1.Expression of CREB in children with recurrent wheezing and its effect on ORMDL3 gene expression.
Rui JIN ; Zhen-Xing ZHANG ; Liang-Hua ZHU ; Li-Li ZHUANG ; Xiao-Qing CHEN
Chinese Journal of Contemporary Pediatrics 2020;22(9):980-983
OBJECTIVE:
To study the expression level of cAMP response element-binding protein (CREB) in children with recurrent wheezing under three years of age and its effect on the expression of the serum orosomucoid 1-like protein 3 (ORMDL3) gene.
METHODS:
Thirty-six children with recurrent wheezing under three years of age who visited the hospital from June 2017 to June 2019 were selected as the recurrent wheezing group. Twenty-four healthy children from physical examination were selected as the control group. The CREB expression level in peripheral blood was measured by quantitative real-time PCR. Human bronchial epithelial cells (BEAS-2B) were cultured, and dual-luciferase reporter assay and quantitative real-time PCR were used to investigate the effects of overexpression and siRNA interference of CREB on the promoter activity and mRNA expression of the ORMDL3 gene in the BEAS-2B cells.
RESULTS:
The expression level of CREB in the recurrent wheezing group was significantly higher than that in the control group (P<0.001). In BEAS-2B cells, overexpression of CREB significantly up-regulated the promoter activity and mRNA expression of the ORMDL3 gene (P<0.05), while siRNA interference of CREB significantly reduced the promoter activity and mRNA expression of the ORMDL3 gene (P<0.05).
CONCLUSIONS
The expression of CREB is increased in children with recurrent wheezing, and CREB may be involved in the pathogenesis of recurrent wheezing by regulating expression of the ORMDL3 gene.
Child, Preschool
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Cyclic AMP Response Element-Binding Protein
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Epithelial Cells
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Humans
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Membrane Proteins
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genetics
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Promoter Regions, Genetic
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Respiratory Sounds
4.Quantitative analysis of SMN gene copies in spinal muscular atrophy.
Hua-xing DING ; Xiao-su YANG ; Bo XIAO ; Zhi-guo WU ; Li-fang ZHANG
Chinese Journal of Medical Genetics 2004;21(2):153-155
OBJECTIVETo study the genetic basis in the patients with clinical diagnosis of spinal muscular atrophy(SMA) but without survival motor neuron telomeric copy (SMN-T) deletion; the relationship between the SMN-C (centromeric) copies and the phenotype; and the distribution of SMN-C and SMN-T copies in the SMA patients, the carriers and the controls.
METHODSQuantitative PCR analysis of SMN-T and SMN-C copies were carried out in 45 patients, 25 consanguineous and 33 control individuals. The patients were identified by clinical manifestation and muscular pathology. Two internal standards of SMN-T and cystic fibrosis transmembrane conductance regulator (CFTR) were constructed. Nonradioactive and nonfluorescence-labelling competitive PCR were used. The numbers of SMN-T and SMN-C copies were determined by calculating the ratios of SMN-T/CFTR and SMN-C/CFTR.
RESULTSQuantitation of SMN-T gene copies in SMA patients revealed that nine cases of type I-III were homozygously deleted. Two cases of type III had only one copy and four cases of type III had two copies. SMA IV and other type cases had two copies. Nine cases of consanguineous individuals had one copy, but other 16 had two copies. All of the normal individuals had two copies. Analysis of SMN-C copies showed that SMA I had < or = 2 copies, II-III had < or = 3 copies, SMA IV and others had 0-3 copies, the consanguineous individuals and normal individuals had 0-3 copies.
CONCLUSIONThe number of copies determined by PCR quantitative assay of SMN-T is in accordance with the result of PCR qualitative assay of homozygous deletion. Quantitative assay of the number of copies can find out the cases and the carriers of heterozygous deletion. The SMA phenotype is related to the number of copies of SMN-C; the smaller the number of copies the patient has, the severer the patient's phenotype will be. The pathogenesis of SMA IV and other types of SMA may not relate to SMN gene.
Cyclic AMP Response Element-Binding Protein ; Gene Dosage ; Humans ; Muscular Atrophy, Spinal ; genetics ; Nerve Tissue Proteins ; genetics ; RNA-Binding Proteins ; SMN Complex Proteins
5.Localization of cAMP-response element binding protein target gene Staufen in mouse hippocampus by situ hybridization.
Guo-zheng LI ; Jing-jing GUO ; Cong-bin PENG
Journal of Zhejiang University. Medical sciences 2014;43(1):66-70
OBJECTIVETo search for an appropriate animal model that is more closely related to human to study cAMP-response element binding protein target gene Staufen and to identify its location.
METHODSThe phylogenetic tree was constructed with Staufen protein (STAUFEN) sequences of different species, and the most suitable animal model was selected by analyzing relativity among them. The Staufen fragments were amplified with reverse transcription-PCR and inserted into a vector and then the sub-clone was transformed into bacteria, selected, amplified, extracted and sequenced. Staufen probes were in vitro transcribed and hybridized in situ on the cryosections of the mouse brain. The cryosections were stained and observed.
RESULTSThe clustering patterns of the phylogenetic tree indicated that the mouse and human Staufen1 had 99.7% protein sequences similarity. The mRNA of Staufen was located in CA1, CA2, CA3 and DG hippocampus regions shown by in situ hybridization.
CONCLUSIONThe mouse is a preferable animal model for research on Staufen transcription in hippocampus.
Animals ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Hippocampus ; metabolism ; In Situ Hybridization ; Mice ; RNA, Messenger ; genetics ; RNA-Binding Proteins ; genetics ; metabolism
6.Association of cyclic adenosine monophosphate response element-binding protein gene and major depressive disorder.
Xiao-hua LIU ; Yi-feng XU ; Dong-hong CUI ; San-duo JIANG ; Yi-ping QIAN ; Shun-ying YU ; Kai-da JIANG
Chinese Journal of Medical Genetics 2010;27(3):263-266
OBJECTIVETo investigate the association between single nucleotide polymorphisms (SNPs) in cyclic adenosine monophosphate response element-binding protein(CREB1) gene and major depressive disorder (MDD).
METHODSWe recruited 105 parent-offspring trios of Chinese descent, extracted whole blood genomic DNA, and genotyped the SNPs in rs10932201 and rs6740584 loci. Single-marker transmission disequilibrium test (TDT), pairwise SNP linkage disequilibrium(LD) and haplotype-based TDT were performed.
RESULTSNo significant association with MDD was observed for SNPs rs10932201 and rs6740584 (P=0.1004 and P=0.4986). However, there was strong positive association between the rs10932201-rs6740584 haplotype and MDD (P=0.00003241), and both haplotypes of A-C and A-T were significantly associated with MDD (P=0.020 and P=0.00022).
CONCLUSIONThe rs10932201-rs6740584 haplotype of the CREB1 gene may play an important role in the pathogenesis of MDD.
Cyclic AMP Response Element-Binding Protein ; genetics ; Depressive Disorder, Major ; genetics ; Female ; Genetic Predisposition to Disease ; Haplotypes ; Humans ; Male ; Polymorphism, Single Nucleotide ; genetics
7.Effects of cAMP-response element binding protein-1 (CREB-1) on transforming growth factor-b3 (TGFb3) mRNA expression and promoter activity in hepatic stellate cells.
Guan-yu ZHOU ; Jin-ming HUANG ; Liang DENG ; Ka-hua LIU ; Qi LI ; Wei QIAN ; Ke-shu XU
Chinese Journal of Hepatology 2012;20(11):822-827
OBJECTIVETo investigate the effects of cAMP-response element binding protein-1 (CREB-1) on transforming growth factor-b3 (TGF b3) mRNA expression and promoter activity in hepatic stellate cells (HSCs).
METHODSFreshly isolated HSCs from rats were divided into six groups: CREB-1 expression plasmid transfected group (C), siRNA-CREB-1 plasmid transfected group (S), negative control group (N), forskolin treated group (F), H-89 treated group (H), and blank group (B). Rats in each group were further sub-divided according to whether (+) or not (-) they were exposed to exogenous TGF b3. TGF b3 mRNA expression was measured by real time quantitative PCR. HSCs of the C, S, N, F, H and B groups were transfected with the TGF b3 promoter luciferase reporter plasmid (PGL3-TGF b3-P; W group), the TGF b3 promoter luciferase reporter plasmid with CRE mutation (PGL3-basic-TGF b3P-mCRE; M group) and the renilla luciferase control plasmid (pRL-SV40; control group). TGF b3 promoter activity was assessed by luciferase reporter assays.
RESULTSCompared to N(-), the TGF b3 mRNA expression was reduced to 0.69+/-0.15 in S(-) (P less than 0.05) and increased to 4.68+/-2.76 in C(-) (P more than 0.05). Compared to B(-), the TGF b3 mRNA expression was reduced to 0.57+/-0.08 in H(-) (P less than 0.05). The differences between N(+) and N(-), S(+) and S(-), B(+) and B(-), and H(+) and H(-) were all significant (P less than 0.05). The values of TGF b3 promoter activity in S(W), N(W), and C(W) were 0.062+/-0.013, 0.122+/-0.011, and 0.165+/-0.016 (P less than 0.05), but the changes of TGF b3 promoter activity in S(M), N(M), and C(M) were not significant (P more than 0.05). The values of TGF b3 promoter activity in H(W), B(W), and F(W) were 0.154+/-0.010, 0.188+/-0.016, and 0.276+/-0.031 (P less than 0.05), but the changes of TGF b3 promoter activity in H(M), B(M), and F(M) were not significant (P more than 0.05).
CONCLUSIONIncreased levels of CREB-1 mRNA or p-CREB-1 up-regulate the TGF b3 mRNA expression and promoter activity in rat HSCs. The CRE site in the TGF b3 promoter is critical for this effect, and the gene's activity becomes significantly decreased when the site is missing. Exogenous TGF b3 enhances expression of endogenous TGF b3 in rat HSCs.
Animals ; Cells, Cultured ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Hepatic Stellate Cells ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; Rats ; Transforming Growth Factor beta3 ; genetics
8.Effect of chronic stress on PKA and P-CREB expression in hippocampus of rats and the antagonism of antidepressors.
Zhe WANG ; Sui-yu HU ; De-liang LEI ; Wei-xi SONG
Journal of Central South University(Medical Sciences) 2006;31(5):767-771
OBJECTIVE:
To observe the effect of chronic unpredicted sequence of mild stress on the expression of cAMP-dependent protein kinase A(PKA) and phosphorylated cAMP-responsive element binding protein (P-CREB) in hippocampus of rats and the antagonism of antidepressors (fluoxetine).
METHODS:
Thirty-six male Sprague Dawley rats were randomly and equally allocated to 3 groups: A normal control group, a model group, and a fluoxetine group. All rats except the control group were singly housed and exposed to an unpredicted sequence of mild stressors. The different distribution and expression of PKA and P-CREB in the hippocampus of rats in different groups were investigated with immunohistochemistry and Westernblot technique.
RESULTS:
The positive PKA and P-CREB cells in the hippocampus of normal controls were the pyramidal cells and the granule cells. The PKA and P-CREB protein expression levels in the hippocampus of model rats were significantly lower than those of the normal controls (P<0.05). The PKA and P-CREB protein expression levels in the hippocampus of the fluoxetine group were significantly higher than those of the model group (P<0.05).
CONCLUSION
Chronic unpredicted mild stress can affect the PKA and P-CREB expression in hippocampus of rats and fluoxetine has antagonism against it.
Animals
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Antidepressive Agents, Second-Generation
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antagonists & inhibitors
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Cyclic AMP Response Element-Binding Protein
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biosynthesis
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genetics
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Cyclic AMP-Dependent Protein Kinases
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biosynthesis
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genetics
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Depression
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etiology
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metabolism
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Fluoxetine
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antagonists & inhibitors
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Hippocampus
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metabolism
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Male
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Random Allocation
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Rats
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Rats, Sprague-Dawley
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Stress, Physiological
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metabolism
9.Downstream molecular events in the altered profiles of lysophosphatidic acid-induced cAMP in senescent human diploid fibroblasts.
Ik Soon JANG ; Ji Heon RHIM ; Sang Chul PARK ; Eui Ju YEO
Experimental & Molecular Medicine 2006;38(2):134-143
Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ia. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.
Time Factors
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Protein Kinase Inhibitors/pharmacology
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Phosphorylation
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Male
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Lysophospholipids/*pharmacology
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Luciferases/genetics/metabolism
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Humans
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Gene Expression/drug effects
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Fibroblasts/cytology/*drug effects/metabolism
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Diploidy
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Cyclic AMP-Dependent Protein Kinases/genetics/metabolism
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Cyclic AMP Response Element-Binding Protein/metabolism
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Cyclic AMP/*metabolism
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Cells, Cultured
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Cell Aging/physiology
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Catalytic Domain/genetics
10.Rapid genetic diagnosis and prenatal diagnosis of spinal muscular atrophy by denaturing high-performance liquid chromatography.
Hai-yan ZHU ; Ling-qian WU ; Qian PAN ; Bei-sha TANG ; De-sheng LIANG ; Zhi-gao LONG ; He-ping DAI ; Kun XIA ; Jia-hui XIA
Chinese Medical Journal 2006;119(14):1222-1225
Chromatography, High Pressure Liquid
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methods
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Cyclic AMP Response Element-Binding Protein
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genetics
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Female
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Humans
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Male
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Nerve Tissue Proteins
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genetics
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Prenatal Diagnosis
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methods
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RNA-Binding Proteins
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genetics
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SMN Complex Proteins
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Sequence Analysis, DNA
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Spinal Muscular Atrophies of Childhood
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diagnosis
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genetics