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1.
Journal of Experimental Hematology ; (6): 1081-1085, 2020.
Article in Chinese | WPRIM | ID: wpr-827157

ABSTRACT

OBJECTIVE@#To investigate the effect of sphingosine-1-phosphate receptor 2 (S1PR2) specific antagonist JTE-013 on the proliferation of human chronic myeloid leukemia (CML) cell line K562.@*METHODS@#K562 cells were treated with JTE-013 (0, 0.5, 1, 5, 10, 20 μmol/L) for 24 and 48 hours respectively, CCK8 assay was used to detect the cell viability. K562 cells were treated with JTE-013 (0, 5, 10, 20 μmol/L) for 24 hours, propidium iodide (PI) DNA staining was used to analyze the cell cycle, Western blot was used to determine the levels of P21 and Cyclin D1 protein expression.@*RESULTS@#JTE-013 inhibited the proliferation of CML cell line K562 in a dose dependent manner (r=-0.971). The proliferation rate of CML cells showed that the activity of CML cells decreased gradually with the increase of JTE-013 concentration (r=-0.971). The detection demonstrated that JTE-013 suppressed tumor cell proliferation through cell cycle arrest in G/G phase. Further detection of the protein expressions of G phase regulators showed that level of P21 increased, and expression of Cyclin D1 decreased.@*CONCLUSION@#JTE-013, a S1PR2 antagonist, can inhibit the proliferation of human CML K562 cells, which may be achieved by arresting the cells in G/G phase.


Subject(s)
Apoptosis , Cell Proliferation , Humans , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Pyrazoles , Pyridines , Receptors, Lysosphingolipid , Sphingosine-1-Phosphate Receptors
2.
Chinese Medical Journal ; (24): 2429-2436, 2020.
Article in English | WPRIM | ID: wpr-877825

ABSTRACT

BACKGROUND@#Endothelial cells play a key role in the cytokine storm caused by influenza A virus. MicroRNA-155 (miR-155) is an important regulator in inflammation. Its role in the inflammatory response to influenza A infection, however, has yet to be elucidated. In this study, we explored the role as well as the underlying mechanism of miR-155 in the cytokine production in influenza A-infected endothelial cells.@*METHODS@#Human pulmonary microvascular endothelial cells (HPMECs) were infected with the influenza A virus strain H1N1. The efficiency of H1N1 infection was confirmed by immunofluorescence. The expression levels of proinflammatory cytokines and miR-155 were determined using real-time polymerase chain reaction. A dual-luciferase reporter assay characterized the interaction between miR-155 and sphingosine-1-phosphate receptor 1 (S1PR1). Changes in the target protein levels were determined using Western blot analysis.@*RESULTS@#MiR-155 was elevated in response to the H1N1 infection in HPMECs (24 h post-infection vs. 0 h post-infection, 3.875 ± 0.062 vs. 1.043 ± 0.013, P = 0.001). Over-expression of miR-155 enhanced inflammatory cytokine production (miR-155 mimic vs. negative control, all P < 0.05 in regard of cytokine levels) and activation of nuclear factor kappa B in infected HPMECs (miR-155 mimic vs. negative control, P = 0.004), and down-regulation of miR-155 had the opposite effect. In addition, S1PR1 was a direct target of miR-155 in the HPMECs. Inhibition of miR-155 enhanced the expression of the S1PR1 protein. Down-regulation of S1PR1 decreased the inhibitory effect of the miR-155 blockade on H1N1-induced cytokine production and nuclear factor kappa B activation in HPMECs.@*CONCLUSION@#MiR-155 maybe modulate influenza A-induced inflammatory response by targeting S1PR1.


Subject(s)
Down-Regulation , Endothelial Cells , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A virus , Influenza, Human/genetics , MicroRNAs/genetics , Sphingosine-1-Phosphate Receptors
3.
National Journal of Andrology ; (12): 110-119, 2017.
Article in Chinese | WPRIM | ID: wpr-812801

ABSTRACT

Objective@#To screen lentiviral vectors carrying siRNA which can specifically down-regulate the gene expression of the sphingosine-1-phosphate receptor 3 (S1PR3) in the corpus cavernosum smooth muscle (CCSM) cells of rats with spontaneous hypertension (SHT) and investigate the influence of the vectors on the signaling pathways of ROCK1, ROCK2 and eNOS in the CCSM cells of SHT rats.@*METHODS@#Using the S1PR3 mRNA sequence of the rat as an interfering target, we designed and synthesized three pairs of siRNA sequences (siRNA1, 2 and 3) targeting S1PR3 and one pair of negative control, and then constructed and packaged them into lentiviral vectors. We cultured the CCSM cells of SHT and Wistar-Kyoto (WKY) rats in vitro and randomly divided them into groups A (SHT untransfected control), B (SHT transfected and carrying negative control virus), C (SHT transfected and carrying siRNA1 targeting S1PR3), D (SHT transfected and carrying siRNA2 targeting S1PR3), E (SHT transfected and carrying siRNA3 targeting S1PR3), and F (WKY untransfected control). With the multiplicity of infection (MOI) = 60, we transfected the CCSM cells of the SHT rats with the lentiviral vector and then determined the expression of the green fluorescent protein (GFP) as well as the mRNA and protein expressions of S1PR3, ROCK1, ROCK2 and eNOS in the CCSM cells of the SHT and WKY rats by RT-PCR and Western blot.@*RESULTS@#Gene sequencing proved the successful construction of the lentiviral vector. The transfection efficiency of the CCSM cells of the rats was >80% in groups B, C, D and E. Compared with group A, the mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 exhibited no significant difference in group B but were remarkably decreased in groups C, D, E and F (P0.05) but remarkably lower than those in group F (P0.05) but markedly increased in groups A, B, C and D (P< 0.05), while those of eNOS remarkably decreased in groups A, B, C, D and E (P< 0.05).@*CONCLUSIONS@#The three constructed lentiviral vectors carrying siRNA targeting different loci of the S1PR3 gene could significantly inhibit the expression of S1P3 as well as RhoA/Rho kinase signaling pathways in the CCSM cells of SHT rats, and the vector carrying siRNA3 exhibited the highest inhibitory effect.


Subject(s)
Animals , Down-Regulation , Gene Expression , Genetic Vectors , Green Fluorescent Proteins , Metabolism , Lentivirus , Genetics , Male , Myocytes, Smooth Muscle , Metabolism , Nitric Oxide Synthase Type III , Metabolism , Penis , Metabolism , RNA, Messenger , RNA, Small Interfering , Genetics , Metabolism , Random Allocation , Rats , Rats, Inbred WKY , Receptors, Lysosphingolipid , Genetics , Metabolism , Signal Transduction , Sphingosine-1-Phosphate Receptors , Transfection , rho-Associated Kinases , Metabolism
4.
Article in Chinese | WPRIM | ID: wpr-814710

ABSTRACT

OBJECTIVE@#To investigate the variation of senescent endothelial function by regulating the sphingosine-1-phosphate receptor type 2 (S1P2) expression in cultured human umbilical vein endothelial cells (HUVECs).@*METHODS@#The S1P2 receptor expression was regulated by transfecting the cDNA or shRNA of S1P2 in cultured HUVECs. The expression levels of S1P2 receptor in HUVECs were detected by RT-PCR and Western blot. EC chemotaxis was measured by the transwell migration assay. The wound healing assay was performed by a scratch wound model on EC monolayer. Matrigel morphogenesis assay was employed to assess the in vitro angiogenic responses.@*RESULTS@#After up-regulating the S1P2 expression in young ECs, the S1P-stimulated formation of a tubular-like network in Matrigel was dramatically diminished in transfected ECs (P<0.05). Quantification of the wound healing assay showed that transfected ECs grew much slower than young ECs (P<0.05). The chemotactic capability was significantly decreased in transfected ECs (P<0.05). Furthermore, the senescent-associated impairments were revoked by the downregulation of S1P2 receptor in senescent HUVECs.@*CONCLUSION@#The impaired functions (chemotactic, wound-healing and morphogenetic responses) in senescent HUVECs in vitro are mediated by S1P2 receptor.


Subject(s)
Cells, Cultured , Cellular Senescence , Genetics , Human Umbilical Vein Endothelial Cells , Cell Biology , Physiology , Humans , RNA Interference , RNA, Small Interfering , Genetics , Receptors, Lysosphingolipid , Genetics , Metabolism , Sphingosine-1-Phosphate Receptors , Transfection , Up-Regulation
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