Sestrin2 Protein Modulates Pyroptosis of Human Coronary Artery Endothelial Cells by Binding to MiR-3160-5p

Sestrin2 Protein Modulates Pyroptosis of Human Coronary Artery Endothelial Cells by Binding to MiR-3160-5p | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sestrin2 Protein Modulates Pyroptosis of Human Coronary Artery Endothelial Cells by Binding to MiR-3160-5p Cencen Ren, Chi Yang, Xu Che, Yungen Jiao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4020648/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Cardiovascular diseases (CVDs) caused by atherosclerosis (AS) are the leading cause of death and a major health concern globally. However, the mechanisms that lead to have not been fully elucidated. Sestrin2 protein plays an important protective role in myocardial I/R by inhibiting cell apoptosis and other pathways. The aim of this study was to explore the role of Sestrin2 protein in modulating pyroptosis in human coronary artery endothelial cells and explore the specific mechanism of this function. Experimental methods H 2 O 2 was used to establish a pyroptosis model. The expression levels of Sestrin2 protein and mir-3160-5p in the HCAECs pyroptosis model were determined. The impact on the apoptosis of HCAECs was clarified by regulating the expression level of mir-3160-5p. The regulatory relationship between Sestrin2 protein and mir-3160-5p was explored in the pyroptosis model. Conclusion The findings showed that Sestrin2 protein inhibited H 2 O 2 -induced pyroptosis of HCAECs and inhibited miR-3160-5p expression, indicating that Sestrin2 protein inhibited H 2 O 2 -induced pyroptosis of HCAECs by binding to miR-3160-5p. Biological sciences/Cell biology Biological sciences/Molecular biology Health sciences/Cardiology Health sciences/Molecular medicine miR-3160-5P HCAECs Sestrin2 pyroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Introduction Cardiovascular diseases (CVDs) caused by atherosclerosis (AS) are the leading cause of death and a major health concern globally. However, the mechanism of AS has not been fully elucidated. Therefore, exploring the pathogenesis of AS and controlling its development is a potential approach to reduce AS-related CVDs incidence and mortality. AS pathogenesis is a complex process involving several factors and pathways. Oxidative stress and chronic persistent inflammation play an important role in development and progression of AS. Chronic oxidative stress and inflammatory stimulation cause vascular endothelial dysfunction, leading to vascular damage. This traumatic reaction can be manifested as autophagy, apoptosis, pyroptosis, necrosis. Unlike other modes of cell injury, pyroptosis is a programmed mode of cell death associated with inflammation, and the inflammatory response is observed throughout the AS process. Several pyroptotic cells are observed in AS plaques, and in vitro studies demonstrated that many AS-induced pathological factors can induce cell pyroptosis, resulting in the occurrence of AS [ 1 ] . These findings indicate that pyroptosis is highly associated with AS. Sestrin2 protein plays an important role in inhibiting release of reactive oxygen species (ROS) and maintaining oxidation-reduction balance. In addition, Sestrin2 protein is associated with cancer, type II diabetes and neurological related diseases [ 2 – 4 ] . Sestrin2 protein alleviates LPS-induced apoptosis by inhibiting NADPH, suppressing ROS production, and abrogating inflammatory responses through Toll-like receptors [ 5 ] . Studies reported that Sestrin2 protein plays an important protective role in brain and myocardial cell I/R by inhibiting cell apoptosis and modulation of various inflammatory factors [ 6 – 7 ] . Cell pyroptosis is a mode of cell death closely associated with inflammatory response. Therefore, we speculated that the Sestrin2 protein modulates pyroptosis of endothelial cells. MicroRNAs are single stranded ribonucleic acids with a length of approximately 22 nt. These RNAs negatively regulates gene expression by binding to the 3'- UTR of the target gene. The binding of miRNA to the gene induces mRNA degradation or inhibits translation [ 8 – 10 ] . Therefore, miRNA activity can directly modulate several fundamental biological and cellular processes, including stem cell differentiation, cell differentiation, cell proliferation and apoptosis [ 11 ] . miR-3160-5p plays a role in inhibiting prostate cancer cell proliferation [ 12 ] , and enhancing cytotoxicity and apoptosis in human diffuse B-cell lymphoma [ 13 ] . However, the role of miR-3160-5p in endothelial cell apoptosis has not yet been elucidated. Bioinformatics analysis showed that Sestrin2 have bindings sites for miR-3160-5p. This implies that Sestrin2 protein may participate in endothelial cell pyroptosis by binding to miR-3160-5P. In summary, we hypothesized that Sestrin2 protein modulates pyroptosis of human coronary artery endothelial cells (HCAECs) by binding to miR-3160-5P. Therefore, a pyroptosis model of HCAECs was established using hydrogen peroxide (H 2 O 2 ) to explore the effect and regulatory mechanism of Sestrin2 protein on the pyroptosis and the relationship with miR-3160-5P. Materials and Methods All protocols in this study were approved by the Committee on the Ethics of Animal Experiments of Yangzhou University, in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no.85 − 23, revised 1996). The study was performed in accordance with ARRIVE guidelines too. This study mainly used the following reagents: HCAECs purchased from Shanghai Zeye Biotechnology(Shanghai,China).Fetal bovine Serum (FB55011) were bought from ClarkBioscience(Virginia,USA).Anti-β-actin(#4970), anti-caspase1 (#24232), anti-NLRP3 (#15101), and anti-ASC (#67824) antibodies were from Cell Signaling Technology (USA), BeyoClick™, propidium iodide (PI; ST511), the Beyotime Reactive Oxygen Species Assay Kit(S0033),Crystal Violet Staining Solution(C0121) were bought from Beyotime Biotechnology. Mounting Medium With DAPI -Aqueous,Fluoroshield (ab104139)was purchased from Abcam(Boston,USA). Hoechst33342/PI apoptosis detection kit (R21807-100) purchased from Shanghai Shangbao Biological, H2O2 staining kit (S0038), cell cycle detection kit (KGA512)were bought from KeygenBiotech(Jiangsu,China). Cell-Light™ Edu Apollo In Vitro Kit was bought from RiboBio(Guangzhou,China). 2.1 Establishment of HCAECs pyroptosis model The prepared HCAECs were revived and replenished with fresh medium and pyroptosis was initiated with 600 µmol /L H 2 O 2 . The relevant experiments were then performed on the cells at the corresponding time points. After exposing the HCAECs to 600 µmol /L H 2 O 2 for 0h, 1h, 2h, 4h and 8h, the LDH levels in HCAECs culture medium were determined using a kit. In addition, the changes in the expression level of pyroptosis-related proteins NLRP3, Caspase1 and ASC, was assessed by Western blot analysis. HCAECs were treated with H 2 O 2 for 8 hours and the levels of ROS in the cells were assessed using DFCH-DA probes. 2.2 Changes in the expression levels of Sestrin2 protein and miR-3160-5p in HCAECs culture medium after treatment with H 2 O 2 HCAECs were incubated with 600 µmol/L H 2 O 2 for different treatment durations (0, 1h, 2h, 4h, 8h) and the expression levels of Sestrin2 protein and miR-3160-5p were determined. 2.3 Establishment of high and low expression models of Sestrin2 in HCAECs The expression levels of Sestrin2 protein and miR-3160-5p in HCAECs exhibited an inverse relationship under pyroptosis state. Further analysis using gene prediction webservers revealed that Sestrin2 protein can specifically bind to miR-3160, with the corresponding binding sites illustrated in Fig. 1 . Consequently, we hypothesized that Sestrin2 protein modulated the level of pyroptosis in HCAECs through its interaction with miR-3160. The target sequence of Sestrin2 siRNA was identified as GCAGAGACCCATTGAACAA using gene prediction software. We established high expression and low expression models of Sestrin2 protein in HCAECs through lentivirus transfection. Sestrin2, sh-Sestrin2, and Sestrin2-NC were used to represent the high expression group, low expression group, and plasmid control group, respectively. The cells were incubated with puromycin and stable total RNA was extracted from the cells in the Sestrin2, sh-Sestrin2, and Sestrin2-NC groups. The expression level of Sestrin2 protein was assessed by qRT-PCR. 2.4 Determination of LDH levels, expression profiles of pyroptosis-related proteins, and Hoechst33342/PI staining in HCAECs transfected with Sestrin2 protein after treatment with H 2 O 2 The LDH cytotoxicity detection kit was used to assess the changes in LDH levels in the Sestrin2, sh-Esterin2, and Sestrin2-NC groups after treatment of HCAECs with 600 µmol/L H 2 O 2 for 8 hours. Western blot analysis was performed to evaluate the expression levels of pyroptosis-related proteins in HCAECs in the Sestrin2, sh-Sestrin2, and Sestrin2-NC groups. Additionally, Hoechst33342/PI kit was used to determine the number of PI + cells in HCAECs in the Sestrin2, sh Estrin2, and Sestrin2-NC groups. Fluorescence microscopy was used to determine the number of PI + cells in each group. These experiments were conducted to explore the effect of Sestrin2 protein on the pyroptosis of HCAECs after treatment with H 2 O 2 . 2.5 qRT-PCR analysis The changes in miR-3160-5p levels in HCAECs in the Sestrin2-NC, Sestrin2, and sh- Sestrin2 groups were evaluated using qRT-PC to explore whether the Sestrin2 protein modulated the expression of miR-3160-5. 2.6 Dual-luciferase assay A dual-luciferase assay was performed to explore the potential targeting relationship between miR-3160-5p and the Sestrin2 protein. TargetScan database ( http://www.targetscan.org ) was used to predict the potential binding sites between Sestrin2 protein and miR-3160-5p (Fig. 1 ). Subsequently, a reporter gene plasmid containing the Sestrin2 protein sequence was constructed using the Gemma gene. This plasmid was co-transfected with miR-3160-5p mimics or miR-NC into 293T cells then dual-luciferase assays were conducted after 24 hours. 2.7 Construction of HCAEC models with high and low expression of miR-3160-5p and qRT-PCR analysis miR-3160-5p mimics and miR-3160-5p inhibitor were transfected into HCAECs to construct HCAECs models exhibiting high expression and low expression miR-3160-5p to explore the effect of miR-3160-5p on the pyroptosis of HCAECs. The forward and reverse primers used in this study are presented below: forward: 5'-TCAGGGGATGTGAGAGTGTGG-3', reverse: 5'-TCAGGGGATGTGAGAGTGTGG-3', β-actin, forward: 5'-CATGTACGTTGCTATCCAGGC-3', reverse: 5'-CTCCTTAATGTCACGCACGAT-3'. miR-NC, miR-3160-5p mimics, and miR-3160-5p inhibitor were used to represent the NC group, high expression group, and low expression group, respectively. qRT-PCR was performed after 24 hours of transfection. 2.8 Assessment of LDH levels in HCAECs transfected with miR-3160-5p under the action of H 2 O 2 The pyrogenic model of HCAECs was constructed by stimulating HCAECs with 600 µM H 2 O 2 for 8 h. LDH cytotoxicity detection kit was used to evaluate the levels of LDH in HCAECs transfected with miR-NC, miR-3160-5p mimics, and miR-3160-5p inhibitor to explore the effect of miR-3160-5p on LDH levels in H 2 O 2 -treated HCAECs. 2.9 Changes in Hoechst33342/PI staining of HCAECs transfected with miR-3160-5p after treatment with H 2 O 2 The Hoechst33342/PI assay kit was used to evaluate the number of PI + cells in HCAECs transfected with miR-NC, miR-3160-5p inhibitor, and miR-3160-5p mimics to determine the effect of miR-3160-5p on the pyroptosis of HCAECs treated with H 2 O 2 . 2.10 Determination of the expression levels of pyroptosis-related proteins in HCAECs transfected with miR-3160-5p after treatment with H 2 O 2 Western blot was performed to assess the expression profile of pyroptosis related proteins in HCAECs transfected with miR-NC, miR-3160-5p inhibitor, and miR-3160-5p mimics to explore the effect of miR-3160-5p on the expression profile of pyroptosis-related proteins. Results 3.1 Changes in LDH levels, ROS levels, and pyroptosis-related proteins in HCAECs treated with H 2 O 2 HCAECs were treated with H 2 O 2 for 0h, 1h, 2h, 4h, and 8h (Figs. 2 , 3 , and 4). The LDH level in the HCAECs increased in a time-dependent manner after H 2 O 2 treatment compared with the control group. The expression of pyroptosis-related proteins NLRP3, Caspase1, and ASC also exhibited a time-dependent increase. The level of ROS in HCAECs was significantly higher after treatment with H 2 O 2 for 8 hours compared to the control group, indicating that H 2 O 2 stimulation significantly increased the level of ROS in HCAECs. The results showed that H 2 O 2 stimulation significantly increased intracellular ROS production and activated NLRP3 inflammasomes in HCAECs, indicating that the cell pyroptosis model was established successfully. 3.2 Expression levels of Sestrin2 protein and mir-3160-5p during pyroptosis of HCAECs The expression level of Sestrin2 protein in H 2 O 2 -stimulated HCAECs decreased in a time-dependent manner compared with the control group (Fig. 5 ). Conversely, the expression of miR-3160-5p in H 2 O 2 stimulated HCAECs increased in a time-dependent manner compared with the control (Fig. 6 ). 3.3 Identification of HCAEC models transfected with Sestrin2 protein Sestrin2, sh-Sestrin2, and Sestrin2-NC models were constructed by lentivirus transfection and the expression levels of sestrin2 determined (Fig. 7 ). The expression of Sestrin2 protein was significantly upregulated in the Sestrin2 group compared with the control group. Conversely, the expression of Sestrin2 protein was significantly downregulated in the sh-Sestrin2 group compared with the control group. No significant difference in the expression level of Sestrin2 protein was observed between the Sestrin2-NC group and the control group. These results indicate the successful establishment of high and low expression models of Sestrin2 protein. 3.4 LDH levels, expression levels of pyroptosis-related proteins, and intensity of Hoechst33342/PI staining in HCAECs transfected with Sestrin2 protein after treatment with H 2 O 2 LDH cytotoxicity assay kit was used to detect the LDH levels of HCAECs treated with H 2 O 2 (Fig. 8 ). The LDH levels in the H 2 O 2 + Sestrin2-NC group were not significantly different compared with the H 2 O 2 group. H 2 O 2 + Sestrin2 group exhibited higher LDH levels, whereas the LDH levels in the H2O2 + sh-Sestrin2 group were higher compared with the control (p < 0.01). The expression levels of pyroptosis-related proteins in HCAECs transfected with Sestrin2, sh-Sestrin2, and Sestrin2-NC were detected by western blot (Fig. 9 ). The expression level of pyroptosis-related proteins NLRP3, caspase-1, and ASC in the H 2 O 2 + Sestrin2-NC group was not significantly different compared with the H 2 O 2 group. The expression level of NLRP3, caspase-1, and ASC in the H 2 O 2 + sh-Sestrin2 group lower, whereas the expression level of NLRP3, caspase-1, and ASC was higher compared with the H 2 O 2 group (p < 0.01). The Hoechst33342/PI assay kit was used to detect the number of PI + cells in HCAECs transfected with lentivirus Sestrin2, sh-Sestrin2, and Sestrin2-NC after incubation with H 2 O 2 (Fig. 10 ). The number of PI + cells in the group overexpressing Sestrin2 decreased, whereas the number of PI + cells in the sh-Sestrin2 group increased compared with the Sestrin2-NC group. 3.5 Expression levels of miR-3160-5p in HCAECs after transfection with Sestrin2 The expression levels of miR-3160-5p in HCAECs were determined after transfection with different levels of Sestrin2 (Fig. 11 ). No significant change in the expression level of miR-3160-5p was observed in the Sestrin2-NC group compared with the control group. The expression level of miR-3160-5p in the overexpression Sestrin2 group was downregulated, whereas the expression level of miR-3160-5p in the low-expression Sestrin2 group was significantly upregulated compared with control group (p < 0.01). 3.6 The luciferase activity in the co-transfection group of Sestrin2 and miR-3160-5p mimics was significantly lower that the NC group (p < 0.01, Fig. 12 ) (excitation wavelength 580 nm, * * p < 0.01 vs miR-NC + Sestrin2). 3.7 qRT PCR detection was performed after 24 hours of transfection (Fig. 13 ). No significant change in the expression level of miR-3160-5p was observed in the miR-NC group compared with the control group. The expression level of miR-3160-5p in the miR-3160-5p mimics group was significantly higher, whereas the expression level of miR-3160-5p in the miR-3160-5p inhibitor group was significantly lower than the control group. These results indicated successful establishment of the high- and low-expression model of miR-3160-5p. qRT-PCR was performed to detect the expression levels of miR-3160-5p in HCAECs 24h after transfection with miR-NC, miR-3160-5p mimics and miR-3160-5p inhibitor (* p<0.05 vs control, ** p<0.01 vs control). 3.8 H 2 O 2 increased the release of LDH in HCAECs (Fig. 14 ). The H 2 O 2 + miR-3160-5p inhibitor group exhibited lower LDH level, whereas the H 2 O 2 + miR-3160-5p mimics group showed increased LDH level compared with the H 2 O 2 + NC group (p < 0.01). LDH levels of HCAECs were measured by the LDH cytotoxicity test in the NC group, miR-3160-5p inhibitor group and miR-3160-5p mimics group after treatment with 600 µM H 2 O 2 for 8h (** p<0.01 vs. NC, ## p<0.01 vs. H 2 O 2 + NC). 3.9 The miR-3160-5p inhibitor group exhibited a decrease in the number of PI + cells, whereas the miR-3160-5p mimics group showed an increase in the number of PI + cells after treatment with 600 µM H 2 O 2 compared with the miR-NC group (Fig. 15 ). 3.10 The results are shown in Fig. 16 ,H 2 O 2 treatment increased the expression of NLRP3, caspase-1, and ASC in HCAECs. The expression levels of NLRP3, caspase-1, and ASC in the H 2 O 2 + miR-3160-5p inhibitor group decreased, whereas the expression of NLRP3, caspase-1, and ASC in the H 2 O 2 + 3160-5p mimics group increased compared with the H 2 O 2 + NC group. MiR-3160-5p targeted regulation of HCAECs pyroptosis Discussion Cell pyroptosis is characterized by the formation of inflammasomes, gas dermin D (GSDMD) dependent cell perforation, and release of inflammatory factors. NLRP3 containing inflammasomes is a common cell pyroptosis pathway that has been extensively studied. NLRP3 inflammasomes are mainly activated by reactive oxygen species (ROS) and ox-LDL, inducing oxidative stress in endothelial cells. Oxidative stress is involved in the occurrence of cell necrosis and the development of atherosclerosis. Therefore, it is imperative to abrogate in the occurrence and development of endothelial pyroptosis induced by oxidative stress to present and treat AS. Although pharmacological studies report that multiple drugs, such as trimetazidine, can be used as antipyroptotic therapy, gene intervention therapy is a better alternative in inhibiting cell pyroptosis due to its precise targets. Cerebellar degeneration-related protein 1 antisense RNA (CDR1as) is implicated in cell proliferation, apoptosis and the regulation of inflammatory factors. Knock down of ciRS-7 promotes IL-1 β-induced release of inflammatory cytokines and cell apoptosis [ 14 ] . In addition, CDR1as plays a regulatory role in vascular endothelial cells and smooth muscle cells. Consequently, CDR1as may inhibit the release of LDH in HCAECs treated with H 2 O 2 , and abrogate the release of inflammatory factors and cell pyroptosis induced by H 2 O 2 . Previous findings indicate that CDR1as can inhibit oxidative stress-induced pyroptosis of HCAECs [ 1 ] . However, studies have not explored whether CDR1as has a synergistic relationship with Sestrin2 protein. In our study, we predicted that the binding of miR-3160-5P to Sestrin2 and observed that the binding sites are highly complementary. This implies that miR-3160-5P can affect the expression of Sestrin2. Lentivirus transfection HCAECs was performed to construct high-expression and low-expression Sestrin2 protein models to confirm the predicted relationship between miR-3160-5P and the protein. Further, we evaluated the impact of miR-3160-5p on pyroptosis of HCAECs. The findings showed that Sestrin2 protein inhibited H 2 O 2 -induced pyroptosis of HCAECs by binding miR-3160-5p. HCAECs pyroptosis under hypoxia conditions is caused by a combination of multiple pathways and multiple factors. Therefore, inhibiting the expression of Sestrin2 protein and miR-3160-5p only partially improves the pyroptosis of HCAECs. Therefore, further research should be conducted to explore the pathological and physiological changes associated with pyroptosis. Conclusion The findings indicated that Sestrin2 protein inhibits H 2 O 2 -induced pyroptosis of HCAECs, implying that Sestrin2 protein is implicated in modulation of the pyroptosis of HCAECs. The expression of Sestrin2 protein was inhibited by binding to miR-3160-5p, whereas expression of miR-3160-5p promoted pyroptosis of HCAECs, indicating that the Sestrin2 protein inhibited H 2 O 2 -induced pyroptosis of HCAECs through interaction with miR-3160-5p. These results show that exploring the mechanism of inhibiting oxidative stress to modulate pyroptosis of HCAECs by regulating the expression of miR-3160-5p may provide a new approach for the treatment of cardiovascular diseases such as AS. Declarations Author Contribution C.R. and C.Y. wrote the main manuscript text and C.Y. prepared figures, X.C.collected important background information,and Y.J.Participate in experimental design and manuscript review.All authors reviewed the manuscript. Data availability Data is provided within the manuscript or supplementary information files. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4020648","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":283573266,"identity":"5b4e0739-6e18-4d83-bc90-1bc12e8d9333","order_by":0,"name":"Cencen Ren","email":"","orcid":"","institution":"Department of Cardiology, The Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou","correspondingAuthor":false,"prefix":"","firstName":"Cencen","middleName":"","lastName":"Ren","suffix":""},{"id":283573267,"identity":"b56206f7-4bb6-4a70-afed-9b2eb56c1f43","order_by":1,"name":"Chi Yang","email":"","orcid":"","institution":"Xuzhou No.6 People's Hospital,Xuzhou","correspondingAuthor":false,"prefix":"","firstName":"Chi","middleName":"","lastName":"Yang","suffix":""},{"id":283573268,"identity":"9ec51f87-94f4-4644-a3e6-1a4571736e28","order_by":2,"name":"Xu Che","email":"","orcid":"","institution":"Department of Cardiology, The Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Che","suffix":""},{"id":283573269,"identity":"51cd02b8-c46a-46f7-b539-6a44fd3ab0dd","order_by":3,"name":"Yungen Jiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACxgYQWWEjx8/MfPgBCVrOpBlLtrOlGZBgVcuhRIPzPAoSRKlmnpF+8XFhw4EE48M8DAYMNTbRhC2YkVNsPHPHnTyzw7wHHjAcS8ttIEJLmjTvmWfFZof5EgwYGw4TpSX9N2/b4cTNzTwGEkRqST/GDNKygZloLT1vmKV5gIEscRgYyAnE+MWwPf3hZx5QVPYfPvzgQ40NEVom5CBFYAIh5SAgz3/8ATHqRsEoGAWjYCQDAKjiQy/ZSzbNAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Cardiology, The Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou","correspondingAuthor":true,"prefix":"","firstName":"Yungen","middleName":"","lastName":"Jiao","suffix":""}],"badges":[],"createdAt":"2024-03-06 11:22:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4020648/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4020648/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53528340,"identity":"76d5afeb-7f42-44b1-9a9d-2fbe3c386a42","added_by":"auto","created_at":"2024-03-27 05:35:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":144607,"visible":true,"origin":"","legend":"\u003cp\u003eSpecific binding site map of Sestrin2 protein to miR-3160\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/a37d78d33fdebe0e07d5971a.png"},{"id":53528341,"identity":"ec25aa1a-6ffc-462c-b98f-b1d66ad998ba","added_by":"auto","created_at":"2024-03-27 05:35:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17252,"visible":true,"origin":"","legend":"\u003cp\u003elevels in HCAECs treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at different time points (600 μmol /L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, * p＜0.05 vs. control, ** p＜0.01 vs. control)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/142125c1df89738cf88b0d74.png"},{"id":53528337,"identity":"f4c324cb-e9cb-4a3f-866f-7d6fd4b1237e","added_by":"auto","created_at":"2024-03-27 05:35:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":113050,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression levels of pyroptosis-related proteins in HCAECs treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at different time points (** p\u0026lt;0.01 vs control).\u003c/p\u003e\n\u003cp\u003e(A) Western blot detected levels of pyroptosis-related proteins NLRP3, Caspase1 and ASC in HCAECs treated with 600 μmol /L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for different durations (0h, 1h, 2h, 4h, 8h); (B) Quantitative analysis of NLRP3, Caspase1, and ASC protein bands (** p＜0.01 vs. control).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/ee71beb8c591c7d8ad22c078.png"},{"id":53528336,"identity":"05b3b320-1ac9-4873-b722-98b7d74b0ee6","added_by":"auto","created_at":"2024-03-27 05:35:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":158249,"visible":true,"origin":"","legend":"\u003cp\u003eROS levels in HCAECs after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/2923c0322118a24ee8f084b8.png"},{"id":53528330,"identity":"999d4caf-c04d-41d6-bcc7-514303d334d3","added_by":"auto","created_at":"2024-03-27 05:35:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31629,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of Sestrin2 protein in HCAECs treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for different durations (** p\u0026lt;0.01 vs control).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/5a6415fe32d485266ca1a687.png"},{"id":53528966,"identity":"582415bd-a9a5-470b-b9b4-18f5f2eb4c76","added_by":"auto","created_at":"2024-03-27 05:43:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36671,"visible":true,"origin":"","legend":"\u003cp\u003eExpression level of miR-3160-5p in HCAECs treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for different durations.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/bf9b7f9fffe20d3fa81f51be.png"},{"id":53528343,"identity":"1ef60c41-8638-44e1-92ca-e5a51e8af5bf","added_by":"auto","created_at":"2024-03-27 05:35:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":39955,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of Sestrin2 protein in HCAECs transfected with Sestrin2, sh Sestrin2, and Sestrin2-NC (* p＜0.05 vs. control, ** p＜0.01 vs. control).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/4fafa3ee3d0d824195dbb458.png"},{"id":53528338,"identity":"d02b22e7-7575-4ce0-a841-a57c543009f0","added_by":"auto","created_at":"2024-03-27 05:35:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":48539,"visible":true,"origin":"","legend":"\u003cp\u003eLDH levels in HCAECs transfected with Sestrin2-NC, Sestrin2, and sh-Sestrin2 after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/82e90c913981d4db8c2260c8.png"},{"id":53528344,"identity":"d4723e12-290a-4a6c-88d8-ed1ae757f183","added_by":"auto","created_at":"2024-03-27 05:35:49","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":121809,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of pyroptosis-related proteins in HCAECs transfected with Sestrin2, sh-Sestrin2, and Sestrin2-NC after t H\u003csub\u003e2\u003c/sub\u003eO\u003csup\u003e2\u003c/sup\u003e treatment.\u003c/p\u003e\n\u003cp\u003e(A) The expression levels of NLRP3, Caspase1, and ASC in the blank group, Sestrin2 group, sh-Sestrin2 group, and Sestrin2-NC group after stimulation of HCAECs with 600 μm/L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as determined by western blot; (B) Quantitative analysis of bands of NLRP3, Caspase1, and ASC proteins in the different groups (** p＜0.01vs control).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/4e72ea4a472dc051e6400919.png"},{"id":53528334,"identity":"e2170bc7-c3a5-4739-bb0e-0c03ada45b80","added_by":"auto","created_at":"2024-03-27 05:35:48","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":367406,"visible":true,"origin":"","legend":"\u003cp\u003eHoechst33342/PI staining of HCAECs transfected with Sestrin2, sh-Sestrin2, and Sestrin2-NC after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (PI: red fluorescence, Hoechst33342: blue fluorescence, Merge is the superimposed image of Hoechst33342 and PI)\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/ce13b143f1eb363e8bff28d0.png"},{"id":53528333,"identity":"6f5def0e-f667-4c2e-a147-8b746c5c957e","added_by":"auto","created_at":"2024-03-27 05:35:48","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":49522,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression levels of miR-3160-5p in HCAECs transfected with Sestrin2-NC, Sestrin2, and sh-Sestrin2 (** p＜0.01 vs. control).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/7224842c8a5bf4599dc56cce.png"},{"id":53528339,"identity":"607552ef-5d9b-46c2-9f85-43fa12989709","added_by":"auto","created_at":"2024-03-27 05:35:49","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":51045,"visible":true,"origin":"","legend":"\u003cp\u003eLuciferase activity after 24h (excitation wavelength 580nm, **p \u0026lt; 0.01 vs miR-NC+ Sestrin2)\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/61f91005edc0532f67dab415.png"},{"id":53528331,"identity":"8952e3b5-6efd-46f4-8095-921e089ecf1b","added_by":"auto","created_at":"2024-03-27 05:35:48","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":33924,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression levels of miR-3160-5p in HCAECs after transfection with miR-NC, miR-3160-5p mimics and miR-3160-5p inhibitor.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/f6348c90147f86b075ecc000.png"},{"id":53528345,"identity":"fa59199d-9ec0-4a07-a13f-114dd3f62dcc","added_by":"auto","created_at":"2024-03-27 05:35:49","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":40949,"visible":true,"origin":"","legend":"\u003cp\u003eLDH levels in HCAECs transfected with miR-NC, miR-3160-5p mimics, and miR-3160-5p inhibitor after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/607937a0a4c8055f11c2b635.png"},{"id":53528965,"identity":"4f348825-ec97-4269-9f15-342809ea1f24","added_by":"auto","created_at":"2024-03-27 05:43:48","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":305828,"visible":true,"origin":"","legend":"\u003cp\u003eHoechst33342/PI staining of HCAECs transfected with miR-NC, miR-3160-5p mimics and miR-3160-5p inhibitor after treatment with 600 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 8h. Fluorescence microscopy was used to observe the number of PI+cells representing pyroptosis in the miR-NC group, miR-3160-5p mimics group and miR-3160-5p inhibitor group (Hoechst33342: blue-fluorescence; PI: red fluorescence; Merge is the superimposed image of Hoechst33342 and PI), with a scale =50 μm.\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/6ab63865773964dac39557f6.png"},{"id":53528332,"identity":"ebcf8613-ea51-422e-8cee-cfa7d84323f5","added_by":"auto","created_at":"2024-03-27 05:35:48","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":125523,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression levels of pyroptosis-related proteins in HCAECs transfected with miR-NC, miR-3160-5p inhibitor, and miR-3160-5p mimics after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (A) Expression levels of NLRP3, Caspase1 and ASC in the NC group, miR-3160-5p inhibitor group and miR-3160-5p mimics group 8h after treatment with 600 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e; (B) Quantitative analysis of the expression levels of NLRP3, Caspase1, and ASC (** p＜0.01vs. NC, # p＜0.01vs. H2O2+NC, ##p＜0.01vs. H2O2+NC)\u003c/p\u003e\n\u003cp\u003eMiR-3160-5p targeted regulation of HCAECs pyroptosis\u003c/p\u003e","description":"","filename":"16.png","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/cb3033e9cd110677e171e82b.png"},{"id":55265180,"identity":"80bb6bd0-cb96-4452-b4a6-a859dc6c4591","added_by":"auto","created_at":"2024-04-25 01:57:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2037790,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4020648/v1/92c2093d-2c1e-4441-a768-a3a49fe65abb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sestrin2 Protein Modulates Pyroptosis of Human Coronary Artery Endothelial Cells by Binding to MiR-3160-5p","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular diseases (CVDs) caused by atherosclerosis (AS) are the leading cause of death and a major health concern globally. However, the mechanism of AS has not been fully elucidated. Therefore, exploring the pathogenesis of AS and controlling its development is a potential approach to reduce AS-related CVDs incidence and mortality. AS pathogenesis is a complex process involving several factors and pathways. Oxidative stress and chronic persistent inflammation play an important role in development and progression of AS.\u003c/p\u003e\n\u003cp\u003eChronic oxidative stress and inflammatory stimulation cause vascular endothelial dysfunction, leading to vascular damage. This traumatic reaction can be manifested as autophagy, apoptosis, pyroptosis, necrosis. Unlike other modes of cell injury, pyroptosis is a programmed mode of cell death associated with inflammation, and the inflammatory response is observed throughout the AS process. Several pyroptotic cells are observed in AS plaques, and \u003cem\u003ein vitro\u003c/em\u003e studies demonstrated that many AS-induced pathological factors can induce cell pyroptosis, resulting in the occurrence of AS \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. These findings indicate that pyroptosis is highly associated with AS.\u003c/p\u003e\n\u003cp\u003eSestrin2 protein plays an important role in inhibiting release of reactive oxygen species (ROS) and maintaining oxidation-reduction balance. In addition, Sestrin2 protein is associated with cancer, type II diabetes and neurological related diseases \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Sestrin2 protein alleviates LPS-induced apoptosis by inhibiting NADPH, suppressing ROS production, and abrogating inflammatory responses through Toll-like receptors \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Studies reported that Sestrin2 protein plays an important protective role in brain and myocardial cell I/R by inhibiting cell apoptosis and modulation of various inflammatory factors \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Cell pyroptosis is a mode of cell death closely associated with inflammatory response. Therefore, we speculated that the Sestrin2 protein modulates pyroptosis of endothelial cells.\u003c/p\u003e\n\u003cp\u003eMicroRNAs are single stranded ribonucleic acids with a length of approximately 22 nt. These RNAs negatively regulates gene expression by binding to the 3'- UTR of the target gene. The binding of miRNA to the gene induces mRNA degradation or inhibits translation \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Therefore, miRNA activity can directly modulate several fundamental biological and cellular processes, including stem cell differentiation, cell differentiation, cell proliferation and apoptosis \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. miR-3160-5p plays a role in inhibiting prostate cancer cell proliferation \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, and enhancing cytotoxicity and apoptosis in human diffuse B-cell lymphoma \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, the role of miR-3160-5p in endothelial cell apoptosis has not yet been elucidated. Bioinformatics analysis showed that Sestrin2 have bindings sites for miR-3160-5p. This implies that Sestrin2 protein may participate in endothelial cell pyroptosis by binding to miR-3160-5P.\u003c/p\u003e\n\u003cp\u003eIn summary, we hypothesized that Sestrin2 protein modulates pyroptosis of human coronary artery endothelial cells (HCAECs) by binding to miR-3160-5P. Therefore, a pyroptosis model of HCAECs was established using hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) to explore the effect and regulatory mechanism of Sestrin2 protein on the pyroptosis and the relationship with miR-3160-5P.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eAll protocols in this study were approved by the Committee on the Ethics of Animal Experiments of Yangzhou University, in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no.85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996). The study was performed in accordance with ARRIVE guidelines too.\u003c/p\u003e\n\u003cp\u003eThis study mainly used the following reagents: HCAECs purchased from Shanghai Zeye Biotechnology(Shanghai,China).Fetal bovine Serum (FB55011) were bought from ClarkBioscience(Virginia,USA).Anti-\u0026beta;-actin(#4970), anti-caspase1 (#24232), anti-NLRP3 (#15101), and anti-ASC (#67824) antibodies were from Cell Signaling Technology (USA), BeyoClick\u0026trade;, propidium iodide (PI; ST511), the Beyotime Reactive Oxygen Species Assay Kit(S0033),Crystal Violet Staining Solution(C0121) were bought from Beyotime Biotechnology. Mounting Medium With DAPI -Aqueous,Fluoroshield (ab104139)was purchased from Abcam(Boston,USA). Hoechst33342/PI apoptosis detection kit (R21807-100) purchased from Shanghai Shangbao Biological, H2O2 staining kit (S0038), cell cycle detection kit (KGA512)were bought from KeygenBiotech(Jiangsu,China). Cell-Light\u0026trade; Edu Apollo In Vitro Kit was bought from RiboBio(Guangzhou,China).\u003c/p\u003e\n\u003cp\u003e2.1 Establishment of HCAECs pyroptosis model\u003c/p\u003e\n\u003cp\u003eThe prepared HCAECs were revived and replenished with fresh medium and pyroptosis was initiated with 600 \u0026micro;mol /L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The relevant experiments were then performed on the cells at the corresponding time points. After exposing the HCAECs to 600 \u0026micro;mol /L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 0h, 1h, 2h, 4h and 8h, the LDH levels in HCAECs culture medium were determined using a kit. In addition, the changes in the expression level of pyroptosis-related proteins NLRP3, Caspase1 and ASC, was assessed by Western blot analysis. HCAECs were treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 8 hours and the levels of ROS in the cells were assessed using DFCH-DA probes.\u003c/p\u003e\n\u003cp\u003e2.2 Changes in the expression levels of Sestrin2 protein and miR-3160-5p in HCAECs culture medium after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eHCAECs were incubated with 600 \u0026micro;mol/L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for different treatment durations (0, 1h, 2h, 4h, 8h) and the expression levels of Sestrin2 protein and miR-3160-5p were determined.\u003c/p\u003e\n\u003cp\u003e2.3 Establishment of high and low expression models of Sestrin2 in HCAECs\u003c/p\u003e\n\u003cp\u003eThe expression levels of Sestrin2 protein and miR-3160-5p in HCAECs exhibited an inverse relationship under pyroptosis state. Further analysis using gene prediction webservers revealed that Sestrin2 protein can specifically bind to miR-3160, with the corresponding binding sites illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Consequently, we hypothesized that Sestrin2 protein modulated the level of pyroptosis in HCAECs through its interaction with miR-3160.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe target sequence of Sestrin2 siRNA was identified as GCAGAGACCCATTGAACAA using gene prediction software. We established high expression and low expression models of Sestrin2 protein in HCAECs through lentivirus transfection. Sestrin2, sh-Sestrin2, and Sestrin2-NC were used to represent the high expression group, low expression group, and plasmid control group, respectively. The cells were incubated with puromycin and stable total RNA was extracted from the cells in the Sestrin2, sh-Sestrin2, and Sestrin2-NC groups. The expression level of Sestrin2 protein was assessed by qRT-PCR.\u003c/p\u003e\n\u003cp\u003e2.4 Determination of LDH levels, expression profiles of pyroptosis-related proteins, and Hoechst33342/PI staining in HCAECs transfected with Sestrin2 protein after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eThe LDH cytotoxicity detection kit was used to assess the changes in LDH levels in the Sestrin2, sh-Esterin2, and Sestrin2-NC groups after treatment of HCAECs with 600 \u0026micro;mol/L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 8 hours. Western blot analysis was performed to evaluate the expression levels of pyroptosis-related proteins in HCAECs in the Sestrin2, sh-Sestrin2, and Sestrin2-NC groups. Additionally, Hoechst33342/PI kit was used to determine the number of PI\u0026thinsp;+\u0026thinsp;cells in HCAECs in the Sestrin2, sh Estrin2, and Sestrin2-NC groups. Fluorescence microscopy was used to determine the number of PI\u0026thinsp;+\u0026thinsp;cells in each group. These experiments were conducted to explore the effect of Sestrin2 protein on the pyroptosis of HCAECs after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e2.5 qRT-PCR analysis\u003c/p\u003e\n\u003cp\u003eThe changes in miR-3160-5p levels in HCAECs in the Sestrin2-NC, Sestrin2, and sh- Sestrin2 groups were evaluated using qRT-PC to explore whether the Sestrin2 protein modulated the expression of miR-3160-5.\u003c/p\u003e\n\u003cp\u003e2.6 Dual-luciferase assay\u003c/p\u003e\n\u003cp\u003eA dual-luciferase assay was performed to explore the potential targeting relationship between miR-3160-5p and the Sestrin2 protein. TargetScan database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.targetscan.org\u003c/span\u003e\u003c/span\u003e) was used to predict the potential binding sites between Sestrin2 protein and miR-3160-5p (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Subsequently, a reporter gene plasmid containing the Sestrin2 protein sequence was constructed using the Gemma gene. This plasmid was co-transfected with miR-3160-5p mimics or miR-NC into 293T cells then dual-luciferase assays were conducted after 24 hours.\u003c/p\u003e\n\u003cp\u003e2.7 Construction of HCAEC models with high and low expression of miR-3160-5p and qRT-PCR analysis\u003c/p\u003e\n\u003cp\u003emiR-3160-5p mimics and miR-3160-5p inhibitor were transfected into HCAECs to construct HCAECs models exhibiting high expression and low expression miR-3160-5p to explore the effect of miR-3160-5p on the pyroptosis of HCAECs.\u003c/p\u003e\n\u003cp\u003eThe forward and reverse primers used in this study are presented below:\u003c/p\u003e\n\u003cp\u003eforward: 5'-TCAGGGGATGTGAGAGTGTGG-3',\u003c/p\u003e\n\u003cp\u003ereverse: 5'-TCAGGGGATGTGAGAGTGTGG-3',\u003c/p\u003e\n\u003cp\u003e\u0026beta;-actin,\u003c/p\u003e\n\u003cp\u003eforward: 5'-CATGTACGTTGCTATCCAGGC-3',\u003c/p\u003e\n\u003cp\u003ereverse: 5'-CTCCTTAATGTCACGCACGAT-3'.\u003c/p\u003e\n\u003cp\u003emiR-NC, miR-3160-5p mimics, and miR-3160-5p inhibitor were used to represent the NC group, high expression group, and low expression group, respectively. qRT-PCR was performed after 24 hours of transfection.\u003c/p\u003e\n\u003cp\u003e2.8 Assessment of LDH levels in HCAECs transfected with miR-3160-5p under the action of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eThe pyrogenic model of HCAECs was constructed by stimulating HCAECs with 600 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 8 h. LDH cytotoxicity detection kit was used to evaluate the levels of LDH in HCAECs transfected with miR-NC, miR-3160-5p mimics, and miR-3160-5p inhibitor to explore the effect of miR-3160-5p on LDH levels in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated HCAECs.\u003c/p\u003e\n\u003cp\u003e2.9 Changes in Hoechst33342/PI staining of HCAECs transfected with miR-3160-5p after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eThe Hoechst33342/PI assay kit was used to evaluate the number of PI\u0026thinsp;+\u0026thinsp;cells in HCAECs transfected with miR-NC, miR-3160-5p inhibitor, and miR-3160-5p mimics to determine the effect of miR-3160-5p on the pyroptosis of HCAECs treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e2.10 Determination of the expression levels of pyroptosis-related proteins in HCAECs transfected with miR-3160-5p after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot was performed to assess the expression profile of pyroptosis related proteins in HCAECs transfected with miR-NC, miR-3160-5p inhibitor, and miR-3160-5p mimics to explore the effect of miR-3160-5p on the expression profile of pyroptosis-related proteins.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e3.1 Changes in LDH levels, ROS levels, and pyroptosis-related proteins in HCAECs treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eHCAECs were treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 0h, 1h, 2h, 4h, and 8h (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, and 4). The LDH level in the HCAECs increased in a time-dependent manner after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment compared with the control group. The expression of pyroptosis-related proteins NLRP3, Caspase1, and ASC also exhibited a time-dependent increase. The level of ROS in HCAECs was significantly higher after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 8 hours compared to the control group, indicating that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulation significantly increased the level of ROS in HCAECs.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results showed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulation significantly increased intracellular ROS production and activated NLRP3 inflammasomes in HCAECs, indicating that the cell pyroptosis model was established successfully.\u003c/p\u003e\n\u003cp\u003e3.2 Expression levels of Sestrin2 protein and mir-3160-5p during pyroptosis of HCAECs\u003c/p\u003e\n\u003cp\u003eThe expression level of Sestrin2 protein in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated HCAECs decreased in a time-dependent manner compared with the control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Conversely, the expression of miR-3160-5p in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulated HCAECs increased in a time-dependent manner compared with the control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e3.3 Identification of HCAEC models transfected with Sestrin2 protein\u003c/p\u003e\n\u003cp\u003eSestrin2, sh-Sestrin2, and Sestrin2-NC models were constructed by lentivirus transfection and the expression levels of sestrin2 determined (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). The expression of Sestrin2 protein was significantly upregulated in the Sestrin2 group compared with the control group. Conversely, the expression of Sestrin2 protein was significantly downregulated in the sh-Sestrin2 group compared with the control group. No significant difference in the expression level of Sestrin2 protein was observed between the Sestrin2-NC group and the control group. These results indicate the successful establishment of high and low expression models of Sestrin2 protein.\u003c/p\u003e\n\u003cp\u003e3.4 LDH levels, expression levels of pyroptosis-related proteins, and intensity of Hoechst33342/PI staining in HCAECs transfected with Sestrin2 protein after treatment with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eLDH cytotoxicity assay kit was used to detect the LDH levels of HCAECs treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). The LDH levels in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;Sestrin2-NC group were not significantly different compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;Sestrin2 group exhibited higher LDH levels, whereas the LDH levels in the H2O2\u0026thinsp;+\u0026thinsp;sh-Sestrin2 group were higher compared with the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003cp\u003eThe expression levels of pyroptosis-related proteins in HCAECs transfected with Sestrin2, sh-Sestrin2, and Sestrin2-NC were detected by western blot (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). The expression level of pyroptosis-related proteins NLRP3, caspase-1, and ASC in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;Sestrin2-NC group was not significantly different compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group. The expression level of NLRP3, caspase-1, and ASC in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;sh-Sestrin2 group lower, whereas the expression level of NLRP3, caspase-1, and ASC was higher compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003cp\u003eThe Hoechst33342/PI assay kit was used to detect the number of PI\u0026thinsp;+\u0026thinsp;cells in HCAECs transfected with lentivirus Sestrin2, sh-Sestrin2, and Sestrin2-NC after incubation with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e). The number of PI\u0026thinsp;+\u0026thinsp;cells in the group overexpressing Sestrin2 decreased, whereas the number of PI\u0026thinsp;+\u0026thinsp;cells in the sh-Sestrin2 group increased compared with the Sestrin2-NC group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.5 Expression levels of miR-3160-5p in HCAECs after transfection with Sestrin2\u003c/p\u003e\n\u003cp\u003eThe expression levels of miR-3160-5p in HCAECs were determined after transfection with different levels of Sestrin2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e). No significant change in the expression level of miR-3160-5p was observed in the Sestrin2-NC group compared with the control group. The expression level of miR-3160-5p in the overexpression Sestrin2 group was downregulated, whereas the expression level of miR-3160-5p in the low-expression Sestrin2 group was significantly upregulated compared with control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003cp\u003e3.6\u003c/p\u003e\n\u003cp\u003eThe luciferase activity in the co-transfection group of Sestrin2 and miR-3160-5p mimics was significantly lower that the NC group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e) (excitation wavelength 580 nm, * * p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs miR-NC\u0026thinsp;+\u0026thinsp;Sestrin2).\u003c/p\u003e\n\u003cp\u003e3.7\u003c/p\u003e\n\u003cp\u003eqRT PCR detection was performed after 24 hours of transfection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e). No significant change in the expression level of miR-3160-5p was observed in the miR-NC group compared with the control group. The expression level of miR-3160-5p in the miR-3160-5p mimics group was significantly higher, whereas the expression level of miR-3160-5p in the miR-3160-5p inhibitor group was significantly lower than the control group. These results indicated successful establishment of the high- and low-expression model of miR-3160-5p.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eqRT-PCR was performed to detect the expression levels of miR-3160-5p in HCAECs 24h after transfection with miR-NC, miR-3160-5p mimics and miR-3160-5p inhibitor (* p\u0026lt;0.05 vs control, ** p\u0026lt;0.01 vs control).\u003c/p\u003e\n\u003cp\u003e3.8\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e increased the release of LDH in HCAECs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e). The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;miR-3160-5p inhibitor group exhibited lower LDH level, whereas the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;miR-3160-5p mimics group showed increased LDH level compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NC group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLDH levels of HCAECs were measured by the LDH cytotoxicity test in the NC group, miR-3160-5p inhibitor group and miR-3160-5p mimics group after treatment with 600 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 8h (** p\u0026lt;0.01 vs. NC, ## p\u0026lt;0.01 vs. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NC).\u003c/p\u003e\n\u003cp\u003e3.9\u003c/p\u003e\n\u003cp\u003eThe miR-3160-5p inhibitor group exhibited a decrease in the number of PI\u0026thinsp;+\u0026thinsp;cells, whereas the miR-3160-5p mimics group showed an increase in the number of PI\u0026thinsp;+\u0026thinsp;cells after treatment with 600 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e compared with the miR-NC group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e3.10\u003c/p\u003e\n\u003cp\u003eThe results are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e16\u003c/span\u003e,H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment increased the expression of NLRP3, caspase-1, and ASC in HCAECs. The expression levels of NLRP3, caspase-1, and ASC in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;miR-3160-5p inhibitor group decreased, whereas the expression of NLRP3, caspase-1, and ASC in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;3160-5p mimics group increased compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NC group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMiR-3160-5p targeted regulation of HCAECs pyroptosis\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCell pyroptosis is characterized by the formation of inflammasomes, gas dermin D (GSDMD) dependent cell perforation, and release of inflammatory factors. NLRP3 containing inflammasomes is a common cell pyroptosis pathway that has been extensively studied. NLRP3 inflammasomes are mainly activated by reactive oxygen species (ROS) and ox-LDL, inducing oxidative stress in endothelial cells. Oxidative stress is involved in the occurrence of cell necrosis and the development of atherosclerosis. Therefore, it is imperative to abrogate in the occurrence and development of endothelial pyroptosis induced by oxidative stress to present and treat AS. Although pharmacological studies report that multiple drugs, such as trimetazidine, can be used as antipyroptotic therapy, gene intervention therapy is a better alternative in inhibiting cell pyroptosis due to its precise targets.\u003c/p\u003e\n\u003cp\u003eCerebellar degeneration-related protein 1 antisense RNA (CDR1as) is implicated in cell proliferation, apoptosis and the regulation of inflammatory factors. Knock down of ciRS-7 promotes IL-1 \u0026beta;-induced release of inflammatory cytokines and cell apoptosis \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In addition, CDR1as plays a regulatory role in vascular endothelial cells and smooth muscle cells. Consequently, CDR1as may inhibit the release of LDH in HCAECs treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and abrogate the release of inflammatory factors and cell pyroptosis induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Previous findings indicate that CDR1as can inhibit oxidative stress-induced pyroptosis of HCAECs \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. However, studies have not explored whether CDR1as has a synergistic relationship with Sestrin2 protein.\u003c/p\u003e\n\u003cp\u003eIn our study, we predicted that the binding of miR-3160-5P to Sestrin2 and observed that the binding sites are highly complementary. This implies that miR-3160-5P can affect the expression of Sestrin2. Lentivirus transfection HCAECs was performed to construct high-expression and low-expression Sestrin2 protein models to confirm the predicted relationship between miR-3160-5P and the protein. Further, we evaluated the impact of miR-3160-5p on pyroptosis of HCAECs. The findings showed that Sestrin2 protein inhibited H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced pyroptosis of HCAECs by binding miR-3160-5p. HCAECs pyroptosis under hypoxia conditions is caused by a combination of multiple pathways and multiple factors. Therefore, inhibiting the expression of Sestrin2 protein and miR-3160-5p only partially improves the pyroptosis of HCAECs. Therefore, further research should be conducted to explore the pathological and physiological changes associated with pyroptosis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe findings indicated that Sestrin2 protein inhibits H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced pyroptosis of HCAECs, implying that Sestrin2 protein is implicated in modulation of the pyroptosis of HCAECs.\u003c/p\u003e\n\u003cp\u003eThe expression of Sestrin2 protein was inhibited by binding to miR-3160-5p, whereas expression of miR-3160-5p promoted pyroptosis of HCAECs, indicating that the Sestrin2 protein inhibited H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced pyroptosis of HCAECs through interaction with miR-3160-5p.\u003c/p\u003e\n\u003cp\u003eThese results show that exploring the mechanism of inhibiting oxidative stress to modulate pyroptosis of HCAECs by regulating the expression of miR-3160-5p may provide a new approach for the treatment of cardiovascular diseases such as AS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.R. and C.Y. wrote the main manuscript text and C.Y. prepared figures, X.C.collected important background information,and Y.J.Participate in experimental design and manuscript review.All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCao Qidong.The Mechanism of CircRNA CDR1as Regulating H2O2-induced Pyrotosis of Human Coronary Endothelial Cells through MiR-135a/ SIRT1 Axis[D].Jilin University,2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe Q, Huang W, Li D, et al. 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Int Immunopharmacol, 2019, 71: 233\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"miR-3160-5P, HCAECs, Sestrin2, pyroptosis","lastPublishedDoi":"10.21203/rs.3.rs-4020648/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4020648/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCardiovascular diseases (CVDs) caused by atherosclerosis (AS) are the leading cause of death and a major health concern globally. However, the mechanisms that lead to have not been fully elucidated. Sestrin2 protein plays an important protective role in myocardial I/R by inhibiting cell apoptosis and other pathways. The aim of this study was to explore the role of Sestrin2 protein in modulating pyroptosis in human coronary artery endothelial cells and explore the specific mechanism of this function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was used to establish a pyroptosis model. The expression levels of Sestrin2 protein and mir-3160-5p in the HCAECs pyroptosis model were determined. The impact on the apoptosis of HCAECs was clarified by regulating the expression level of mir-3160-5p. The regulatory relationship between Sestrin2 protein and mir-3160-5p was explored in the pyroptosis model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe findings showed that Sestrin2 protein inhibited H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced pyroptosis of HCAECs and inhibited miR-3160-5p expression, indicating that Sestrin2 protein inhibited H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced pyroptosis of HCAECs by binding to miR-3160-5p.\u003c/p\u003e","manuscriptTitle":"Sestrin2 Protein Modulates Pyroptosis of Human Coronary Artery Endothelial Cells by Binding to MiR-3160-5p","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-27 05:35:39","doi":"10.21203/rs.3.rs-4020648/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9caaca57-035b-4652-a1b0-ff032218b495","owner":[],"postedDate":"March 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29849927,"name":"Biological sciences/Cell biology"},{"id":29849928,"name":"Biological sciences/Molecular biology"},{"id":29849929,"name":"Health sciences/Cardiology"},{"id":29849930,"name":"Health sciences/Molecular medicine"}],"tags":[],"updatedAt":"2024-04-22T06:18:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-27 05:35:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4020648","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4020648","identity":"rs-4020648","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}