scholarly journals MiR-34a regulates blood–tumor barrier function by targeting protein kinase Cε

2015 ◽  
Vol 26 (10) ◽  
pp. 1786-1796 ◽  
Author(s):  
Wei Zhao ◽  
Ping Wang ◽  
Jun Ma ◽  
Yun-Hui Liu ◽  
Zhen Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Protein Expression ◽  
Tight Junction ◽  
Target Genes ◽  
Vascular Endothelial Cells ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Expression Levels ◽  
Related Proteins

MicroRNA-34a (miR-34a) functions to regulate protein expression at the posttranscriptional level by binding the 3′ UTR of target genes and regulates functions of vascular endothelial cells. However, the role of miR-34a in regulating blood–tumor barrier (BTB) permeability remains unknown. In this study, we show that miR-34a overexpression leads to significantly increased permeability of BTB, whereas miR-34a silencing reduces the permeability of the BTB. In addition, miR-34a overexpression significantly down-regulates the expression and distribution of tight junction–related proteins in glioma endothelial cells (GECs), paralleled by protein kinase Cε (PKCε) reduction. Moreover, luciferase reporter gene analysis shows that PKCε is the target gene of miR-34a. We also show that cotransfection of miR-34a and PKCε inversely coregulates BTB permeability and protein expression levels of tight junction–related proteins. Pretreatment of ψεRACK, a PKCε-specific activator, decreases BTB permeability in miR-34a–overexpressed GECs and up-regulates expression levels of tight junction proteins. In contrast, pretreatment of εV1-2, a specific PKCε inhibitor, gives opposite results. Collectively, our findings indicate that miR-34a regulates BTB function by targeting PKCε; after phosphorylation, PKCε is activated and contributes to regulation of the expression of tight junction–related proteins, ultimately altering BTB permeability.

Abstract 267: Hemoglobin, Endothelial Cells, and TLRs. Common Connection?

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Christina Lisk ◽  
David Irwin
Keyword(s):  
Endothelial Cells ◽  
Messenger Rna ◽  
Time Course ◽  
Vascular Endothelial Cells ◽  
Vascular Diseases ◽  
Luciferase Reporter ◽  
Signaling Cascade ◽  
Pulmonary Vascular Disease ◽  
Luciferase Reporter Gene ◽  
Pulmonary Arterial

Introduction: Patients suffering from chronic hereditary hemolytic anemic syndromes, such as sickle cell disease (SCD) and thalassemia, are often at risk for systemic and pulmonary vascular disease. It has been suggested that chronic exposure to cell free hemoglobin (CFH) may contribute to some vascular diseases associated with these syndromes such as pulmonary arterial hypertension. To date, the vasculotoxic effects of CFH have mostly been attributed to its pro-oxidant and nitric oxide scavenging characteristics. However, emerging evidence suggests CFH may contribute to inflammation by directly activating a signaling cascade event by binding to a pattern recognition receptor (PRR) or a toll like receptor (TLR) on vascular endothelial cells. Hypothesis: We hypothesized that CFH would increase the activity of transcription factors, NF-κb and HIF-1α, via a MyD88-dependent pathway. Methods: Human microvascular endothelial cells (HMEC) were transfected with either an NF-κB or HIF-1α luciferase reporter gene and treated with CFH (ferrous, ferric, and ferryl forms) in the presence or absence of SOD, catalase, dexamethasome, MyD88 inhibitor, or, the PHD inhibitor, DMOG. Messenger RNA for HIF-1α and HIF-2 were also measured after treatments. Results: All three states of hemoglobin increased NF-κB and HIF-1α activity in a dose response fashion, with ferryl inducing the greatest activity of both NF-κB and HIF-1α. Time course studies showed that NF-κB and HIF-1α activity tracked together. A unique synergy was noted with co-treatment of ferryl and DMOG. Co-treatment with SOD or catalase did not inhibit the CFH-induced NF-κB or HIF-1α response. Dexamthasome and MyD88 inhibition reduced the CFH-induced NF-κB and HIF-1α activity. Conclusion: Our results support the hypothesis, that CFH may activate a TLR or PRR signaling cascade subsequently activating MyD88-NF-κB and HIF-1α. Our data, that showed SOD and/or catalase did not block CFH effects, suggests that this event is not mediated by CFH pro-oxidant characteristics. CFH-induced HIF-1α was blocked by NF-κB inhibition with either, Dexamethasome or MyD88 inhibition emphasizing the importance of NF-κB in the HIF-1α pathway.

scholarly journals Effects of IL-1β and TNF-α on the Expression of P311 in Vascular Endothelial Cells and Wound Healing in Mice

2020 ◽  
Vol 11 ◽  
Author(s):  
Daijun Zhou ◽  
Tengfei Liu ◽  
Song Wang ◽  
Weifeng He ◽  
Wei Qian ◽  
...  
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Protein Expression ◽  
Vascular Endothelial Cells ◽  
Control Group ◽  
Vascular Endothelial ◽  
Expression Levels ◽  
Relative Expression ◽  
Tnf Α

ObjectiveThis study aimed to define the role of interleukine-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the expression of P311 in vascular endothelial cells (VECs) and in wound healing.MethodsDAPI staining, a CCK-8 assay, cell migration assay, and an angiogenesis assay were used to assess the effects exerted by TNF-α and IL-1β at various concentrations on morphology, proliferation, migration, and angiogenesis of VECs. Western blot (WB) and reverse transcription-polymerase chain reaction (RT-PCR) models were employed to observe the effects exerted by proteins related to the nuclear factor-kappa B (NF-κB) signaling pathway and P311 mRNA expression. Bioinformatics analysis was performed on the binding sites of P311 and NF-κB. Finally, to investigate the effects of IL-1β and TNF-α on wound healing and the length of new epithelium in mice, we established a full-thickness wound defect model in mice. Immunohistochemistry was used to measure changes in P311, proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), CD31 (platelet endothelial cell adhesion molecule-1, PECAM-1/CD31), as well as other related proteins.ResultsWhen levels of TNF-α and IL-1β were both 20 ng/ml, their effects on cell proliferation, cytoskeleton protein expression, cell migration, and angiogenesis were the greatest (P < 0.05). IL-1β and TNF-α at moderate concentrations effectively promoted P311 mRNA and p-NF-κB protein expression (P < 0.05), while p-NF-K b protein expression was decreased (P < 0.05). Luciferase assays showed that P311 expression was also relatively greater when stimulated at moderate concentrations (P < 0.05), while relative expression was significantly lower when the p-NF-K b inhibitor CAPE was added (P < 0.05). On 7-day wound healing rate comparison, the control, IL-1β, IL-1βab, TNF-α, and TNF-αab groups were 18, 37, 35, 39, and 36%, respectively, while control group + P311 siRNA was 31% (P < 0.05). New epithelial length, granulation tissue thickness, and number of blood vessels trends were also the same. In the control group, P311 showed lower relative expression levels than the others (P < 0.05). P311 relative expression levels trended as follows: control group > IL-1βab > IL-1β > TNF-αab > TNF-α (P < 0.05).ConclusionWhen IL-1β and TNF-α concentrations are moderate, they effectively promote the proliferation, expression, migration, and angiogenesis of VECs, possibly by promoting the expression of the NF-K b pathway and thereby promoting the expression of P311. In vitro experiments on mice suggest that P311 effectively promotes wound healing, and its mechanism may be closely related to PCNA, CD31, and VEGF.

miR-27b-3p Down-Regulation Prevents Hypoxia-Induced Cardiomyocyte Apoptosis Through Regulating Yes-Associated Protein 1 (YAP1) Expression

2021 ◽  
Vol 11 (5) ◽  
pp. 948-956
Author(s):  
Lilin Wang ◽  
Bo Feng ◽  
Shu Zhu
Keyword(s):  
Cell Viability ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
H9c2 Cells ◽  
Luciferase Reporter Gene ◽  
Western Blot Assay ◽  
Heart Protection ◽  
Gene Assay ◽  
Related Proteins

Background: Congenital heart disease (CHD) is one of the most common birth defects. MicroR-NAs (miRNAs) are a group of endogenous, non-coding small RNAs and mediate the target genes expression. An increasing evidence showed that in recent years, miRNAs have given rise to more and more attention in heart protection and development. In our research, the main purpose was to determine the effect of miR-27b-3p in CHD and analyze related mechanisms. Methods: We performed qRT-PCR analysis to examine miR-27b-3p expression in myocardial tissue from 30 patients with CHD and hypoxia-induced H9C2 cells. Then, we performed biological software TargetScan to predict the relationship of miR-27b-3p and YAP1, and dual luciferase reporter gene assay was used to verify the results. H9C2 cells were transfected with inhibitor control, miR-27b-3p inhibitor, miR-27b-3p inhibitor + control-siRNA or miR-27b-3p inhibitor + YAP1-siRNA for 6 hours and then induced by hypoxia for 72 hours. Subsequently, we performed MTT and FCM analysis to detect cell viability and apoptosis. Finally, we used western blot assay to measure the expression of apoptosis-related proteins. Results: Our study indicated that miR-27b-3p expression in myocardial samples of cyanotic CHD patients was significantly higher than that of the acyanotic CHD patients. miR-27b-3p expression was gradually up-regulated with the increase of hypoxia induction time in H9C2 cells. Besides, we confirmed that YAP1 was a target gene of miR-27b-3p. Moreover, our results showed that miR-27b-3p inhibitor improved cell viability, decreased apoptosis, and affected apoptosis-related proteins expression in hypoxia induced H9C2 cells. These changes were reversed by YAP1-siRNA. All data demonstrated that miR-27b-3p/YAP1 might be new potential bio-marker and therapeutic target for CHD treatment.

scholarly journals Histamine reduces ZO-1 tight-junction protein expression in cultured retinal microvascular endothelial cells

1996 ◽  
Vol 320 (3) ◽  
pp. 717-721 ◽  
Author(s):  
Thomas W. GARDNER ◽  
Treena LESHER ◽  
Sonny KHIN ◽  
Cuong VU ◽  
Alistair J. BARBER ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Tight Junction ◽  
Protein Content ◽  
Vascular Endothelial Cells ◽  
Tight Junction Protein ◽  
Vascular Endothelial ◽  
Tight Junction Protein Expression ◽  
Junction Protein ◽  
Protein Alterations

We examined ZO-1 protein content in cultured retinal vascular endothelial cells to test the hypothesis that histamine alters tight-junction-protein expression. Histamine (10-9 –10-4 M) causes a reversible concentration-dependent reduction of ZO-1 protein content, mediated by both H1 and H2 receptors. Histamine reduces ZO-1 expression within the time associated with increased paracellular permeability. Tight-junction-protein alterations may be a novel explanation for the mechanism by which vasoactive agents increase microvascular permeability.

scholarly journals miR-99a-3p Targeting EIF4EBP1 Affects B Lymphocytes Function Through Autophagy and Aggravates SLE Disease Progression

2021 ◽  
Author(s):  
meng yang ◽  
Deng Danqi
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Protein Expression ◽  
Reporter Gene ◽  
Immune Cells ◽  
Target Genes ◽  
Kidney Tissue ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mrna And Protein Expression

Abstract Background:Overactivation of immune cells plays a key role in the pathogenesis of systemic lupus erythematosus (SLE). The regulation of immune cells by miRNA is a research hotspot.In this study, the second-generation high-throughput sequencing found that the expression of miR-99a-3p in SLE decreased, but the specific mechanism is still unclear.The purpose of this study is to explore the potential target genes, target cells of miR-99a-3p and their potential mechanisms affecting the progression of SLE.Methods: Isolate PBMC from healthy individulas and SLE patients, transfect Ball-1, Jurkat, THP-1 and K562 cells with miR-99a-3p agomir and antagomir,detect miR-99a-3p, predict target genes and autophagy pathway mRNA and protein expression by RT-qPCR and Western blotting.CCK-8 method detects cell proliferation, PI method detects cell cycle, flow cytometry detects cell apoptosis, and luciferase reporter gene experiment determines miR-99a-3p target genes.With C57BL/6J mice as control,construct miR-99a-3p overexpression and interference model based on MRL/lpr mice,ELISA detects plasma ANA, dsDNA, IgE, IgM, IL-6, IL-10, BLyS.Pathological analysis of HE staining and C3 immunofluorescence(IF) deposition in mouse kidney tissue,Immunohistochemistry(IHC) detects changes in target genes and pathway proteins in kidney tissue,isolate B cells to verify the differential expression of miR-99a-3p, target genes, pathway mRNA and protein.Results: Compared with healthy individulas, miR-99a-3p in SLE was down-regulated, while EIF4EBP1, LC3II, LAMP-2A mRNA and protein expression were up-regulated.After Ball-1 was transfected with miR-99a-3p agomir, cell proliferation decreased and apoptosis increased.After transfected with miR-99a-3p antagomir, the effect was opposite;Luciferase reporter gene detection proved that miR-99a-3p directly targets EIF4EBP1. Rescue experiments confirmed the interaction model between miR-99a-3p and EIF4EBP1. Clinical, in vitro, and in vivo experiments further confirmed that miR-99a-3p agomir can reduce the expression of EIF4EBP1, LC3-Ⅱ, and LAMP-2A, while miR-99a-3p antagomir had the opposite effect.In vivo experiment antagomir group mice serum ANA, dsDNA, IgE, IgM, IL-6, IL-10, BLyS were higher than those in the MRL/lpr group, EIF4EBP1, LC3-Ⅱ, LAMP-2A mRNA, protein and IHC levels also increased, and the urinary protein and C3 IF deposition of mice in the antagomir group were increased, and the related indexes of mice in the agomir group were lower than those in the MRL/lpr group.Conclusion:The expression of miR-99a-3p in SLE PBMC was down-regulated. Up-regulation of miR-99a-3p by transfection can protect B cells from autophagy mediated by EIF4EBP1.The down-regulation of miR-99a-3p induces autophagy by regulating the autophagy signaling pathway mediated by EIF4EBP1 in SLE B cells. These results indicate that miR-99a-3p and EIF4EBP1 may be potential targets of SLE.

scholarly journals Study on the Mechanism of miR-34a Affecting the Proliferation, Migration, and Invasion of Human Keloid Fibroblasts by Regulating the Expression of SATB1

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Lei Jiang ◽  
Xiufang Shi ◽  
Meng Wang ◽  
Huaxia Chen
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Normal Skin ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Expression Levels ◽  
Gene Test ◽  
Satb1 Expression ◽  
Keloid Fibroblasts

Objectives. To explore the effect and mechanism of miR-34a on the proliferation, migration, and invasion of keloid fibroblasts (KFB). Methods. Isolate and culture KFB and normal skin fibroblast (NFB), detect the mRNA expression levels of miR-34a and integrin β5 (SATB1) in KFB and NFB by RT-qPCR, and detect SATB1 by western blot. The level of protein expression, MTT method, Transwell method, RT-qPCR, and western blot were used to detect the effects of overexpression of miR-34a or inhibition of SATB1 expression on the proliferation, migration, and invasion of KFB cells and the expression of related proteins. The dual luciferase reporter gene test verifies the targeting relationship between miR-34a and SATB1. Results. Compared with NFB, the expression of miR-34a was downregulated in KFB and the mRNA and protein expression levels of SATB1 were upregulated. Overexpression of miR-34a or inhibition of SATB1 expression inhibited the proliferation, migration, and invasion of KFB. miR-34a can negatively regulate the expression of SATB1, and overexpression of SATB1 reverses the effects of overexpression of miR-34a on the proliferation, migration, and invasion of KFB. Conclusions. miR-34a inhibits the proliferation, migration, and invasion of keloid fibroblasts by downregulating the expression of SATB1.

scholarly journals Cervical cancer-derived exosomal miR-663b promotes angiogenesis by inhibiting vinculin expression in vascular endothelial cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xuewu You ◽  
Wenxiong Sun ◽  
Ying Wang ◽  
Xiaoli Liu ◽  
Aihong Wang ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Endothelial Cells ◽  
Tumor Growth ◽  
Target Genes ◽  
Vascular Endothelial Cells ◽  
Antiangiogenic Therapy ◽  
Tube Formation ◽  
Luciferase Reporter ◽  
Cancer Tissue ◽  
Vascular Endothelial

Abstract Background Angiogenesis provides essential nutrients and oxygen for tumor growth and has become the main mechanism of tumor invasion and metastasis. Exosomes are nanoscale membrane vesicles containing proteins, lipids, mRNA and microRNA (miRNA), which mediate intercellular communication and play an important role in tumor progression. Accumulated evidence indicates that tumor-derived exosomal miRNAs participate in the tumor microenvironment and promote angiogenesis. Methods Bioinformatic target prediction and dual luciferase reporter assays were performed to identify the binding site between miR-663b and the 3′-UTR of vinculin (VCL). VCL overexpression lentivirus and miR-663b overexpression/inhibition lentivirus were used to create a VCL overexpression model and miR-663b overexpression/inhibition model in-vitro. Immunohistochemistry (IHC) assays and western blot assays were used to detect protein expression. Exosome-cell cocultures, wound healing assays, tube formation assays and transwell assays were used to measure the migration and tube formation ability of vascular endothelial cells [human umbilical vein endothelial cells (HUVECs)]. siRNA targeted VCL was used to knockdown VCL. Results In the present study, we found that miR-663b was elevated in cervical cancer tissue and exosomes. miR-663b could bind the 3′-UTR of VCL and inhibit its expression. VCL is downregulated in cervical cancer, and decreased VCL has a negative correlation with a high level of miR-663b. Further studies demonstrated that exosomes secreted by cervical cancer cells can deliver miR-663b to HUVECs and inhibit the expression of VCL, thereby promoting angiogenesis and tumor growth. Conclusions miR-663b derived from cancer cell exosomes acts as a driving factor for angiogenesis and a potential target of antiangiogenic therapy in cervical cancer. Our findings illustrated a new signaling pathway, including exosomes, miRNAs and target genes, which provides potential targets for antiangiogenic therapy.

scholarly journals MiR-326 mediates malignant biological behaviors of lung adenocarcinoma by targeting ZEB1

2021 ◽  
Vol 104 (2) ◽  
pp. 003685042110093
Author(s):  
Mingxin Liu ◽  
Hong Wu ◽  
Yiqiang Liu ◽  
Yan Tan ◽  
Songtao Wang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Invasion And Migration ◽  
Transwell Invasion Assay ◽  
Enhancer Binding Protein ◽  
Adenocarcinoma Cells ◽  
And Migration ◽  
Related Proteins

MiR-326 functions as an antioncogene in the several types of cancer. However, the underling mechanisms through which miRNA-326 regulates the anti-carcinogenesis of lung adenocarcinoma have remained elusive. The aim of this study was to explore the role and regulatory mechanism of miR-326 in cell proliferation, invasion, migration and apoptosis in lung adenocarcinoma. Quantitative real-time PCR (qRT-PCR) was used to detect the expression pattern of miR-326 in human bronchial epithelial cells (HBES-2B), 4 kinds of lung adenocarcinoma cell lines (H23, H1975, H2228, H2085) and 20 lung adenocarcinoma tissues. Then, H23 cells were infected with miR-326 mimics, miR-326 inhibitors and si-ZEB1 to build up-regulated miR-326 cell lines, down-regulated ZEB1(zinc-finger-enhancer binding protein 1)cell lines, simultaneous down-regulated ZEB1 and miR-326 cell lines. Moreover, CCK-8 assay, transwell invasion assay, wound healing assay and flow cytometry assay were employed to examine the effects of miR-326 and ZEB1 on the proliferation, invasion, migration and apoptosis abilities of H23 cells. Western blot was performed to explore the effects of miR-326 and ZEB1 on the expression of invasion and migration related proteins N-cadherin, E-cadherin, MMP7, MMP13, SLUG and apoptotic proteins PARP, BAX. On the mechanism, a dual-luciferase reporter gene was used to measure the target relationship between miR-326 and ZEB1. MiR-326 expression was significantly downregulated in lung adenocarcinoma tissues and cells. Overexpression of miR-326 significantly inhibited the malignant behaviors of H23 cells. Mechanically, luciferase reporter assay showed that ZEB1 was a direct target of miR-326. MiR-326 mimic downregulated the expression of ZEB1. Furthermore, knocking down ZEB1 strongly inhibited the proliferation, invasion and migration of H23 cells but promoted apoptosis. MiR-326 could target ZEB1 to inhibit the proliferation, invasion and migration of lung adenocarcinoma cells and promote apoptosis, which is a potential therapeutic target for lung adenocarcinoma.

scholarly journals Mechanism of MicroRNA-375 Promoter Methylation in Promoting Ovarian Cancer Cell Malignancy

2021 ◽  
Vol 20 ◽  
pp. 153303382098011
Author(s):  
Junjun Shu ◽  
Ling Xiao ◽  
Sanhua Yan ◽  
Boqun Fan ◽  
Xia Zou ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Protein Expression ◽  
Cell Lines ◽  
Cell Invasion ◽  
Promoter Methylation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Cell Malignancy

Objective: Ovarian cancer (OC) ranks one of the most prevalent fatal tumors of female genital organs. Aberrant promoter methylation triggers changes of microRNA (miR)-375 in OC. Our study aimed to evaluate the mechanism of methylated miR-375 promoter region in OC cell malignancy and to seek the possible treatment for OC. Methods: miR-375 promoter methylation level in OC tissues and cells was detected. miR-375 expression in OC tissues and cell lines was compared with that in demethylated cells. Role of miR-375 in OC progression was measured. Dual-luciferase reporter gene assay was utilized to verify the targeting relationship between miR-375 and Yes-associated protein 1 (YAP1). Then, Wnt/β-catenin pathway-related protein expression was tested. Moreover, xenograft transplantation was applied to confirm the in vitro experiments. Results: Highly methylated miR-375 was seen in OC tissues and cell lines, while its expression was decreased as the promoter methylation increased. Demethylation in OC cells brought miR-375 back to normal level, with obviously declined cell invasion, migration and viability and improved apoptosis. Additionally, miR-375 targeted YAP1 to regulate the Wnt/β-catenin pathway protein expression. Overexpressed YAP1 reversed the protein expression, promoted cell invasion, migration and viability while reduced cell apoptosis. Overexpressed miR-375 in vivo inhibited OC progression. Conclusion: Our study demonstrated that demethylated miR-375 inhibited OC growth by targeting YAP1 and downregulating the Wnt/β-catenin pathway. This investigation may offer novel insight for OC treatment.

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