scholarly journals miR-450a-5p eliminats MGO-induced insulin resistance via targeting CREB

2019 ◽  
Author(s):  
Cuifeng Wei ◽  
Li Meng ◽  
Yuting Zhang
Keyword(s):  
Insulin Resistance ◽  
Endothelial Cells ◽  
Cell Invasion ◽  
High Glucose ◽  
Target Gene ◽  
Cell Activity ◽  
Luciferase Reporter ◽  
Akt Pathway ◽  
Potential Target ◽  
In Cells

Abstract Background miR-450a-5p was involved in fat formation, but its role in insulin resistance remains unclear. This study further investigated the effects of miR-450a-5p in endothelial cells, with the aim of finding a potential target for diabetes mellitus. Methods Human umbilical vein endothelial cells (HUVECs) were severally treated with low-glucose, high-glucose, methylglyoxal (MGO), and insulin only or plus MGO. miR-450a-5p was up-regulated or down-regulated in treated HUVECs. miR-450a-5p expression in cells was measured by quantitative real-time polymerase chain reaction (qRT-PCR) assays. The cell activity was determined through MTT experiments. Transwell assay and oil red O staining were used for the detection of cell invasion and fat formation. The expressions of eNOS/AKT pathway-related proteins in cells were assessed by Western blot (WB) analysis. Furthermore, the target gene of miR-450a-5p was analyzed by double-luciferase reporter analysis, and its influence in eNOS/AKT pathway was estimated. Results miR-450a-5p decreased obviously in endothelial cells with high-glucose and MGO. Through in vitro cell experiments, we knew that MGO could not only intensify the activity of endothelial cells, but also accelerate cell invasion and fat accumulation, which could be reversed by up-regulated miR-450a-5p. Moreover, MGO inhibited eNOS/AKT pathway activation and NO release mediated by insulin, which were eliminated by up-regulated miR-450a-5p. Furthermore, CREB was the target gene of miR-450a-5p that had an activation effect on the eNOS/AKT pathway. Conclusions Up-regulated miR-450a-5p eliminated MGO-induced insulin resistance via targeting CREB, which might be a potential target to improve insulin resistance and benefit patients with related diseases.

scholarly journals MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4

2021 ◽  
Vol 20 ◽  
pp. 153303382098586
Author(s):  
Xuhui Wu ◽  
Gongzhi Wu ◽  
Huaizhong Zhang ◽  
Xuyang Peng ◽  
Bin Huang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Invasion ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Invasion And Migration ◽  
And Migration ◽  
Dual Luciferase Reporter Assay

Objective: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells. Methods: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells. Results: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration. Conclusion: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

scholarly journals Circ_CLASP2 Regulates High Glucose-Induced Dysfunction of Human Endothelial Cells Through Targeting miR-140-5p/FBXW7 Axis

2021 ◽  
Vol 12 ◽  
Author(s):  
Qin Zhang ◽  
Jing Long ◽  
Nannan Li ◽  
Xuelian Ma ◽  
Lisheng Zheng
Keyword(s):  
Endothelial Cells ◽  
High Glucose ◽  
Colony Formation ◽  
Umbilical Vein ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Proliferation And Apoptosis ◽  
The Stability ◽  
Umbilical Vein Endothelial Cells ◽  
Related Proteins

Hyperglycemia exposure results in the dysfunction of endothelial cells (ECs) and the development of diabetic complications. Circular RNAs (circRNAs) have been demonstrated to play critical roles in EC dysfunction. The current study aimed to explore the role and mechanism of circRNA CLIP–associating protein 2 (circ_CLASP2, hsa_circ_0064772) on HG-induced dysfunction in human umbilical vein endothelial cells (HUVECs). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the levels of circ_CLASP2, miR-140-5p and F-box, and WD repeat domain-containing 7 (FBXW7). The stability of circ_CLASP2 was identified by the actinomycin D and ribonuclease (RNase) R assays. Cell colony formation, proliferation, and apoptosis were measured by a standard colony formation assay, colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay, and flow cytometry, respectively. Western blot analysis was performed to determine the expression of related proteins. Targeted correlations among circ_CLASP2, miR-140-5p, and FBXW7 were confirmed by dual-luciferase reporter assay. High glucose (HG) exposure downregulated the expression of circ_CLASP2 in HUVECs. Circ_CLASP2 overexpression or miR-140-5p knockdown promoted proliferation and inhibited apoptosis of HUVECs under HG conditions. Circ_CLASP2 directly interacted with miR-140-5p via pairing to miR-140-5p. The regulation of circ_CLASP2 overexpression on HG-induced HUVEC dysfunction was mediated by miR-140-5p. Moreover, FBXW7 was a direct target of miR-140-5p, and miR-140-5p regulated HG-induced HUVEC dysfunction via FBXW7. Furthermore, circ_CLASP2 mediated FBXW7 expression through sponging miR-140-5p. Our current study suggested that the overexpression of circ_CLASP2 protected HUVEC from HG-induced dysfunction at least partly through the regulation of the miR-140-5p/FBXW7 axis, highlighting a novel therapeutic approach for the treatment of diabetic-associated vascular injury.

scholarly journals SOX30, a target gene of miR-653-5p, represses the proliferation and invasion of prostate cancer cells through inhibition of Wnt/β-catenin signaling

2019 ◽  
Vol 24 (1) ◽  
Author(s):  
Qiang Fu ◽  
Zhenye Sun ◽  
Fan Yang ◽  
Tianci Mao ◽  
Yanyao Gao ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Target Gene ◽  
Prostate Cancer Cell ◽  
Luciferase Reporter ◽  
Prostate Cancer Cells ◽  
Normal Prostate ◽  
Proliferation And Invasion ◽  
In Cells

Abstract Background Sex-determining region Y-box containing gene 30 (SOX30) is a newly identified tumor-associated gene in several types of cancer. However, whether SOX30 is involved in the development and progression of prostate cancer remains unknown. This study investigated the potential role of SOX30 in prostate cancer. Methods Prostate cancer cell lines and a normal prostate epithelial cell line were used for the experiments. The expression of SOX30 was determined using quantitative real-time PCR and western blot analysis. The malignant cellular behaviors of prostate cancer were assessed using the Cell Counting Kit-8, colony formation and Matrigel invasion assays. The miRNA–mRNA interaction was validated using the dual-luciferase reporter assay. Results SOX30 expression was lower in cells of prostate cancer lines than in cells of the normal prostate epithelial line. Its overexpression repressed the proliferation and invasion of prostate cancer cells. SOX30 was identified as a target gene of microRNA-653-5p (miR-653-5p), which is upregulated in prostate cancer tissues. MiR-653-5p overexpression decreased SOX30 expression, while its inhibition increased SOX30 expression in prostate cancer cells. MiR-653-5p inhibition also markedly restricted prostate cancer cell proliferation and invasion. SOX30 overexpression or miR-653-5p inhibition significantly reduced β-catenin expression and downregulated the activation of Wnt/β-catenin signaling. SOX30 knockdown significantly reversed the miR-653-5p inhibition-mediated inhibitory effect on the proliferation, invasion and Wnt/β-catenin signaling in prostate cancer cells. Conclusions These results reveal a tumor suppressive function for SOX30 in prostate cancer and confirmed the gene as a target of miR-653-5p. SOX30 upregulation due to miR-653-5p inhibition restricted the proliferation and invasion of prostate cancer cells, and this was associated with Wnt/β-catenin signaling suppression. These findings highlight the importance of the miR-653-5p–SOX30–Wnt/β-catenin signaling axis in prostate cancer progression.

scholarly journals PTP4A3, A Novel Target Gene of HIF-1alpha, Participates in Benzene-Induced Cell Proliferation Inhibition and Apoptosis through PI3K/AKT Pathway

2020 ◽  
Vol 17 (3) ◽  
pp. 910
Author(s):  
Yunqiu Pu ◽  
Fengxia Sun ◽  
Rongli Sun ◽  
Zhaodi Man ◽  
Shuangbin Ji ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Target Gene ◽  
Akt Pathway ◽  
Proliferation Inhibition ◽  
Cell Proliferation Inhibition ◽  
A Cell ◽  
Novel Target ◽  
In Cells

Benzene, a commonly used chemical, has been confirmed to specifically affect the hematopoietic system as well as overall human health. PTP4A3 is overexpressed in leukemia cells and is related to cell proliferation. We previously found that HIF-1alpha was involved in benzene toxicity and PTP4A3 may be the target gene of HIF-1alpha via ChIP-seq. The aim of this study is to confirm the relationship between HIF-1alpha and PTP4A3 in benzene toxicity, as well as the function of PTP4A3 on cell toxicity induced by 1,4-benzoquinone (1,4-BQ). Our results indicate that HIF-1alpha could regulate PTP4A3 with in vivo and in vitro experiments. A cell line with suppressed PTP4A3 was established to investigate the function of PTP4A3 in 1,4-BQ toxicity in vitro. The results revealed that cell proliferation inhibition was more aggravated in PTP4A3 low-expression cells than in the control cells after 1,4-BQ treatment. The relative oxygen species (ROS) significantly increased in cells with inhibited PTP4A3, while the rise was inferior to the control cells at the 20 μM 1,4-BQ group. An increase in DNA damage was seen in PTP4A3 down-regulated cells at the 10 μM 1,4-BQ group, whereas the results reversed at the concentration of 20 μM. Moreover, the apoptosis rate increased higher in down-regulated PTP4A3 cells after 1,4-BQ exposure. In addition, PI3K/AKT pathway was significantly restrained in cells with inhibited PTP4A3 after 1,4-BQ treatment. Our results indicate that HIF-1alpha may regulate PTP4A3 to be involved in benzene toxicity. Inhibition of PTP4A3 could aggravate cell proliferation suppression and apoptosis by regulating PI3K/AKT pathway after 1,4-BQ treatment.

scholarly journals Attenuation of Glomerular Endothelial Cells from High Glucose-Induced Injury by Blockade of MAD2B

2015 ◽  
Vol 35 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Yu-Mei Wang ◽  
Yu Hao ◽  
Xian-Fang Meng ◽  
Fang-Fang He ◽  
Shan Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
High Glucose ◽  
Cell Monolayer ◽  
No Production ◽  
Protein Levels ◽  
Glomerular Endothelial Cells ◽  
Monolayer Permeability ◽  
Glucose Treatment ◽  
In Cells

Background/Aims: To assess the role of mitotic arrest-deficient 2-like protein 2 (MAD2B) in high glucose-induced injury in mouse glomerular endothelial cells (GEnCs). Methods: GEnCs were cultured in vitro, and MAD2B protein levels were measured by Western blot in cells stimulated with high glucose (30 mM) for various periods of time. MAD2B and scrambled shRNA were introduced into GEnCs by liposomal transfection. Cell proliferation, apoptosis, nitric oxide (NO) production, and monolayer permeability were then measured in cells grown in the following conditions: control, high glucose treatment, MAD2B shRNA transfection with high glucose treatment, and scrambled shRNA transfection with high glucose treatment. Results: High glucose increased the protein levels of MAD2B in GEnCs. Compared with control cells, apoptosis was increased by high glucose treatment, which was attenuated by transfection with MAD2B shRNA transfection. Cells treated with high glucose produced less NO than control cells, whereas MAD2B shRNA transfection increased NO production. Cell monolayer permeability was enhanced in high glucose treated cells, but MAD2B shRNA transfection reduced permeability. Conclusion: High glucose levels induced the expression of MAD2B in GEnCs, whereas suppressing its expression reduced high glucose-induced endothelial cell apoptosis and high permeability, and promoted cell proliferation and NO production.

scholarly journals Down-regulated miR-145 rescues insulin resistance in endothelial cells

2019 ◽  
Author(s):  
Jing Wang ◽  
Zhichun Dong ◽  
Liying Lou
Keyword(s):  
Insulin Resistance ◽  
Endothelial Cells ◽  
High Glucose ◽  
Umbilical Vein ◽  
Glucose Consumption ◽  
Over Expression ◽  
Qrt Pcr ◽  
Starting Point ◽  
Polymerase Chain ◽  
Umbilical Vein Endothelial Cells

Abstract Background: MiR-145 is involved in insulin resistance (IR) in liver cells, but its effects in human umbilical vein endothelial cells (HUVECs) induced by IR remains unclear. This study took this as the starting point, aiming to find a potential target for the treatment of related disease. Methods: HUVECs were respectively treated with glucose of 15, 30, 45 mmol/L, or insulin of 1, 2, 3, 4, 5 μmol/L on the basis of high-glucose (33.3 mmol/L). MiR-145 mimics and miR-145 inhibitor were severally transfected into HUVECs with or without IR (4 μmol/L insulin + high-glucose). Quantitative real-time polymerase chain reaction (qRT-PCR) assay determined the miR-145 expression in HUVECs after treatment and transfection. The glucose consumption and glycogen contents of cells were appraised by glucose oxidase-peroxidase and anthranone-sulfuric acid methods, respectively. The apoptotic rates were ascertained using the flow cytometry. The expressions of apoptosis-related indicators Bcl-2 and Bax were analyzed by western blot (WB) and qRT-PCR assays. Results: The expression of miR-145 was increased in IR models and incremental glucose concentrations. The glucose consumption and glycogen content were down-regulated in IR-induced HUVECs, which were enhanced by over-expressed miR-145 but reversed by down-regulation. Moreover, over-expression of miR-145 aggravated the apoptosis of IR-induced HUVECs, while the inhibition of miR-145 had a completely opposite effect. Accordingly, up-regulated miR-145 obviously reduced Bcl-2 level and enhanced Bax expression in IR models, which was contrary to the down-regulated miR-145. Conclusion: Down-regulated miR-145 rescued IR in endothelial cells, which might be a conceivable treatment for IR of endothelial cells.

scholarly journals MicroRNA-132, Delivered by Mesenchymal Stem Cell-Derived Exosomes, Promote Angiogenesis in Myocardial Infarction

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Teng Ma ◽  
Yueqiu Chen ◽  
Yihuan Chen ◽  
Qingyou Meng ◽  
Jiacheng Sun ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Endothelial Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Target Gene ◽  
Tube Formation ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Ischemic Diseases

Background. To cure ischemic diseases, angiogenesis needs to be improved by various strategies in ischemic area. Considering that microRNA-132 (miR-132) regulates endothelial cell behavior during angiogenesis and the safe and efficacious delivery of microRNAs in vivo is rarely achieved, an ideal vehicle for miR-132 delivery could bring the promise for ischemic diseases. As a natural carrier of biological molecules, exosomes are more and more developed as an ideal vehicle for miRNA transfer. Meanwhile, mesenchymal stem cells could release large amounts of exosomes. Thus, this study aimed to investigate whether MSC-derived exosomes can be used for miR-132 delivery in the treatment of myocardial ischemia. Methods. MSC-derived exosomes were electroporated with miR-132 mimics and inhibitors. After electroporation, miR-132 exosomes were labelled with DiI and added to HUVECs. Internalization of DiI-labelled exosomes was examined by fluorescent microscopy. Expression levels of miR-132 in exosomes and HUVECs were quantified by real-time PCR. The mRNA levels of miR-132 target gene RASA1 in HUVECs were quantified by real-time PCR. Luciferase reporter assay was performed to examine the targeting relationship between miR-132 and RASA1. The effects of miR-132 exosomes on the angiogenic ability of endothelial cells were evaluated by tube formation assay. Matrigel plug assay and myocardial infarction model were used to determine whether miR-132 exosomes can promote angiogenesis in vivo. Results. miR-132 mimics were effectively electroporated and highly detected in MSC-derived exosomes. The expression level of miR-132 was high in HUVECs preincubated with miR-132 mimic-electroporated exosomes and low in HUVECs preincubated with miR-132 inhibitor-electroporated exosomes. The expression level of RASA1, miR-132 target gene, was reversely correlated with miR-132 expression in HUVECs pretreated with exosomes. Luciferase reporter assay further confirmed that RASA1 was a direct target of miR-132. Exosomes loaded with miR-132, as a vehicle for miRNA transfer, significantly increased tube formation of endothelial cells. Moreover, subcutaneous injection of HUVECs pretreated with miR-132 exosomes in nude mice significantly increased their angiogenesis capacity in vivo. In addition, transplantation of miR-132 exosomes in the ischemic hearts of mice markedly enhanced the neovascularization in the peri-infarct zone and preserved heart functions. Conclusions. The findings suggest that the export of miR-132 via MSC-derived exosomes represents a novel strategy to enhance angiogenesis in ischemic diseases.

scholarly journals Adaptive induction of NF-E2-related factor-2-driven antioxidant genes in endothelial cells in response to hyperglycemia

2011 ◽  
Vol 300 (4) ◽  
pp. H1133-H1140 ◽  
Author(s):  
Zoltan Ungvari ◽  
Lora Bailey-Downs ◽  
Tripti Gautam ◽  
Rosario Jimenez ◽  
Gyorgy Losonczy ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
High Glucose ◽  
Target Genes ◽  
Cell Model ◽  
Luciferase Reporter ◽  
Standard Diet ◽  
Related Factor ◽  
Antioxidant Genes ◽  
Gene Assay

Hyperglycemia in diabetes mellitus promotes oxidative stress in endothelial cells, which contributes to development of cardiovascular diseases. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a transcription factor activated by oxidative stress that regulates expression of numerous reactive oxygen species (ROS) detoxifying and antioxidant genes. This study was designed to elucidate the homeostatic role of adaptive induction of Nrf2-driven free radical detoxification mechanisms in endothelial protection under diabetic conditions. Using a Nrf2/antioxidant response element (ARE)-driven luciferase reporter gene assay we found that in a cultured coronary arterial endothelial cell model hyperglycemia (10–30 mmol/l glucose) significantly increases transcriptional activity of Nrf2 and upregulates the expression of the Nrf2 target genes NQO1, GCLC, and HMOX1. These effects of high glucose were significantly attenuated by small interfering RNA (siRNA) downregulation of Nrf2 or overexpression of Keap-1, which inactivates Nrf2. High-glucose-induced upregulation of NQO1, GCLC, and HMOX1 was also prevented by pretreatment with polyethylene glycol (PEG)-catalase or N-acetylcysteine, whereas administration of H2O2 mimicked the effect of high glucose. To test the effects of metabolic stress in vivo, Nrf2+/+ and Nrf2−/− mice were fed a high-fat diet (HFD). HFD elicited significant increases in mRNA expression of Gclc and Hmox1 in aortas of Nrf2+/+ mice, but not Nrf2−/− mice, compared with respective standard diet-fed control mice. Additionally, HFD-induced increases in vascular ROS levels were significantly greater in Nrf2−/− than Nrf2+/+ mice. HFD-induced endothelial dysfunction was more severe in Nrf2−/− mice, as shown by the significantly diminished acetylcholine-induced relaxation of aorta of these animals compared with HFD-fed Nrf2+/+ mice. Our results suggest that adaptive activation of the Nrf2/ARE pathway confers endothelial protection under diabetic conditions.

Sign in / Sign up

Export Citation Format

Share Document