1.The study of the influence of different shear stress on the mRNA expression of scavenger receptor class B type 1 in endothelial cells.
Fengxu YU ; Ying ZHANG ; Shenglin LING ; Yingkang SHI ; Bin LIAO ; Jiang WU
Journal of Biomedical Engineering 2011;28(1):81-120
The present paper is to research the expression level of the mRNA of scavenger receptor class B type 1-receptor of high-density lipoprotein in endothelial cells after being treated by different shear stress. The second to fourth generations of the cultured human umbilical vein endothelial cells (HUVECs) were used in the experiment. The cells were divided into two groups. The first group was the control group which was not dealt with shear stress; the second group was the experimental group which concluded low shear stress group (4.2 dyne/cm2), moderate shear stress group (8.4 dyne/cm2) and high shear stress group (15 dyne/cm2). The load time was 1h, 2h, 4h and 8h, respectively. Harvesting the cells and extracting total RNA after being treated by different shear stresses, the expression level of the SR-B1 mRNA was detected by semi-quantitative RT-PCR technic. It was found that the expression of SR-B1 mRNA became weaker and weaker compared to the control group when it was stimulated continuously by the low shear stress, the lowest expression of SR-B1 mRNA appeared at 8h. In the moderate shear stress group, the expression of SR-B1 mRNA increased obviously. Compared to the control group, there was significant difference after being treated with 2h. In the high shear stress group, the expression of SR-B1 mRNA increased immediately when it was stimulated by the shear stress. And the expression of SR-B1 mRNA arrived peak value at 4h. Compared to the control group, there was significant difference after being treated for 1h. It was concluded that the harmful mechanism of the low shear stress is that it can increase the incidence of the atherosclerosis by reducing the reverse cholesterol transport and endothelial protection through decreasing the expression of the SR-B1. Otherwise, the high shear stress prevent the genesis of atherosclerosis by the contrary mechanism.
Atherosclerosis
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etiology
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Cells, Cultured
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Human Umbilical Vein Endothelial Cells
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cytology
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metabolism
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Humans
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RNA, Messenger
;
genetics
;
metabolism
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Scavenger Receptors, Class B
;
genetics
;
metabolism
;
Stress, Mechanical
2.Association Analysis Between Methylation of SCARB1 Gene Promoter and Coronary Heart Disease.
Wei LI ; Zhen-Hua WANG ; Peng SHI ; Song XUE
Acta Academiae Medicinae Sinicae 2023;45(3):405-409
Objective To explore the relationship between scavenger receptor class B member 1 (SCARB1) gene promoter methylation and the pathogenesis of coronary artery disease. Methods A total of 120 patients with coronary heart disease treated in Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine from December 2018 to May 2020 were selected as the case group,while 140 gender and age matched healthy participants were randomly selected as the control group for a case-control study.The methylation status was detected by high-throughput target sequencing after bisulfite converting,and the methylation of CpG sites in the promoter region of SCARB1 gene was compared between the two groups. Results The case group showed higher methylation level of SCARB1+67 and lower methylation level of SCARB1+134 than the control group (both P<0.001),and the differences remained statistically significant in men (both P<0.001) and women (both P<0.001).The overall methylation level in the case group was lower than that in the control group [(80.27±2.14)% vs.(81.11±1.27)%;P=0.006],while this trend was statistically significant only in men (P=0.002). Conclusion The methylation of SCARB1 gene promotor is associated with the pathogenesis and may participate in the occurrence and development of coronary heart disease.
Male
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Humans
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Female
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Methylation
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Case-Control Studies
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China
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Coronary Artery Disease/genetics*
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Promoter Regions, Genetic
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DNA Methylation
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Scavenger Receptors, Class B/genetics*
3.Establishment of a drug screening model for identifying up-regulator of human high density lipoprotein receptor.
Xiao-hui LIU ; Bin HONG ; Li-fei WANG ; Yuan YANG ; Shu-yi SI ; Yuan LI
Acta Academiae Medicinae Sinicae 2004;26(4):354-358
<b>OBJECTIVEb>To establish a new drug screening model based on transcriptional regulation of human high density lipoprotein (HDL) receptor gene CD36 and LIMPII analogous-1 (CLA-1) for discovering up-regulator of this receptor.
<b>METHODSb>The upstream regulatory sequence of CLA-1 was obtained by polymerase chain reaction. A recombinant reporter plasmid pGL3-CLAP was constructed by inserting the regulatory sequence upstream of luciferase gene of pGL3-Basic. Human hepatoma cell line BEL-7402 was transfected with pGL3-CLAP. Samples were detected by testing luciferase activity of transfected BEL-7402 cells in microtiter wells.
<b>RESULTSb>The drug screening model was established and optimized. Significant difference was present between pGL3-CLAP and pGL3-Basic transfected BEL-7402 cells (P< 0.001), and coefficient of variation was less than 10%. After primary and secondary screening, 1 compounds and 3 fermentation extracts had up-regulating activities.
<b>CONCLUSIONb>This new drug screening model may be efficiently used to screen up-regulators of human HDL receptor expression, which might become lead compounds for new anti-atherosclerosis drugs.
CD36 Antigens ; Cholesterol Esters ; metabolism ; Drug Evaluation, Preclinical ; methods ; Gene Expression Regulation ; drug effects ; Humans ; Hypolipidemic Agents ; chemical synthesis ; pharmacology ; Lipoproteins, HDL ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA-Binding Proteins ; Receptors, Immunologic ; genetics ; Receptors, Lipoprotein ; genetics ; Receptors, Scavenger ; Scavenger Receptors, Class B ; Transcription, Genetic ; drug effects ; Up-Regulation
4.H19 recruited N 6 -methyladenosine (m 6 A) reader YTHDF1 to promote SCARB1 translation and facilitate angiogenesis in gastric cancer.
Rumeng BAI ; Miaomiao SUN ; Yuanyuan CHEN ; Shuaishuai ZHUO ; Guoxin SONG ; Tianjun WANG ; Zhihong ZHANG
Chinese Medical Journal 2023;136(14):1719-1731
BACKGROUND:
Angiogenesis is described as a complex process in which new microvessels sprout from endothelial cells of existing vasculature. This study aimed to determine whether long non-coding RNA (lncRNA) H19 induced the angiogenesis of gastric cancer (GC) and its possible mechanism.
METHODS:
Gene expression level was determined by quantitative real-time polymerase chain reaction and western blotting. Cell counting kit-8, transwell, 5-Ethynyl-2'-deoxyuridine (EdU), colony formation assay, and human umbilical vein endothelial cells (HUVECs) angiogenesis assay as well as Matrigel plug assay were conducted to study the proliferation, migration, and angiogenesis of GC in vitro and in vivo . The binding protein of H19 was found by RNA pull-down and RNA Immunoprecipitation (RIP). High-throughput sequencing was performed and next Gene Ontology (GO) as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted to analyze the genes that are under H19 regulation. Methylated RIP (me-RIP) assay was used to investigate the sites and abundance among target mRNA. The transcription factor acted as upstream of H19 was determined through chromatin immunoprecipitation (ChIP) and luciferase assay.
RESULTS:
In this study, we found that hypoxia-induced factor (HIF)-1α could bind to the promoter region of H19, leading to H19 overexpression. High expression of H19 was correlated with angiogenesis in GC, and H19 knocking down could inhibit cell proliferation, migration and angiogenesis. Mechanistically, the oncogenic role of H19 was achieved by binding with the N 6 -methyladenosine (m 6 A) reader YTH domain-containing family protein 1 (YTHDF1), which could recognize the m 6 A site on the 3'-untransated regions (3'-UTR) of scavenger receptor class B member 1 (SCARB1) mRNA, resulting in over-translation of SCARB1 and thus promoting the proliferation, migration, and angiogenesis of GC cells.
CONCLUSION
HIF-1α induced overexpression of H19 via binding with the promoter of H19, and H19 promoted GC cells proliferation, migration and angiogenesis through YTHDF1/SCARB1, which might be a beneficial target for antiangiogenic therapy for GC.
Humans
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Cell Line, Tumor
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Cell Proliferation/genetics*
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Endothelial Cells/metabolism*
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Gene Expression Regulation
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Gene Expression Regulation, Neoplastic/genetics*
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Hypoxia
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MicroRNAs/genetics*
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RNA
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RNA, Long Noncoding/metabolism*
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RNA-Binding Proteins/metabolism*
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Scavenger Receptors, Class B/metabolism*
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Stomach Neoplasms/genetics*
5.Hepatitis C virus: virology and life cycle.
Chang Wook KIM ; Kyong Mi CHANG
Clinical and Molecular Hepatology 2013;19(1):17-25
Hepatitis C virus (HCV) is a positive sense, single-stranded RNA virus in the Flaviviridae family. It causes acute hepatitis with a high propensity for chronic infection. Chronic HCV infection can progress to severe liver disease including cirrhosis and hepatocellular carcinoma. In the last decade, our basic understanding of HCV virology and life cycle has advanced greatly with the development of HCV cell culture and replication systems. Our ability to treat HCV infection has also been improved with the combined use of interferon, ribavirin and small molecule inhibitors of the virally encoded NS3/4A protease, although better therapeutic options are needed with greater antiviral efficacy and less toxicity. In this article, we review various aspects of HCV life cycle including viral attachment, entry, fusion, viral RNA translation, posttranslational processing, HCV replication, viral assembly and release. Each of these steps provides potential targets for novel antiviral therapeutics to cure HCV infection and prevent the adverse consequences of progressive liver disease.
Antigens, CD81/metabolism
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Genome, Viral
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Hepacivirus/genetics/*physiology
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Humans
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RNA, Viral/metabolism
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Scavenger Receptors, Class B/metabolism
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Viral Envelope Proteins/chemistry/metabolism
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Viral Nonstructural Proteins/chemistry/metabolism
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Virus Assembly
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Virus Internalization
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Virus Replication
6.Effect of olive antihyperlipidemia capsule on low and high density lipoprotein receptor in rat liver gene expression in hyperlipmia rat liver.
China Journal of Chinese Materia Medica 2007;32(6):519-522
<b>OBJECTIVEb>To study on the regulatory mechanism of lipid metabolism disorders in the blood fat of hyperlipemia rat model with Olive Antihyperlipidemia capsule, and do systematic observation on the functions of this medicine on low And high density lipoprotein receptor in rat liver gene expression, and then to clarify the mechanism of action of this medicine on treating hyperlipemia.
<b>METHODb>To select SD rat as investigated subject. The hyperlipemia rat models were made with feeding high-fat forage and were randomly divided into six groups based on the total cholesterol level at the ratsfasting for 12 hours: group A, B, C, D, E and group F. The samples in the research were collected and analyzed the changes of LDLR/SR-B1 gene expression in rat's liver by RT-PCR.
<b>RESULTb>Olive Antihyperlipidemia capsule can markedly enhance LDLR/SR-B1 gene expression in rat's liver and finally accomplish the purpose of reducing blood fat. The experiment shows this medicine has the remarkable effect on hyperlipidemia and proved the theoretical system of treating hyperlipemia for curing the liver is correct.
<b>CONCLUSIONb>Olive Antihyperlipidemia capsule has an applicable value on preventing the cause, enhance LDLR/SR-B1 gene expression in rat's liver and finally accomplish the purpose of reducing blood fat and development of hyperlipemia and its complications.
Animals ; Capsules ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Gene Expression Regulation ; drug effects ; Hyperlipidemias ; genetics ; pathology ; prevention & control ; Hypolipidemic Agents ; isolation & purification ; pharmacology ; Lipoproteins, HDL ; genetics ; Liver ; metabolism ; Male ; Olea ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, LDL ; genetics ; Receptors, Lipoprotein ; genetics ; Scavenger Receptors, Class B ; genetics
7.Development of a new high-throughput screening model for human high density lipoprotein receptor (CLA-1) agonists.
De-Feng TIAN ; Bin HONG ; Shu-Yi SI
Biomedical and Environmental Sciences 2005;18(4):265-272
<b>OBJECTIVEb>To develop a new high-throughput screening model for human high-density lipoprotein (HDL) receptor (CD36 and LIMPII analogous-1, CLA-1) agonists using CLA-1-expressing insect cells.
<b>METHODSb>With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA (cDNA) of CLA-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) into donor vector pFastBac1 and recombinant pFastBac1-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacmid DNA. Recombinant bacmid-CLA-1 was transfected into Spodoptera frugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA-1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized.
<b>RESULTSb>Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries.
<b>CONCLUSIONb>Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.
Animals ; Baculoviridae ; genetics ; metabolism ; Biological Assay ; Carbocyanines ; metabolism ; Cell Line, Tumor ; Cholesterol, HDL ; metabolism ; DNA, Complementary ; genetics ; metabolism ; Fluorescent Dyes ; metabolism ; Gene Expression ; Humans ; Lipoproteins, HDL ; agonists ; genetics ; metabolism ; Lipoproteins, LDL ; metabolism ; Receptors, Lipoprotein ; agonists ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Scavenger Receptors, Class B ; agonists ; genetics ; metabolism ; Spodoptera ; genetics ; metabolism
8.Sirt1 regulates testosterone biosynthesis in Leydig cells via modulating autophagy.
Muhammad Babar KHAWAR ; Chao LIU ; Fengyi GAO ; Hui GAO ; Wenwen LIU ; Tingting HAN ; Lina WANG ; Guoping LI ; Hui JIANG ; Wei LI
Protein & Cell 2021;12(1):67-75
Animals
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Autophagy/genetics*
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Cholesterol/metabolism*
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Gene Expression Regulation
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Integrases/metabolism*
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Leydig Cells/metabolism*
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Male
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Mice, Knockout
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Multienzyme Complexes/metabolism*
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Phosphoproteins/metabolism*
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Primary Cell Culture
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Progesterone Reductase/metabolism*
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RNA Splicing Factors/metabolism*
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Scavenger Receptors, Class B/metabolism*
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Sequestosome-1 Protein/metabolism*
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Signal Transduction
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Sirtuin 1/genetics*
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Sodium-Hydrogen Exchangers/metabolism*
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Steroid 17-alpha-Hydroxylase/metabolism*
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Steroid Isomerases/metabolism*
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Testosterone/genetics*
9.Effect of Antrodia cinnamomea on gene expression related to aortal endothelial injury in rats with hyperlipidemia.
Jie QI ; Yun TAO ; Jun ZHANG ; Jian FU
China Journal of Chinese Materia Medica 2014;39(9):1670-1674
<b>OBJECTIVEb>To investigate the effect of Antrodia cinnamomea on gene expression related to aortal endothelial injury of rats with hyperlipidemia.
<b>METHODb>Fifty SD rats were randomly divided into five groups: the normal control group (NG), the model group (MG), the antrodia cinnamomea groups of low, middle and high doses (AC-LG, AC-MG, AC-HG, 250, 500, 1 000 mg x kg(-1)). The rats were fed with high-fat diets to establish the hyperlipidemia model. After the drug administration for 10 weeks, their serum lipid, SOD, MDA and ox-LDL, LOX-1, P38 MAPK and NF-kappaB mRNA and protein expression were respectively determined, and the aortal endothelial injury was observed under electron microscope.
<b>RESULTb>In the model group, the contents of TC, TG and LDL-C significant increased (P < 0.01), whereas the content of HDL-C significant decreased (P < 0.01). Compared with the model group, both the AC-M group and the AC-H group showed reduction in endothelial injury and significant decrease in the content of TC, TG and LDL-C (P < 0.05 or P < 0.01). The content of HDL-C increased, but with no significant difference. SOD activity in serum remarkably increased (P < 0.05 or P < 0.01), MDA and ox-LDL levels dramatically decreased (P < 0.05 or P < 0.01).
<b>CONCLUSIONb>A. cinnamomea can alleviate endothelial lipid injury by inhibiting the expressions of LOX-1, P38MAPK and NF-kappaB in aorta and better protect aortal endothelial cells from oxidative lipid injury.
Animals ; Antrodia ; chemistry ; Aorta ; drug effects ; metabolism ; ultrastructure ; Atherosclerosis ; blood ; genetics ; prevention & control ; Biological Products ; pharmacology ; Cholesterol ; blood ; Cholesterol, HDL ; blood ; Cholesterol, LDL ; blood ; Endothelium, Vascular ; drug effects ; metabolism ; pathology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; drug effects ; Hyperlipidemias ; blood ; genetics ; prevention & control ; Lipoproteins, LDL ; blood ; Male ; Malondialdehyde ; blood ; Microscopy, Electron ; NF-kappa B ; blood ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Scavenger Receptors, Class E ; blood ; genetics ; metabolism ; Superoxide Dismutase ; blood ; Triglycerides ; blood ; p38 Mitogen-Activated Protein Kinases ; blood ; genetics ; metabolism