scholarly journals Baicalin inhibits recruitment of GATA1 to the HSD3B2 promoter and reverses hyperandrogenism of PCOS

2019 ◽  
Vol 240 (3) ◽  
pp. 497-507
Author(s):  
Jin Yu ◽  
Yuhuan Liu ◽  
Danying Zhang ◽  
Dongxia Zhai ◽  
Linyi Song ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Polycystic Ovary ◽  
Ovarian Tissue ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Before And After ◽  
Key Enzyme ◽  
H295r Cells ◽  
Underlying Mechanisms ◽  
Androgen Levels

High androgen levels in patients suffering from polycystic ovary syndrome (PCOS) can be effectively reversed if the herb Scutellaria baicalensis is included in traditional Chinese medicine prescriptions. To characterize the effects of baicalin, extracted from S. baicalensis, on androgen biosynthesis in NCI-H295R cells and on hyperandrogenism in PCOS model rats and to elucidate the underlying mechanisms. The optimum concentration and intervention time for baicalin treatment of NCI-H295R cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and ELISA. The functional genes affected by baicalin were studied by gene expression profiling (GEP), and the key genes were identified using a dual luciferase assay, RNA interference technique and genetic mutations. Besides, hyperandrogenic PCOS model rats were induced and confirmed before and after baicalin intervention. As a result, baicalin decreased the testosterone concentrations in a dose- and time-dependent manner in NCI-H295R cells. GEP revealed that 3β-hydroxysteroid dehydrogenase type II (HSD3B2) was the key enzyme of androgen biosynthesis, and baicalin inhibited the expression of HSD3B2 by regulating the binding of transcription factor GATA-binding factor 1 (GATA1) to the HSD3B2 promoter. Hyperandrogenic PCOS model rats treated with baicalin significantly reversed the high androgen levels of serum and the abnormal ovarian status, restored the estrous cyclicity and decreased the expression of HSD3B2 in ovarian. In summary, our data revealed that GATA1 is an important transcription factor activating the HSD3B2 promoter in steroidogenesis, and baicalin will potentially be an effective therapeutic agent for hyperandrogenism in PCOS by inhibiting the recruitment of GATA1 to the HSD3B2 promoter in ovarian tissue.

scholarly journals Specification of the germline by Nanos-dependent down-regulation of the somatic synMuvB transcription factor LIN-15B

2017 ◽  
Author(s):  
Chih-Yung S. Lee ◽  
Tu Lu ◽  
Geraldine Seydoux
Keyword(s):  
Transcription Factor ◽  
Germ Cells ◽  
Rna Binding ◽  
Primordial Germ Cells ◽  
Somatic Cells ◽  
Rna Binding Protein ◽  
Dependent Manner ◽  
Embryonic Cells ◽  
C Elegans ◽  
Underlying Mechanisms

AbstractThe Nanos RNA-binding protein has been implicated in the specification of primordial germ cells (PGCs) in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of PGCs lacking the nanos homologues nos-1 and nos-2 iC. elegans. nos-1nos-2 PGCs fail to silence hundreds of genes normally expressed in oocytes and somatic cells, a phenotype reminiscent of PGCs lacking the repressive PRC2 complex. The nos-1nos-2 phenotype depends on LIN-15B, a broadly expressed synMuvB class transcription factor known to antagonize PRC2 activity in somatic cells. LIN-15B is maternally-inherited by all embryonic cells and is down-regulated specifically in PGCs in a nos-1nos-2-dependent manner. Consistent with LIN-15B being a critical target of Nanos regulation, inactivation of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These studies demonstrate a central role for Nanos in reprogramming the transcriptome of PGCs away from an oocyte/somatic fate by down-regulating an antagonist of PRC2 activity.

Abstract 101: The Role of MicroRNAs in the Regulation of Human Aldosterone Synthase Gene Expression

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Shreekrishna Maharjan ◽  
Brahmaraju Mopidevi ◽  
Varunkumar G. Pandey ◽  
Sudhir Jain ◽  
Ashok Kumar
Keyword(s):  
Gene Expression ◽  
Luciferase Activity ◽  
Mrna Level ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Aldosterone Synthase ◽  
Small Noncoding Rnas ◽  
Molecular Variants ◽  
Key Enzyme ◽  
H295r Cells

The human aldosterone synthase ( hCYP11B2 ) gene encodes a key enzyme for the biosynthesis of aldosterone which is part of the renin-angiotensin-aldosterone system. Molecular variants in this gene have been associated with essential hypertension. MicroRNAs (miRNAs) are a family of small, noncoding RNAs that bind to the 3’-untranslated region (3’UTR) of the gene and regulate its expression. Our main objective in this study was to investigate whether miRNAs bind at the polymorphic sites present in the 3’UTR of hCYP11B2 gene and modulate its expression. Previous studies have suggested that -344T/C (rs1799998) polymorphism in the hCYP11B2 promoter is associated with hypertension. Here, we compared nucleotide sequences of the promoter and 3’UTR of hCYP11B2 gene from hypertensive and normotensive subjects and found that rs1799998 is in linkage disequilibrium with 735G/A (rs28491316). Thus, variant -344T almost always occurs with variant 735G; whereas variant -344C almost always occurs with variant 735A. Bioinformatic algorithms of the full-length 3’UTR for miRNAs revealed that hsa-miR-766 was possibly binding at the 735G allele. To examine if this miRNA regulates hCYP11B2 gene expression, reporter constructs were made where luciferase gene was attached to 1.4 kb of hCYP11B2 3’UTR containing either 735G, or 735A and co-transfected into HEK293 cells with either pre-miR-766 or anti-pre-miR-766. Our results showed that pre-miR-766 reduced luciferase activity in a dose dependent manner in the presence of 735G allele. There was a significant decrease (65+2%) in the luciferase activity with pre-miR-766 (50 nM) in the presence of 735G allele, whereas it was insignificant (4+5%) in the presence of 735A allele. In H295R cells that express hCYP11B2 gene, pre-miR-766 (50nM) reduced luciferase activity by 81+1% in the presence of 735G allele. We also showed that transfection of pre-mir-766 reduced hCYP11B2 mRNA level by about 23.6% in H295R cells. In corroboration with these results, anti-pre-miR-766 did not decrease luciferase activity in presence of either allele in both cell cultures. Our results suggest that hCYP11B2 gene expression may be regulated by differential binding of miR-766 at 735G/A polymorphism present in its 3’UTR.

scholarly journals UCA1 and microRNA-18a signaling pathway mediates the irisin-lowering effect of met in the management of polycystic ovary syndrome (PCOS)

2021 ◽  
Author(s):  
Wei Wang ◽  
Tian Hua ◽  
Xiaodong Li ◽  
Xinxian Zhang ◽  
Wei Hao
Keyword(s):  
Polycystic Ovary Syndrome ◽  
Real Time ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Polycystic Ovary ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Ovary Syndrome ◽  
Lowering Effect ◽  
Pcos Group

IntroductionThe present study aimed to clarify the underlying mechanism of metformin (met) in the management of polycystic ovary syndrome (PCOS) and to explore the role of UCA1/ microRNA-18a signaling pathway in the control of PCOS.Material and methodsReal-time PCR was performed to compare the level of irisin, blood glucose, UCA1 and miR-18a among PCOS, PCOS + Met, and control groups using area under curve (AUC) values. In-silicon analysis and luciferase assay were performed to explore the regulatory relationship among UCA1, miR-18a and irisin. Real-time PCR and Western-blot analysis were carried out to detect the effect of met on the expression of UCA1, miR-18a and irisin.ResultsAUC of UCA1 was the highest while AUC of irisin was the lowest. Also, irisin and UCA1 levels in the PCOS group were much higher than those in the PCOS + Met group, while miR-18a level in the PCOS group was much lower than PCOS + Met group. Through the luciferase assay, miR-18a was proved to directly bound to irisin 3’UTR. Additionally, irisin was identified to be a target gene of miR-18a. Finally, the treatment with met at the increasing concentration reduced the level of UCA1 and irisin but increased the level of miR-18a in a dose dependent manner.ConclusionsIn the management of PCOS, the irisin-lowering effect of met is regulated by the UCA1/miR-18a/RhoB signaling pathway.

scholarly journals Ethanol Extract ofLycopus lucidusTurcz. ex Benth Inhibits Metastasis by Downregulation of Runx-2 in Mouse Colon Cancer Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Kwang-Youn Kim ◽  
Tae Woo Oh ◽  
Jin-Yeul Ma ◽  
Kwang-Il Park
Keyword(s):  
Colon Cancer ◽  
Transcription Factor ◽  
Blood Stasis ◽  
Ethanol Extract ◽  
Migration And Invasion ◽  
Dependent Manner ◽  
Mouse Colon ◽  
Related Transcription Factor ◽  
Underlying Mechanisms ◽  
Ethanol Extracts

Lycopus lucidusTurcz. ex Benth (LT) has been broadly used as a traditional medicinal herb in Asia including Korea, China, and Japan due to its noted ability to promote blood circulation and remove blood stasis. However, its anticancer mechanism is not understood. This study aims to elucidate the effects of ethanol extracts of LT (ELT) relative to the role of Runt-related transcription factor- (Runx-) 2 in the invasive and metastatic potentials of mouse colon cancer to determine the underlying mechanisms involved. ELT was evaluated for the antimetastasis activity using CT-26 colon cancer using wound healing, transwell matrigel, and western blot analysis. We used Runx-2-specific siRNA to further determine the relationship between Runx-2 and matrix metalloprotease- (MMP-) 9 in the migration and invasion of CT-26 cells. Runx-2 was first demonstrated to be a transcription factor that plays a remarkable role in diverse biological processes of chondrocytes and osteoblasts, but recently, Runx-2 has been reported to be associated with the progression of certain human cancers. ELT was not altered in its effects on growth inhibition. However, ELT significantly inhibited wound closure and cell invasion in a dose-dependent manner. ELT decreased the metastasis by regulating the activity of MMP-9 and Runx-2 at the translational levels. Our results demonstrate that ELT decreases metastasis by inhibiting the Runx-2–MMP-9 axis. We suggest that it can be used as a novel agent in therapeutic strategies for combating colon cancer.

scholarly journals Lipopolysaccharide regulates thymic stromal lymphopoietin expression via TLR4/MAPK/Akt/NF-κB signaling pathways in nasal fibroblasts: differential inhibitory effects of macrolide and corticosteroid

2020 ◽  
Author(s):  
Ju-Hyung Kang ◽  
Hyun-Woo Yang ◽  
Joo-Hoo Park ◽  
Jae-Min Shin ◽  
Tae-Hoon Kim ◽  
...  
Keyword(s):  
Western Blot ◽  
Inferior Turbinate ◽  
Thymic Stromal Lymphopoietin ◽  
Luciferase Assay ◽  
Treatment Modalities ◽  
Dependent Manner ◽  
Tlr4 Expression ◽  
New Treatment ◽  
Underlying Mechanisms ◽  
Sinonasal Mucosa

Abstract BackgroundChronic rhinosinusitis (CRS) is inflammatory disease of sinonasal mucosa. Thymic stromal lymphopoietin (TSLP) is associated with Th-2 response and induced by pathogen, allergen, Toll-like receptor (TLR) ligands, and cytokines. Fibroblasts have known to modulators of wound healing, from inflammation to tissue remodeling. We examined effect of lipopolysaccharide (LPS) on TSLP production and underlying mechanisms. We aimed to determine whether effects of commonly used medications in CRS, corticosteroids and macrolides, are related to LPS-induced TSLP in nasal fibroblasts.ResultsFibroblasts were isolated from inferior turbinate tissues of CRS patients. TSLP and TLR4 expression was determined by RT-PCR, western blot, ELISA, and immunofluorescence staining. MAPK, Akt, and NF-κB phosphorylation was determined by western blot and/or luciferase assay. LPS increased TSLP expression in a dose- and time-dependent manner. LPS antagonist and corticosteroids inhibited TLR4 expression in LPS-stimulated fibroblasts. LPS-RS, macrolides, corticosteroids, and specific inhibitors suppressed LPS-induced alterations. Ex vivo culture showed similar results.ConclusionsLPS induces TSLP production via TLR4, MAPK, Akt, and NF-κB pathways. Effects of corticosteroids and macrolides are related to LPS-induced TSLP expression. We would explore new treatment modalities targeting LPS-induced TSLP production that could replace current usage of corticosteroid and macrolides in treatment of CRS.

scholarly journals Hoxa5 Promotes Adipose Differentiation via Increasing DNA Methylation Level and Inhibiting PKA/HSL Signal Pathway in Mice

2018 ◽  
Vol 45 (3) ◽  
pp. 1023-1033 ◽  
Author(s):  
Weina Cao ◽  
Yatao Xu ◽  
Dan Luo ◽  
Muhammad Saeed ◽  
Chao Sun
Keyword(s):  
Transcription Factor ◽  
Adipose Tissue ◽  
Transcriptional Activation ◽  
Methylation Level ◽  
Type Ii Diabetes ◽  
Luciferase Assay ◽  
Type Ii ◽  
Underlying Mechanisms

Background/Aims: Impaired adipogenesis may be the underlying cause in the development of obesity and type II diabetes. Mechanistically, the family of Homeobox transcription factors is implicated in the regulation of adipocyte fate. Hoxa5 is highly expressed in adipocytes, and its mRNA expression is decreased during differentiation. However, the function of Hoxa5 in adipose tissue has been poorly understood. The aim of this study is to unveil the role of Hoxa5 on adipocyte differentiation and its underlying mechanisms. Methods: Quantitative real-time PCR (qPCR) and western blot were performed to determine Hoxa5 expression in primary adipocytes and in adipose tissues from mice. Lipid accumulation was evaluated by bodipy staining. Dual luciferase assay was applied to explore the transcription factor of Hoxa5 and the transcriptional target gene modulated by Hoxa5. All measurements were performed at least for three times at least. Results: A significant reduction of Hoxa5 expression was observed in adipose tissue of High Fat Diet (HFD) induced obesity mice. We determined Hoxa5 increased adipocytes differentiation and mitochondrial biogenesis in adipocytes in vitro. CEBPβ was determined a transcription factor of Hoxa5 and inhibited methylation level of Hoxa5 by combining on the promoter of Hoxa5. Importantly, we found Fabp4, a known positive regulator of adipocytes differentiation, was transcriptional activation by Hoxa5. In addition, Hoxa5 promotes adipocytes differentiation by inhibiting PKA/HSL pathway. Conclusion: Our study demonstrated the promoting role of Hoxa5 in adipocytes differentiation and therefore bringing a new therapeutic mean to the treatment of obesity and type II diabetes.

scholarly journals Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Takehiro Ko ◽  
Yutaka Kakizoe ◽  
Naoki Wakida ◽  
Manabu Hayata ◽  
Kohei Uchimura ◽  
...  
Keyword(s):  
Serine Protease ◽  
Channel Blocker ◽  
Systemic Circulation ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Kinase C ◽  
Aldosterone Production ◽  
H295r Cells ◽  
Dose Dependent

A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells). Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC) inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

scholarly journals Total Flavonoids from Leaves of Carya Cathayensis Ameliorate Renal Fibrosis via the miR-21/Smad7 Signaling Pathway

2018 ◽  
Vol 49 (4) ◽  
pp. 1551-1563 ◽  
Author(s):  
Xiaoli Wu ◽  
Xiaoqiang Ding ◽  
Zhishan Ding ◽  
Ping  Jia
Keyword(s):  
Signaling Pathway ◽  
Renal Fibrosis ◽  
Luciferase Assay ◽  
Total Flavonoids ◽  
Dependent Manner ◽  
Collagen Iii ◽  
Carya Cathayensis ◽  
Underlying Mechanisms ◽  
Tgf Β1

Background/Aims: Renal tubulointerstitial fibrosis is the most common pathway of progressive kidney injury, leading to end-stage renal disease. At present, no effective prophylactic treatment method is available. This study investigated the anti-fibrotic effects of total flavonoids (TFs) extracted from leaves of Carya Cathayensis in vivo and in vitro, and explored the underlying mechanisms. Methods: Anti-fibrotic effects of TFs were measured using a mouse model of unilateral ureteral obstruction (UUO) and in transforming growth factor-β1 (TGF-β1)-treated mouse tubular epithelial cells (mTECs). mRNA expression and protein levels of Collagen I, Collagen III, and α-smooth muscle actin (α-SMA) were also tested by real-time reverse transcription PCR and western blot analysis. To elucidate the underlying mechanisms, expression of miR-21 was examined in mTECs treated with TFs using miR-21 mimics transfected into mTECs before TGF-β1 and TFs treatment. Regulation of mothers against decapentaplegic homolog (Smad) signaling by miR-21 was subsequently validated via overexpression and deletion of miR-21 followed by a luciferase assay. Results: TFs treatment attenuated renal fibrosis, and inhibited expression of collagens and α-SMA in the kidneys of mice subjected to UUO. In vitro, the TFs significantly decreased expression of fibrotic markers in TGF-β1-treated mTECs. Moreover, TFs reduced miR-21 expression in a time- and dose-dependent manner in mTECs, increased expression of Smad7, and decreased phosphorylation of Smad3. Treatment with miR-21 mimics abolished the anti-fibrotic effects of the TFs on the TGF-β1-treated mTECs. In addition, genetic deletion of miR-21 upregulated expression of Smad7 and suppressed phosphorylation of Smad3, attenuating renal fibrosis in mice. Bioinformatics predictions revealed the potential binding site of miR-21 in the 3′-untranslated region of Smad7, and this was further confirmed by the luciferase assay. Conclusion: TFs ameliorate renal fibrosis via a miR-21/Smad7 signaling pathway, indicating a potential therapy for the prevention of renal fibrosis.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

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