-5p inhibitor and the mRNA expression of GPX4 after transfection were detected by qRT-PCR,and the protein expression of GPX4 after transfection was detected by WB.（4）Dual luciferase reporter gene assay was used to verify the direct regulation of miR-1909-5p on GPX4.HUVECs were treated with a concentration gradient of 10^-7-10^-3M nicotine for 24h,and the mRNA expression levels of miR-1909-5p and GPX4 were detected by qRT-PCR.Fluorescence in situ hybridization assay（FISH）combined with IHF was used to detect the expression and localization of miR-1909-5p in the aortae of long-term smoking AD patients and nicotine-treated AD mice.（5）After HUVECs were transfected with miR-1909-5p mimics or miR-1909-5p inhibitor and co-treated with nicotine for 24 h,ferroptosis was detected by WB and reactive oxygen species（ROS）,malondialdehyde（MDA）and reduced glutathione（GSH）detection kits.（6）Nicotine-treated AD mice were injected with miR-1909-5p antagomir or antagomir NC into the tail vein as the treatment group and the control group,respectively.The incidence of AD was monitored and the survival rate was calculated.The dissected aorta was photographed and its diameter was measured.The maximum diameter of aorta was measured by ultrasound.Hematoxylin-eosin（H&E）staining was used to observe the morphology of aorta.Collagen fiber fragmentation was detected by Masson’s trichrome staining.Elastin fragmentation was detected by Verhoeff-Van Gieson（EVG）staining.IHF combined with FISH was used to detect the expression of miR-1909-5p.WB and IHC were used to detect the expression of GPX4.MDA detection kit was used to detect lipid peroxides.Results（1）WB results showed that the expression of GPX4 in the aorta of long-term smoking AD patients and nicotine-treated AD mice was significantly down-regulated compared with that of healthy control arteries,IHC and IHF showed that GPX4 was mainly enriched in the intima of arteries（2）HUVECs were selected as the study cells,and GPX4was significantly down-regulated after 24h treatment with 10^-3M nicotine.Fer-1upregulated nicotine-downregulated GPX4,leading to ferroptosis in HUVECs.（3）MiR-1909-5p,the upstream target gene of GPX4,was screened by bioinformatics online websites.The knockdown or overexpression efficiency of miR-1909-5p in HUVECs was high,and miR-1909-5p negatively regulated GPX4 at the transcriptional and translational levels.Dual luciferase reporter gene assay confirmed the direct binding of miR-1909-5p to GPX4 and targeted negative regulation of GPX4.After HUVECs were treated with nicotine at a gradient concentration,the mRNA level of miR-1909-5p was opposite to that of GPX4.FISH combined with IHF confirmed the abundant expression of miR-1909-5p in the aortic intima of AD.（4）Overexpression of miR-1909-5p promoted nicotine-induced ferroptosis（increased GPX4,increased ROS and MDA production,and decreased GSH production）in HUVECs;（5）Knockdown of miR-1909-5p inhibited nicotine-induced ferroptosis（decreased GPX4,ROS and MDA production,and increased GSH production）in HUVECs;（6）Animal experiments showed that compared with the control group injected with antagomir NC,the incidence and mortality of AD in the treatment group injected with miR-1909-5p antagomir were decreased,and the maximum diameter of the aorta was significantly reduced.Meanwhile,the thickness of aorta and the breakage of collagen and elastic fibers were decreased in the treatment group.Molecular and biochemical assays showed reduced miR-1909-5p expression,inhibited ferroptosis（decreased GPX4）and reduced MDA production in the treatment group.ConclusionsIn this study,we found that miR-1909-5p aggravated nicotine-induced ferroptosis by targeting GPX4 in HUVECs and further promoted the progression of AD with long-term smoking.This finding suggests that inhibition of miR-1909-5p may be a novel strategy to improve aortic function in long-term smokers with AD.