Inhibition of microRNA-181a-5p Protects H9c2 Cells from Hypoxia-Induced Injury Through Up-Regulating Sirtuin 1 Expression

2020 ◽  
Vol 10 (5) ◽  
pp. 682-689
Author(s):  
Qin He ◽  
Rui Dai ◽  
Xiaoju Xiong ◽  
Jinhua Liu ◽  
Zhonghan He ◽  
...  
Keyword(s):  
Cell Injury ◽  
Sirtuin 1 ◽  
Luciferase Reporter ◽  
H9c2 Cell ◽  
Inflammatory Factor ◽  
H9c2 Cells ◽  
Creatine Kinase Mb ◽  
Gene Assay ◽  
Hypoxia Treatment

Myocardial infarction (MI), a life-threatening cardiac event, results in extreme damage to the heart muscle. In this study, we were committed to exploring the role and related mechanisms of microRNA-181a-5p (miR-181a-5p) in MIin vitro. Firstly, we established the MIin vitro cell model by subjecting H9c2 cells to hypoxia. We found that miR-181a-5p was significantly increased in hypoxia-induced H9c2 cells. Then, TargetScan and dual luciferase reporter gene assay confirmed the binding sites between Sirtuin 1 (SIRT1) and miR-181a-5p. SIRT1 was significantly reduced in hypoxia-induced H9c2 cells. Next, we explored the effect of miR-181a-5p inhibitor on hypoxiainduced H9c2 cell injury. The findings indicated that miR-181a-5p inhibitor significantly reduced creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) production enhanced by hypoxia treatment. Moreover, miR-181a-5p inhibitor increased mitochondrial viability in hypoxia-induced H9c2 cells. MTT assay showed that miR-181a-5p inhibitor enhanced hypoxia-induced H9c2 cell viability, and flow cytometry assay indicated that miR-181a-5p inhibitor reduced H9c2 cell apoptosis. ELISA assay indicated that compared with hypoxia treatment group, miR-181a-5p inhibitor decreased the secretion of inflammatory factor such as IL-6, TNF-α and IL-1β . Finally, Western blot assay showed that miR-181a-5p inhibitor decreased the expression of p-p65, indicating the inhibition on NF- κB signaling pathway activation. However, all these effects of miR-181a-5p inhibitor on hypoxia-induced H9c2 cells were reversed by SIRT1-siRNA. Taken together, miR-181a-5p inhibitor protected against hypoxia-induced H9c2 cell injury by targeting SIRT1.

scholarly journals LncRNA PVT1 Promotes Hypoxia-Induced Cardiomyocyte Injury by Inhibiting miR-214-3p

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Chuanliang Liu ◽  
Jieqiong Zhang ◽  
Xuejie Lun ◽  
Lei Li
Keyword(s):  
Cell Viability ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Flow Cytometry Analysis ◽  
Chain Reaction ◽  
Cell Vitality ◽  
Gene Assay ◽  
Polymerase Chain

Objective. To explore the effect and related mechanism of LncRNA PVT1 on hypoxia-induced cardiomyocyte injury. Methods. PVT1RNA and miR-214-3p levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell vitality and apoptosis were, respectively, evaluated by Cell Counting Kit-8 (CCK-8) and flow cytometry analysis. Starbase and Dual luciferase reporter (DLR) gene assay was employed to validate the interaction between miR-214-3p and PVT1. Results. PVT1 was statistically upregulated, and miR-214-3p was statistically downregulated in hypoxia-induced H9c2 cells. The survival rate of H9c2 cells induced by hypoxia decreased statistically, while the apoptosis rate increased statistically ( P < 0.05 ). PVT1 knockdown upregulated the hypoxia-induced H9c2 cell viability and inhibited apoptosis. DLR assay verified the targeting relationship between PVT1 and miR-214-3p. In addition, miR-214-3p inhibitors reversed the viability of H9c2 cells with PVT1 knockout and promoted apoptosis. Conclusion. Silencing PVT1 can enhance the hypoxia-induced H9c2 cell viability and inhibit apoptosis, providing a potential target for the treatment of cardiovascular diseases.

LncRNA XIST Relieves Hypoxia-Induced Damage in H9C2 Cells by Downregulating miR-429

2021 ◽  
Vol 7 (5) ◽  
pp. 1245-1253
Author(s):  
Na Yu ◽  
Xue Han ◽  
Xueqin Wang ◽  
Wanling Yu ◽  
Liqiu Yan
Keyword(s):  
Caspase 3 ◽  
Luciferase Reporter ◽  
Control Group ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Atp Content ◽  
Xist Expression ◽  
Gene Assay ◽  
Lncrna Xist ◽  
Group A

This paper aimed to investigate LncRNA XIST relieving hypoxia-induced damage in H9C2 cells by downregulating miR-429. Rat H9C2 cell lines were selected and divided into a normal control group, a hypoxia group, a XIST expression group, a XIST blank expression group, a miR-429 interference group and a blank interference group. qPCR was adopted for detecting LncRNA XIST and miR-429 expression. Western blot (WB) was adopted for detecting the expression of AMPK, PDH, FAT, MCPT-1, Caspase-3, Bax and Bcl-2, ATP content, and levels of SOD, MDA and LDH. Dual luciferase reporter gene assay (DLRGA) and RNA pull-down were adopted for verifying the correlation of LncRNA XIST with miR-429. Hypoxia-induced H9C2 cells had low LncRNA XIST expression and high miR-429 expression. LncRNA XIST upregulation or miR-429 downregulation could inhibit AMPK, PDH, Caspase-3 and Bax, upregulate FAT, MCPT-1 and Bcl-2, and increase ATP content and SOD activity, as well as reduce MDA content and LDH activity. miR-429 was the target gene of LncRNA XIST. LncRNA XIST can relieve hypoxia-induced damage in H9C2 cells via binding to and downregulating miR-429

scholarly journals IFN-γ Enhances the Efficacy of Exosomes Derived from Mesenchymal Stem Cells in Myocardial Infarction Rats via miR-21 Stimulated by STAT1

2021 ◽  
Author(s):  
Jian Zhang ◽  
Yao Lu ◽  
Yangming Mao ◽  
Yue Yu ◽  
Tianyu Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Vein ◽  
Luciferase Reporter ◽  
H9c2 Cells ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Ifn Γ

Abstract Background: Mesenchymal stem cells (MSCs) activated with IFN-γ elicit more powerful physical effects. Exosomes (Exos) secreted from MSCs have protective against myocardial injury. The aim of this study was to investigate whether Exsos derived from IFN-γ-pretreated MSCs exhibit more potent cardioprotective function and the underlying mechanisms. Methods: Exos were isolated from MSCs (Ctrl-Exo) and IFN-γ-primed MSCs (IFN-γ-Exo) and were then delivered to H9c2 cells or human umbilical vein endothelial cells (HUVECs) in vitro under oxygen and glucose deprivation (OGD) condition or in vivo in an infarcted rat heart. RNA sequencing was to identify the different expressed functional transcription factor (TF). Quantitative reverse transcription-PCR (qPCR) was to confirm the upregulated TF and miRNA in IFN-γ-primed MSCs. Dual-luciferase reporter gene assay were to analyze the transcriptional regulation of miRNAs by STAT1. The target of miR-21-5p (miR-21) was disclosed by luciferase reporter assays and qPCR. The function of BTG2 was verified in vitro under OGD condition.Result: IFN-γ-Exo accelerated migration, tube-like structure formation, and prevented H9c2 from OGD-induced apoptosis. Similarly, IFN-γ-Exo leaded to further reduction in fibrosis size, reduced cardiomyocyte apoptosis and improved cardiac function compared to Ctrl-Exo. miR-21 was significantly upregulated in both IFN-γ-primed MSCs and IFN-γ-Exo. STAT1 transcriptionally induced miR-21 expression. Up-regulated miR-21 can inhibit the expression of BTG2. BTG2 promoted H9c2 cells apoptosis and reversed the protective effect of miR-21 under OGD environment.Conclusion: IFN-γ-Exo have enhanced therapeutic efficacy against acute MI possibly through promoting angiogenesis and anti-apoptotic effect through increasing the level of miR-21, which directly targeted on BTG2.

scholarly journals miRNA-576 Alleviates the Malignant Progression of Atherosclerosis through Downregulating KLF5

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Jing Wang ◽  
Lihui Zhang ◽  
Ting Wang ◽  
Caige Li ◽  
Lijing Jiao ◽  
...  
Keyword(s):  
Body Weight ◽  
Caspase 3 ◽  
Malignant Progression ◽  
Raw264.7 Cells ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Inflammatory Factor ◽  
Luciferase Reporter Gene ◽  
Gene Assay

Objective. To elucidate the role of microRNA-576 (miRNA-576) in alleviating the deterioration of atherosclerosis (AS) through downregulating krüpple-like factor 5 (KLF5). Materials and Methods. The AS model in mice was first constructed. Body weight, inflammation degrees, blood lipid, and relative levels of KLF5, miRNA-576, caspase-3, and bcl-2 in AS mice and control mice were compared. Dual-luciferase reporter gene assay was performed to evaluate the binding between miRNA-576 and KLF5. RAW264.7 cells were treated with 200 mg/L ox-LDL for establishing in vitro high-fat model. Regulatory effects of miRNA-576/KLF5 on relative levels of β-catenin and inflammatory factors in RAW264.7 cells were explored. Results. Body weight was heavier in AS mice than in controls. Protein levels of KLF5 and caspase-3 were upregulated, while bcl-2 was downregulated in AS mice. In particular, protein level of KLF5 was highly expressed in aortic tissues of AS mice. TC and LDL increased, and HDL decreased in AS mice compared with controls. Inflammatory factor levels were markedly elevated in AS mice. KLF5 was verified to be the target gene binding miRNA-576. Overexpression of miRNA-576 downregulated KLF5, inflammatory factors, and β-catenin in ox-LDL-treated RAW264.7 cells. Regulatory effect of miRNA-576 on the release of inflammatory factors in RAW264.7 cells could be partially abolished by KLF5. Conclusions. miRNA-576 alleviates malignant progression of AS via downregulating KLF5.

scholarly journals Skullcapflavone I protects cardiomyocytes from hypoxia-caused injury through up-regulation of lincRNA-ROR

2019 ◽  
Vol 33 ◽  
pp. 205873841985753 ◽  
Author(s):  
Zhenxiao Zhang ◽  
Hui Li ◽  
Mingyang Liu ◽  
Jianshuai He ◽  
Xiaotian Zhang ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
Cell Injury ◽  
Mitogen Activated Protein Kinase ◽  
Cell Counting ◽  
H9c2 Cell ◽  
Erk Pathway ◽  
H9c2 Cells ◽  
Scutellaria Baicalensis Georgi ◽  
Protein Levels

Myocardial infarction (MI) is a serious heart disease in which cardiomyocytes are damaged, caused by hypoxia. This study explored the possible protective activity of Skullcapflavone I (SF I), a flavonoid isolated from the root of Scutellaria baicalensis Georgi, on hypoxia-stimulated cardiomyocytes cell injury in vitro. Viability and apoptosis of H9c2 cells and primary cardiomyocytes were tested using cell counting kit–8 (CCK-8) assay and Guava Nexin Reagent, respectively. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to measure the long non-coding RNA regulator of reprogramming (lincRNA-ROR) expression. si-ROR was transfected to knockdown lincRNA-ROR. Western blotting was conducted to assess the protein levels of key molecules related to cell proliferation, apoptosis, and mitogen-activated protein kinase/extracellular signal–regulated kinase (MEK/ERK) pathway. We discovered that hypoxia stimulation obviously reduced H9c2 cell and primary cardiomyocytes’ viability and proliferation, but promoted cell apoptosis. SF I treatment mitigated the cell viability and proliferation inhibition, as well as cell apoptosis caused by hypoxia. Moreover, SF I promoted the hypoxia-caused up-regulation of lincRNA-ROR in H9c2 cells and primary cardiomyocytes. Knockdown of lincRNA-ROR reversed the influence of SF I on hypoxia-stimulated H9c2 cells and primary cardiomyocytes. Besides, SF I activated MEK/ERK pathway in H9c2 cells and primary cardiomyocytes via up-regulating lincRNA-ROR. To sum up, our research verified the beneficial activity of SF I on hypoxia-caused cardiomyocytes injury. SF I protected cardiomyocytes from hypoxia-caused injury through up-regulation of lincRNA-ROR and activation of MEK/ERK pathway.

scholarly journals MiR-218 Promotes Adriamycin-Induced H9C2 Apoptosis by Inhibiting Stress-Associated Endoplasmic Reticulum Protein 1

2022 ◽  
Vol 2022 ◽  
pp. 1-10
Author(s):  
Qinghua Chen ◽  
Gang Chen ◽  
Shuofang Zhao
Keyword(s):  
Signaling Pathway ◽  
P38 Mapk ◽  
Cell Apoptosis ◽  
Cell Activity ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Tunel Staining ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Gene Assay

Objective. Adriamycin is a clinically important chemotherapeutic drug, but its use is restricted due to its myocardial toxicity. Therefore, it is especially important to explore the toxicity mechanism of Adriamycin (ADR) to cardiomyocytes. Methods. The myocardial toxicity model of ADR was constructed in vitro, and the effect of miR-218 inhibitor and sh-Serp1 on the activity of H9C2 cells induced by ADR was detected by MTT method. Also, flow cytometry, real-time polymerase chain reaction (RT-PCR), and TUNEL staining were used to detect the cell apoptosis. The activity of LDH was detected by colorimetry, and the interaction of miR-218 with Serp1 was detected by double-luciferase reporter gene assay. Western blotting technique was used to detect the expression level of caspase3 and p38 MAPK signal pathway. Results. miR-218 inhibitor can obviously inhibit ADR-induced decrease in cell activity of H9C2 cells, inhibit cell apoptosis, and inhibit p38 MAPK signaling pathway activation. Conversely, sh-Serp1 aggravated the decrease in H9C2 cell activity and promoted cell apoptosis. Conclusion. Upregulation of miR-218 expression will promote ADR-induced apoptosis of H9C2 cells. At the same time, we confirmed that the mechanism by which miR-218 promotes myocardial apoptosis was through the Serp1/p38 MAPK/caspase-3 signaling pathway.

scholarly journals Berberine Ameliorates Doxorubicin-Induced Cardiotoxicity via a SIRT1/p66Shc-Mediated Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Yan-Zhao Wu ◽  
Lan Zhang ◽  
Zi-Xiao Wu ◽  
Tong-tong Shan ◽  
Chen Xiong
Keyword(s):  
Oxidative Stress ◽  
Specific Inhibitor ◽  
Adaptor Protein ◽  
Cardiomyocyte Apoptosis ◽  
Sirtuin 1 ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Sirt1 Expression

Doxorubicin- (DOX-) induced cardiotoxicity is associated with oxidative stress and cardiomyocyte apoptosis. The adaptor protein p66Shc regulates the cellular redox status and determines cell susceptibility to apoptosis. This study is aimed at investigating the involvement of sirtuin 1- (SIRT1-) mediated p66Shc inhibition in DOX-induced redox signalling and exploring the possible protective mechanisms of berberine (Ber) against DOX-triggered cardiac injury in rats and a cultured H9c2 cell line. Our results showed that the Ber pretreatment markedly increased CAT, SOD, and GSH-PX activities, decreased the levels of MDA, and improved the electrocardiogram and histopathological changes in the myocardium in DOX-treated rats (in vivo). Furthermore, Ber significantly ameliorated the DOX-induced oxidative insult and mitochondrial damage by adjusting the levels of intracellular ROS, ΔΨm, and [Ca2+]m in H9c2 cells (in vitro). Importantly, the Ber pretreatment increased SIRT1 expression following DOX exposure but downregulated p66Shc. Consistent with the results demonstrating the SIRT1-mediated inhibition of p66Shc expression, the Ber pretreatment inhibited DOX-triggered cardiomyocyte apoptosis and mitochondrial dysfunction. After exposing H9c2 cells to DOX, the increased SIRT1 expression induced by Ber was abrogated by a SIRT1-specific inhibitor (EX527) or the use of siRNA against SIRT1. Accordingly, SIRT1 inhibition significantly abrogated the suppression of p66Shc expression and protection of Ber against DOX-induced oxidative stress and apoptosis. These results suggest that Ber protects the heart from DOX injury through SIRT1-mediated p66Shc suppression, offering a novel mechanism responsible for the protection of Ber against DOX-induced cardiomyopathy.

scholarly journals Melatonin Protects Cardiomyocytes from Oxygen Glucose Deprivation And Reperfusion-Induced Injury by Inhibiting Rac1/JNK/Foxo3a/Bim Signaling Pathway

2021 ◽  
Author(s):  
Yulin Wang ◽  
Ying Jian ◽  
Xiaofu Zhang ◽  
Bin Ni ◽  
Mingwei Wang ◽  
...  
Keyword(s):  
Protective Effect ◽  
Signaling Pathway ◽  
Cell Injury ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Oxygen Glucose Deprivation ◽  
H9c2 Cell ◽  
Protective Mechanism ◽  
H9c2 Cells

Abstract Melatonin has been shown to exert protective effect during myocardial ischemia/reperfusion (I/R). However, the underlying mechanism is not completely understood. Using the oxygen-glucose deprivation and reperfusion (OGD/R) model of H9c2 cells in vitro, we found that melatonin alleviated OGD/R-induced H9c2 cell injury via inhibiting Foxo3a/Bim signaling pathway. Inhibition of Rac1 activation contributed to the protective effect of melatonin against OGD/R injury in H9c2 cells. Additionally, melatonin inhibited OGD/R-activated Foxo3a/Bim signaling pathway through inactivation of Rac1. Furthermore, JNK inactivation was responsible for Rac1 inhibition-mediated inactivation of Foxo3a/Bim signaling pathway and decreased cell injury in melatonin-treated H9c2 cells. Taken together, these findings identified a Rac1/JNK/Foxo3a/Bim signaling pathway in melatonin-induced protective effect against OGD/R injury in H9c2 cells. This study provided a novel insight into the protective mechanism of melatonin against myocardial I/R injury.

scholarly journals The Exosomal lncRNA KLF3-AS1 From Ischemic Cardiomyocytes Mediates IGF-1 Secretion by MSCs to Rescue Myocardial Ischemia-Reperfusion Injury

2021 ◽  
Vol 8 ◽  
Author(s):  
Gecai Chen ◽  
Aihuan Yue ◽  
Meixiang Wang ◽  
Zhongbao Ruan ◽  
Li Zhu
Keyword(s):  
Myocardial Ischemia ◽  
Myocardial Injury ◽  
Cell Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Myocardial Ischemia Reperfusion

The purpose of the study was to explore the mechanism by which myocardial ischemia-reperfusion (I/R) injury-induced exosomes modulate mesenchymal stem cells (MSCs) to regulate myocardial injury. In this study, we established an I/R injury model in vivo and a hypoxia-reoxygenation (H/R) model in vitro. Then, exosomes isolated from H/R-exposed H9c2 cells were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot analysis. CCK-8 assays and flow cytometry were performed to assess cell injury. ELISA was applied to determine the level of insulin-like growth factor 1 (IGF-1). Echocardiography was used to assess cardiac function in vivo. HE staining and TUNEL assays were conducted to analyze myocardial injury in vivo. In the present study, H/R-exposed H9c2 cells induced IGF-1 secretion from MSCs to inhibit cell myocardial injury. Moreover, exosomes derived from H/R-exposed H9c2 cells were introduced to MSCs to increase IGF-1 levels. The lncRNA KLF3-AS1 was dramatically upregulated in exosomes derived from H/R-treated H9c2 cells. Functional experiments showed that the exosomal lncRNA KLF3-AS1 promoted IGF-1 secretion from MSCs and increased H9c2 cell viability. In addition, miR-23c contains potential binding sites for both KLF3-AS1 and STAT5B, and miR-23c directly bound to the 3'-UTRs of KLF3-AS1 and STAT5B. Furthermore, the lncRNA KLF3-AS1 promoted IGF-1 secretion from MSCs and rescued myocardial cell injury in vivo and in vitro by upregulating STAT5B expression. The lncRNA KLF3-AS1 may serve as a new direction for the treatment of myocardial I/R injury.

scholarly journals Leflunomide inhibits inflammation and apoptosis of H9c2 cells induced by hydrogen peroxide

2021 ◽  
Vol 20 (9) ◽  
pp. 1887-1893
Author(s):  
Jing Xie ◽  
Yeyu Qin ◽  
Cheng Yu
Keyword(s):  
Myocardial Infarction ◽  
Molecular Mechanisms ◽  
Cell Injury ◽  
Inflammatory Responses ◽  
Control Group ◽  
Tunel Staining ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Tnf Α

 Purpose: To investigate the effects of leflunomide (Lef) on inflammatory response and apoptosis after myocardial infarction, and to explore its molecular mechanisms of action.Methods: H2O2 and H9c2 cells were used to establish myocardial cell injury model in vitro. H9c2 cells were divided into 3 groups: control group, H2O2 group, H2O2 + Lef group. The CCK-8 assay was used to determine the optimal concentration of H2O2 and Lef, while the expressions of TNF-α, IL-6, IL-1β, Bcl-2, Bax, Bad, TLR4, IκB-α, P65 and p-P65 were evaluated by Western blot. PCI was utilized to detect the expression of TNF-α, IL-6, IL-1β, Bcl-2, Bax and Bad mRNA. The levels of TNF-α, IL-6 and IL-1β in supernatant were assessed by ELISA, while apoptosis of the three groups was evaluated by TUNEL staining and flow cytometry.Results: Compared with H2O2 group, TNF-α, IL-6, IL-1β, Bax and Bad expressions in H2O2+Lef group were significantly reduced (p < 0.05), but Bcl-2 expression significantly increased. The levels of TNF-α and IL-6 and IL-1β in supernatant of H2O2 + Lef group were also decreased compared to those in the H2O2 group (p < 0.05). In addition, TUNEL-positive cells and apoptotic rates were significantly reduced after treatment with Lef. Moreover, Lef inhibited expression of TLR4 and p-P65, but activated expression of IκB-α, indicating that Lef inhibited TLR4/NF-κB pathway (p < 0.05).Conclusion: The results show that Lef inhibits H2O2-induced H9c2 cell apoptosis and inflammatory responses by inhibiting TLR4/NF-κB pathway. These findings may provide new targets for the treatment of myocardial infarction.

Sign in / Sign up

Export Citation Format

Share Document