scholarly journals miR-136 Modulates TGF-β1-Induced Proliferation Arrest by Targeting PPP2R2A in Keratinocytes

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Dianbao Zhang ◽  
Jing Wang ◽  
Zhe Wang ◽  
Tao Zhang ◽  
Ping Shi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Skin Wound ◽  
Luciferase Reporter ◽  
Epidermal Keratinocytes ◽  
Dependent Manner ◽  
Hacat Cells ◽  
Skin Wound Healing ◽  
Human Epidermal Keratinocytes ◽  
Reporter Assays ◽  
Normal Human

Keratinocytes proliferation is critical for the capacity to heal wounds and accumulating evidences have proved that dysregulation of microRNAs is involved in proliferation of keratinocytes. However, the molecular mechanisms remain to be completely elucidated. Here, we show that miR-136 was significantly decreased by TGF-β1 treatment in HaCaT cells and normal human epidermal keratinocytes (NHEK), and it was a Smad3-dependent manner. By cell proliferation assay and cell cycle analysis, we found that reintroduction of miR-136 by transfection, as well as PPP2R2A silencing, counteracted TGF-β-induced proliferation arrest in HaCaT cells. Further, PPP2R2A was verified as a direct target of miR-136 by dual-luciferase reporter assays and Western blotting. These data suggest that miR-136 may play an important role during TGF-β1-induced proliferation arrest by targeting PPP2R2A in keratinocytes, which might represent a potential target for improving skin wound healing.

scholarly journals Indirubin attenuates mouse psoriasis-like skin lesion in a CD274-dependent manner: an achievement of RNA sequencing

2018 ◽  
Vol 38 (6) ◽  
Author(s):  
Xiaochun Xue ◽  
Jianhua Wu ◽  
Junhui Li ◽  
Jianguo Xu ◽  
Haiying Dai ◽  
...  
Keyword(s):  
Network Analysis ◽  
Skin Lesion ◽  
Candidate Genes ◽  
Rna Sequencing ◽  
Molecular Mechanisms ◽  
Regulatory Mechanisms ◽  
Epidermal Keratinocytes ◽  
Dependent Manner ◽  
Human Epidermal Keratinocytes ◽  
Ifn Γ

It was previously reported that the expression of CD274 was down-regulated in psoriatic epidermis, leading to immune disorders of psoriasis. However, the regulatory mechanisms of CD274 were rarely elucidated. We aimed to explore the regulatory mechanisms of CD274. Skin samples were collected from 18 patients with psoriasis vulgaris and 9 healthy participants for RNA sequencing. Candidate genes were chosen based on degree and k-core difference of genes in the co-expression network. The relations between candidate genes and CD274 were validated by flow cytometry and real-time PCR in primary human epidermal keratinocytes. The therapeutic effect of indirubin was assessed in an imiquimod-treated mouse model. Interferon-γ (IFN-γ), cyclin-dependent kinase (CDK) 1, Toll-like receptor 3 (TLR3), TLR4 and interleukin (IL)-17A were considered as candidate genes. In primary human epidermal keratinocytes, the level of CD274 was obviously increased under the stimulation of IFN-γ and CDK1 inhibitor (indirubin), independent of TLR4, TLR3 or IL-17A. Indirubin alleviated the severity of psoriatic mice in a CD274-dependent manner. Co-expression network analysis served as an effective method for the exploration of molecular mechanisms. We demonstrated for the first time that CD274 was the regulator of indirubin-mediated effect on mouse psoriasis-like skin lesion based on co-expression network analysis, contributing to the alleviation of mouse psoriasis-like skin lesion.

scholarly journals MicroRNA-26a inhibits wound healing through decreased keratinocytes migration by regulating ITGA5 through PI3K/AKT signaling pathway

2020 ◽  
Vol 40 (9) ◽  
Author(s):  
Zhongping Jiang ◽  
Jie Wei ◽  
Weize Yang ◽  
Wen Li ◽  
Feng Liu ◽  
...  
Keyword(s):  
Wound Healing ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Luciferase Reporter ◽  
Epidermal Keratinocytes ◽  
Dependent Manner ◽  
Hacat Cells ◽  
Akt Signaling Pathway ◽  
Tgf Β1

Abstract Background: Keratinocyte migration is essential for skin wound healing and recent studies demonstrated that microRNAs (miRNAs) are involved in the differentiation, migration and apoptosis in keratinocytes. However, the function of miR-26a in wound healing remains to be largely explored. Methods: Northern blot and quantitative reverse transcriptase PCR (qRT-PCR) were used to detect the miR-26a expression and Western blot was used to detect integrin α-5 (ITGA5), phosphatidylinositol-3-kinase (PI3K), p-PI3K, protein kinase B (AKT) and p-AKT protein expression in immortalized human keratinocyte cell line HaCaT and normal human epidermal keratinocytes (NHEK) after 2 ng/ml transforming growth factor-β1 (TGF-β1) treatment for 0, 6, 12 and 24 h. Transwell assay and Wound healing assay were introduced to measure the cell migration of HaCaT cells. TargetScan online database, luciferase reporter assay and RNA immunoprecipitation (RIP) were employed to confirm the relationship between miR-26a and ITGA5. Results: The RNA expression of miR-26a was down-regulated and ITGA5 protein expression was up-regulated by TGF-β1 treatment in HaCaT and NHEK cells in a time-dependent manner. MiR-26a overexpression inhibited the migration of HaCaT cells induced by TGF-β1 while miR-26a inhibitor enhanced the migration. ITGA5 was a downstream target mRNA and regulated by miR-26a. ITGA5 overexpression reversed the inhibitory effect of miR-26a on migration in HaCaT, while ITGA5 knockdown attenuated the stimulative effect of miR-26a inhibitor in HaCaT via PI3K/AKT signaling pathway. Conclusion: MiR-26a overexpression inhibited TGF-β1 induced HaCaT cells migration via down-regulating ITGA5 through activating the PI3K/AKT signaling pathway.

scholarly journals Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250663
Author(s):  
Yasuko Yoshioka ◽  
Yayoi Kamata ◽  
Mitsutoshi Tominaga ◽  
Yoshie Umehara ◽  
Ikuyo Yoshida ◽  
...  
Keyword(s):  
Skin Diseases ◽  
Epidermal Keratinocytes ◽  
Dependent Manner ◽  
Semaphorin 3A ◽  
Human Epidermal Keratinocytes ◽  
State Dependent ◽  
Gene Assay ◽  
Normal Human ◽  
Culture Supernatants ◽  
Relative Luciferase Activity

In a disease-state-dependent manner, the histamine-resistant itch in dry skin-based skin diseases such as atopic dermatitis (AD) and xerosis is mainly due to hyperinnervation in the epidermis. Semaphorin 3A (Sema3A) is a nerve repulsion factor expressed in keratinocytes and it suppresses nerve fiber elongation in the epidermis. Our previous studies have shown that Sema3A ointment inhibits epidermal hyperinnervation and scratching behavior and improves dermatitis scores in AD model mice. Therefore, we consider Sema3A as a key therapeutic target for improving histamine-resistant itch in AD and xerosis. This study was designed to screen a library of herbal plant extracts to discover compounds with potential to induce Sema3A in normal human epidermal keratinocytes (NHEKs) using a reporter gene assay, so that positive samples were found. Among the positive samples, only the extract of S. baicalensis was found to consistently increase Sema3A levels in cultured NHEKs in assays using quantitative real-time PCR and ELISA. In evaluation of reconstituted human epidermis models, the level of Sema3A protein in culture supernatants significantly increased by application of the extract of S. baicalensis. In addition, we investigated which components in the extract of S. baicalensis contributed to Sema3A induction and found that baicalin and baicalein markedly increased the relative luciferase activity, and that baicalein had higher induction activity than baicalin. Thus, these findings suggest that S. baicalensis extract and its compounds, baicalin and baicalein, may be promising candidates for improving histamine-resistant itch via the induction of Sema3A expression in epidermal keratinocytes.

scholarly journals The advanced lipoxidation end product precursor malondialdehyde induces IL-17E expression and skews lymphocytes to the Th17 subset

2015 ◽  
Vol 20 (4) ◽  
Author(s):  
Kartiga Natarajan ◽  
Gokila Devi Mathialagan ◽  
Somasundaram Raghavan ◽  
Narkunaraja Shanmugam
Keyword(s):  
T Cells ◽  
Molecular Mechanisms ◽  
Human Peripheral Blood ◽  
Peripheral Blood Lymphocytes ◽  
Luciferase Reporter ◽  
Diabetic Patients ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Jurkat T Cells ◽  
Reporter Assays

AbstractMalondialdehyde (MDA) is a highly reactive endogenous product of thromboxane synthesis in the prostagland and lipid peroxidation by reactive oxygen species. Elevated MDA levels occur in diabetes and atherosclerotic plaques. The aim of this study was to examine the molecular mechanisms of MDA-induced IL-17E cytokine expression and its effect on T-cell differentiation. Real-time PCR, RT-PCR and ELISA were used to assess the expression of IL-17 family cytokines in Jurkat T-cells and human peripheral blood lymphocytes (PBLCs) from diabetic subjects. Luciferase reporter assays were used for the promoter activation study. Pharmacological inhibitors were used for signaling pathway experiments. FACS analyses were used to measure the Th1, Th2 and Th17 subset levels. MDA induced significant (2- to 3-fold; p < 0.01) generation of IL-17E mRNA in a dose- and time-dependent manner in Jurkat T-cells and PBLCs. Elevated IL-17E mRNA levels were found in the lymphocytes from diabetic subjects. The increased IL-17E protein and mRNA levels correlate well with serum MDA levels from diabetic patients. Transient transfection of plasmid containing the minimum IL-17E promoter region (pIL-17E-Luc) showed a significant (2-fold; p < 0.01) increase in luciferase activity. Pretreatment of lymphocytes with pharmacological inhibitors showed the involvement of antioxidant, NF-ƙB, p38MAPK, PKC and ERK signaling pathways. Quantification of the Th1, Th2 and Th17 cell population in PBLCs via FACS analyses revealed an increase in the Th17 subset. These results show that MDA transcriptionally upregulates the expression of IL-17E in lymphocytes and alters lymphocyte differentiation towards the pathogenic Th17 subset.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

scholarly journals TEAD4 modulated LncRNA MNX1-AS1 contributes to gastric cancer progression partly through suppressing BTG2 and activating BCL2

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
You Shuai ◽  
Zhonghua Ma ◽  
Weitao Liu ◽  
Tao Yu ◽  
Changsheng Yan ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Mechanistic Model ◽  
Ectopic Expression ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Tissue Samples ◽  
Reporter Assays

Abstract Background Gastric cancer (GC) is the third leading cause of cancer-related mortality globally. Long noncoding RNAs (lncRNAs) are dysregulated in obvious malignancies including GC and exploring the regulatory mechanisms underlying their expression is an attractive research area. However, these molecular mechanisms require further clarification, especially upstream mechanisms. Methods LncRNA MNX1-AS1 expression in GC tissue samples was investigated via microarray analysis and further determined in a cohort of GC tissues via quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. Cell proliferation and flow cytometry assays were performed to confirm the roles of MNX1-AS1 in GC proliferation, cell cycle regulation, and apoptosis. The influence of MNX1-AS1 on GC cell migration and invasion was explored with Transwell assays. A xenograft tumour model was established to verify the effects of MNX1-AS1 on in vivo tumourigenesis. The TEAD4-involved upstream regulatory mechanism of MNX1-AS1 was explored through ChIP and luciferase reporter assays. The mechanistic model of MNX1-AS1 in regulating gene expression was further detected by subcellular fractionation, FISH, RIP, ChIP and luciferase reporter assays. Results It was found that MNX1-AS1 displayed obvious upregulation in GC tissue samples and cell lines, and ectopic expression of MNX1-AS1 predicted poor clinical outcomes for patients with GC. Overexpressed MNX1-AS1 expression promoted proliferation, migration and invasion of GC cells markedly, whereas decreased MNX1-AS1 expression elicited the opposite effects. Consistent with the in vitro results, MNX1-AS1 depletion effectively inhibited the growth of xenograft tumour in vivo. Mechanistically, TEAD4 directly bound the promoter region of MNX1-AS1 and stimulated the transcription of MNX1-AS1. Furthermore, MNX1-AS1 can sponge miR-6785-5p to upregulate the expression of BCL2 in GC cells. Meanwhile, MNX1-AS1 suppressed the transcription of BTG2 by recruiting polycomb repressive complex 2 to BTG2 promoter regions. Conclusions Our findings demonstrate that MNX1-AS1 may be able to serve as a prognostic indicator in GC patients and that TEAD4-activatd MNX1-AS1 can promote GC progression through EZH2/BTG2 and miR-6785-5p/BCL2 axes, implicating it as a novel and potent target for the treatment of GC.

scholarly journals Circular RNA mmu_circ_0005019 inhibits fibrosis of cardiac fibroblasts and reverses electrical remodeling of cardiomyocytes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Na Wu ◽  
Chengying Li ◽  
Bin Xu ◽  
Ying Xiang ◽  
Xiaoyue Jia ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cardiac Fibroblasts ◽  
Circular Rna ◽  
Protective Role ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Candidate Target ◽  
Paired Control ◽  
Reporter Assays ◽  
And Migration

Abstract Background Circular RNA (circRNA) have been reported to play important roles in cardiovascular diseases including myocardial infarction and heart failure. However, the role of circRNA in atrial fibrillation (AF) has rarely been investigated. We recently found a circRNA hsa_circ_0099734 was significantly differentially expressed in the AF patients atrial tissues compared to paired control. We aim to investigate the functional role and molecular mechanisms of mmu_circ_0005019 which is the homologous circRNA in mice of hsa_circ_0099734 in AF. Methods In order to investigate the effect of mmu_circ_0005019 on the proliferation, migration, differentiation into myofibroblasts and expression of collagen of cardiac fibroblasts, and the effect of mmu_circ_0005019 on the apoptosis and expression of Ito, INA and SK3 of cardiomyocytes, gain- and loss-of-function of cell models were established in mice cardiac fibroblasts and HL-1 atrial myocytes. Dual-luciferase reporter assays and RIP were performed to verify the binding effects between mmu_circ_0005019 and its target microRNA (miRNA). Results In cardiac fibroblasts, mmu_circ_0005019 showed inhibitory effects on cell proliferation and migration. In cardiomyocytes, overexpression of mmu_circ_0005019 promoted Kcnd1, Scn5a and Kcnn3 expression. Knockdown of mmu_circ_0005019 inhibited the expression of Kcnd1, Kcnd3, Scn5a and Kcnn3. Mechanistically, mmu_circ_0005019 exerted biological functions by acting as a miR-499-5p sponge to regulate the expression of its target gene Kcnn3. Conclusions Our findings highlight mmu_circ_0005019 played a protective role in AF development and might serve as an attractive candidate target for AF treatment.

scholarly journals Molecular Cloning and Functional Characterization of Three 5-HT Receptor Genes (HTR1B, HTR1E and HTR1F) in Chickens

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 891
Author(s):  
Caiyun Sun ◽  
Yang Qiu ◽  
Qin Ren ◽  
Xiao Zhang ◽  
Baolong Cao ◽  
...  
Keyword(s):  
Serotonin Receptor ◽  
Functional Characterization ◽  
Brain Regions ◽  
Luciferase Reporter ◽  
Molecular Characteristics ◽  
Intracellular Camp ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Serotonin Signaling ◽  
Reporter Assays

The serotonin (5-hydroxytryptamine, 5-HT) signaling system is involved in a variety of physiological functions, including the control of cognition, reward, learning, memory, and vasoconstriction in vertebrates. Contrary to the extensive studies in the mammalian system, little is known about the molecular characteristics of the avian serotonin signaling network. In this study, we cloned and characterized the full-length cDNA of three serotonin receptor genes (HTR1B, HTR1E and HTR1F) in chicken pituitaries. Synteny analyses indicated that HTR1B, HTR1E and HTR1F were highly conserved across vertebrates. Cell-based luciferase reporter assays showed that the three chicken HTRs were functional, capable of binding their natural ligands (5-HT) or selective agonists (CP94253, BRL54443, and LY344864) and inhibiting intracellular cAMP production in a dose-dependent manner. Moreover, activation of these receptors could stimulate the MAPK/ERK signaling cascade. Quantitative real-time PCR analyses revealed that HTR1B, HTR1E and HTR1F were primarily expressed in various brain regions and the pituitary. In cultured chicken pituitary cells, we found that LY344864 could significantly inhibit the secretion of PRL stimulated by vasoactive intestinal peptide (VIP) or forskolin, revealing that HTR1F might be involved in the release of prolactin in chicken. Our findings provide insights into the molecular mechanism and facilitate a better understanding of the serotonergic modulation via HTR1B, HTR1E and HTR1F in avian species.

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