Quantification of the expression levels of lysosomal cysteine proteinases in purified human osteoclastic cells by competitive RT-PCR

Objectives. To observe the role of miR-518d in pregnant women with gestational diabetes mellitus (GDM) and its adjusting effects on PPARα and to explore the regulatory mechanisms of the NF-κB pathway in the development and progression of GDM. Methods. Placenta tissues and peripheral plasma were obtained from pregnant women with normal pregnancy and GDM, respectively, followed by the detections of miR-518d contents by RT-PCR and the expression levels of inflammatory factors using ELISA. Human placenta trophoblast cells (HTR8/SVneo) were cultured under the conditions of physiological glucose (PG group) and high glucose level (HG group). Cells in the HG group were transfected with miR-518d control, mimics, and inhibitors and were separately administered with a PPARα-specific antagonist (GW6471) and PPARα siRNA, and cells were divided into the following groups: HG+miR-518d control group (HGNC group), HG+miR-518d mimic group (HGM group), HG+miR-518d inhibitor group (HGI group), HGI+PPARα antagonist group, and HGI+PPARα siRNA group. The relative expression levels of miR-518d, PPARα, and its downstream genes and NF-κB signalling pathway-related genes were detected by RT-PCR and Western blotting. The contents of inflammatory factors were examined by Western blotting. A dual-luciferase report assay was performed to validate the correlations between miR-518d and PPARα. In this study, mouse GDM models were established to further prove the previous hypothesis with an in vivo experiment. A total of 40 C57BL/6J mice were randomly divided into the following groups: normal diet group (ControlMs), GDM group (GDMMs group), GDM+miR-518d antagomir group, and GDM+miR-518d antagomir+PPARα antagonist group. The mouse model of GDM was established by feeding with combined high-sugar and high-saturated fat diet and injecting streptozotocin (STZ) after 15-day feeding. Female and male mice were cocaged in the number ratio of 2 : 1, and the evidence of vaginal suppository detected in female mice was marked as D0 of pregnancy. The contents of total cholesterol (CH), triglyceride (TG), fast glucose, and insulin (INS) were examined using ELISA, followed by the evaluation of insulin resistance (IR). The related expression levels were also detected with the above methods shown in the previous cell culture. Results. miR-518d has a high expression level in placentas with GDM. As the target gene of miR-518d, PPARα was downregulated with the increased levels of miR-518d. When GDM occurs, inflammatory responses were elevated, stimulating the nuclear transport process of NF-κB. Activated NF-κB triggered the phosphorylation of IKKβ and IκBα. Conclusions. High expression of miR-518d was observed in the development of GDM. In this study, we validated that miR-518d negatively regulates the expression of PPARα and triggers the nuclear transport process of NF-κB and phosphorylation of pathway-associated proteins leading to an inflammatory response and the development of GDM.

Download Full-text

Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0320449 ◽

2004 ◽

Vol 32 (2) ◽

pp. 449-466 ◽

Cited By ~ 84

Author(s):

S Bauersachs ◽

S Rehfeld ◽

SE Ulbrich ◽

S Mallok ◽

K Prelle ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Expression Profiles ◽

Cdna Array ◽

Oestrous Cycle ◽

Cdna Clones ◽

Rt Pcr ◽

Expression Levels ◽

Starting Point ◽

Oviduct Epithelial Cells

The oviduct epithelium undergoes marked morphological and functional changes during the oestrous cycle. To study these changes at the level of the transcriptome we did a systematic gene expression analysis of bovine oviduct epithelial cells at oestrus and dioestrus using a combination of subtracted cDNA libraries and cDNA array hybridisation. A total of 3072 cDNA clones of two subtracted libraries were analysed by array hybridisation with cDNA probes derived from six cyclic heifers, three of them slaughtered at oestrus and three at dioestrus. Sequencing of cDNAs showing significant differences in their expression levels revealed 77 different cDNAs. Thirty-seven were expressed at a higher level at oestrus, for the other 40 genes expression levels were higher at dioestrus. The identified genes represented a variety of functional classes. During oestrus especially genes involved in the regulation of protein secretion and protein modification, and mRNAs of secreted proteins, were up-regulated, whereas during dioestrus particularly transcripts of genes involved in transcription regulation showed a slight up-regulation. The concentrations of seven selected transcripts were quantified by real-time RT-PCR to validate the cDNA array hybridisation data. For all seven transcripts, RT-PCR results were in excellent correlation (r>0.92) with the results obtained by array hybridisation. Our study is the first to analyse changes in gene expression profiles of bovine oviduct epithelial cells during different stages of the oestrous cycle, providing a starting point for the clarification of the key transcriptome changes in these cells.

Download Full-text

Chronic stress promotes gastric cancer progression and metastasis: an essential role for ADRB2

Cell Death and Disease ◽

10.1038/s41419-019-2030-2 ◽

2019 ◽

Vol 10 (11) ◽

Cited By ~ 13

Author(s):

Xuan Zhang ◽

Yi Zhang ◽

Zhongyuan He ◽

Kai Yin ◽

Bowen Li ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Proliferation ◽

Chronic Stress ◽

Adrenergic Receptor ◽

Tumour Growth ◽

Mapk Pathway ◽

Rt Pcr ◽

Expression Levels

Abstract An increasing number of studies indicate that adrenergic signalling plays a fundamental role in chronic stress-induced tumour progression and metastasis. However, its function in gastric cancer (GC) and its potential mechanisms remain unknown. The expression levels of β-adrenergic receptor (ADRB) in GC cell lines were examined by using real-time polymerase chain reaction (RT-PCR) and western blotting. The effects of β2 adrenergic receptor (ADRB2) activation and blockade were investigated in vitro in GC cells by using proliferation, migration, invasion, cell cycle and apoptosis assays. Chronic restraint stress (CRS) increased the plasma levels of catecholamines and cortisol and also induced progression and metastasis of GC in vivo. Furthermore, immunohistochemical staining and a TUNEL assay were employed to observe the regulation of cell viability in vivo. The expression levels of ADRB2 in 100 human GC samples were measured by RT-PCR and immunohistochemistry. The stress hormones epinephrine and norepinephrine significantly accelerated GC cell proliferation, invasion and viability in culture, as well as tumour growth in vivo. These effects were reversed by the ADRB antagonists propranolol and ICI118,551 (an ADRB2-specific antagonist). Moreover, the selective ADRB1 antagonist atenolol had almost no effect on tumour cell proliferation and invasion in vitro and in vivo. ADRB2 antagonists suppressed proliferation, invasion and metastasis by inhibiting the ERK1/2-JNK-MAPK pathway and transcription factors, such as NF-κB, AP-1, CREB and STAT3. Analysis of xenograft models using GC cells revealed that ADRB2 antagonists significantly inhibited tumour growth and metastasis, and chronic stress antagonized these inhibitory effects. In addition, chronic stress increased the expression of VEGF, MMP-2, MMP-7 and MMP-9 in transplanted tumour tissue, and catecholamine hormones enhanced the expression of metastasis-related proteins. The expression of ADRB2 was upregulated in tumour tissues and positively correlated with tumour size, histological grade, lymph node metastasis and clinical stage in human GC samples. Stress hormone-induced activation of the ADRB2 signalling pathway plays a crucial role in GC progression and metastasis. These findings indicate that ADRB2 signalling regulates GC progression and suggest β2 blockade as a novel strategy to complement existing therapies for GC.

Download Full-text

Neural Differentiation of Human Adipose Tissue-Derived Stem Cells Involves Activation of the Wnt5a/JNK Signalling

Stem Cells International ◽

10.1155/2015/178618 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 23

Author(s):

Sujeong Jang ◽

Jong-Seong Park ◽

Han-Seong Jeong

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Neural Differentiation ◽

Neural Induction ◽

Stem Cell Differentiation ◽

Human Adipose Tissue ◽

Pcr Analysis ◽

Rt Pcr ◽

Jnk Pathway ◽

Expression Levels

Stem cells are a powerful resource for cell-based transplantation therapies, but understanding of stem cell differentiation at the molecular level is not clear yet. We hypothesized that the Wnt pathway controls stem cell maintenance and neural differentiation. We have characterized the transcriptional expression of Wnt during the neural differentiation of hADSCs. After neural induction, the expressions of Wnt2, Wnt4, and Wnt11 were decreased, but the expression of Wnt5a was increased compared with primary hADSCs in RT-PCR analysis. In addition, the expression levels of most Fzds and LRP5/6 ligand were decreased, but not Fzd3 and Fzd5. Furthermore, Dvl1 and RYK expression levels were downregulated in NI-hADSCs. There were no changes in the expression of ß-catenin and GSK3ß. Interestingly, Wnt5a expression was highly increased in NI-hADSCs by real time RT-PCR analysis and western blot. Wnt5a level was upregulated after neural differentiation and Wnt3, Dvl2, and Naked1 levels were downregulated. Finally, we found that the JNK expression was increased after neural induction and ERK level was decreased. Thus, this study shows for the first time how a single Wnt5a ligand can activate the neural differentiation pathway through the activation of Wnt5a/JNK pathway by binding Fzd3 and Fzd5 and directing Axin/GSK-3ß in hADSCs.

Download Full-text

Comparison of mRNA Expression Levels Determined with TAQ?Man and Competitive Template RT?PCR

ChemInform ◽

10.1002/chin.200512217 ◽

2005 ◽

Vol 36 (12) ◽

Author(s):

R. Mauritz ◽

W. Schwabe ◽

P. Haeusler ◽

P. Noordhuis ◽

K. Smid ◽

...

Keyword(s):

Mrna Expression ◽

Rt Pcr ◽

Expression Levels ◽

Competitive Template ◽

Mrna Expression Levels

Download Full-text

Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01780-10 ◽

2011 ◽

Vol 55 (6) ◽

pp. 2537-2545 ◽

Cited By ~ 9

Author(s):

Takako Watanabe ◽

Naoya Sakamoto ◽

Mina Nakagawa ◽

Sei Kakinuma ◽

Yasuhiro Itsui ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Real Time ◽

Interferon Regulatory Factor ◽

Mechanisms Of Action ◽

Alpha Interferon ◽

Regulatory Factor ◽

Rt Pcr ◽

Response Element ◽

Expression Levels

ABSTRACTA lack of patient response to alpha interferon (α-IFN) plus ribavirin (RBV) treatment is a major problem in eliminating hepatitis C virus (HCV). We screened chemical libraries for compounds that enhanced cellular responses to α-IFN and identified a triterpenoid, toosendanin (TSN). Here, we studied the effects and mechanisms of action of TSN on HCV replication and its effect on α-IFN signaling. We treated HCV genotype 1b replicon-expressing cells and HCV-J6/JFH-infected cells with TSN, with or without α-IFN, and the level of HCV replication was quantified. To study the effects of TSN on α-IFN signaling, we detected components of the interferon-stimulated gene factor 3 (ISGF3), phosphorylated signal transducer and activator of transcription 1 (STAT1), and STAT2 by Western blotting analysis; expression levels of mRNA of interferon regulatory factor 9 using real-time reverse transcription-PCR (RT-PCR); and interferon-stimulated response element reporter activity and measured the expression levels of interferon-inducible genes for 2′,5′-oligoadenylate synthetase, MxA, protein kinase R, and p56 using real-time RT-PCR. TSN alone specifically inhibited expression of the HCV replicon (50% effective concentration = 20.6 nM, 50% cytotoxic concentration > 3 μM, selectivity index > 146). Pretreatment with TSN prior to α-IFN treatment was more effective in suppressing HCV replication than treatment with either drug alone. Although TSN alone did not activate the α-IFN pathway, it significantly enhanced the α-IFN-induced increase of phosphorylated STATs, interferon-stimulated response element activation, and interferon-stimulated gene expression. TSN significantly increased baseline expression of interferon regulatory factor 9, a component of interferon-stimulated gene factor 3. Antiviral effects of treatment with α-IFN can be enhanced by pretreatment with TSN. Its mechanisms of action could potentially be important to identify novel molecular targets to treat HCV infection.

Download Full-text