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.
Apoptosis ; Cell Proliferation ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Pyrazoles ; Pyridines ; Receptors, Lysosphingolipid ; Sphingosine-1-Phosphate Receptors

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.
Cells, Cultured ; Cellular Senescence ; genetics ; Human Umbilical Vein Endothelial Cells ; cytology ; physiology ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptors, Lysosphingolipid ; genetics ; metabolism ; Sphingosine-1-Phosphate Receptors ; Transfection ; Up-Regulation

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 (P< 0.05), most significantly in group E, with the inhibition rates of the mRNA and protein expressions of S1PR3 of (34.2±2.9) and (77.7±4.7)%, those of ROCK1 of (33.3±1.4) and (51.1±7.3)%, and those of ROCK2 of (30.8±3.6) and (58.32±5.5)%, respectively. The mRNA and protein expressions of eNOS in group A showed no significant difference from those in groups B, C, D and E (P>0.05) but remarkably lower than those in group F (P< 0.05). Compared with group F, the mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 were not significantly different from those in group E (P>0.05) 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.
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

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Chlorpromazine ; Humans ; Lymphoma ; Sphingosine-1-Phosphate Receptors

Transmembrane protein, also known as integral membrane protein, can be distributed in the lipid bilayer or across the entire membrane, and it plays an important role in cell signal transduction. It has been discovered that multiple transmembrane proteins are involved in the regulation of tumor signals. Recent studies have revealed that the abnormal expression of some transmembrane protein is closely related to the occurrence, development and prognosis of non-Hodgkin's lymphoma (NHL), including programmed cell death protein 1 (PD-1), TMEM30A, NOTCH1, TOLL-like receptor (TLR), sphingosine-1-phosphate receptor, TRAIL, etc. The study on these transmembrane proteins and related genes has important clinical significance for the treatment and prognosis of NHL, and it may become a new therapeutic target. At present, there have been some research results in this field at home and abroad. This article reviewed the research progress of transmembrane protein that has inhibitory effects on NHL in recent years.
Humans ; Lipid Bilayers ; Lymphoma, Non-Hodgkin/therapy* ; Prognosis ; Programmed Cell Death 1 Receptor ; Sphingosine-1-Phosphate Receptors

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.
Down-Regulation ; Endothelial Cells ; Humans ; Influenza A Virus, H1N1 Subtype/genetics* ; Influenza A virus ; Influenza, Human/genetics* ; MicroRNAs/genetics* ; Sphingosine-1-Phosphate Receptors

BACKGROUNDThe sphingosine 1-phosphate (S1P) receptors (S1PRs) are a group of G protein-coupled receptors expressed on the surface of lymphocytes. The interaction between S1P and S1PRs plays a significant role in the migration and distribution of lymphocytes.

OBJECTIVETo investigate the influence of S1PR5 defect on the lymphocytes distribution in mice.

METHODSThe distribution of different subsets of lymphocyte in the mice with S1PR5 defect was examined by flow cytometry.

RESULTSCompared with wild type mice, the number of NK cells in the peripheral blood (PB) and spleen (SP) from the mice with S1PR5 defect decreased very significantly 〔PB: 6.4±0.45% vs 2.2±0.47(P<0.01,n=3)；SP: 3.0±0.91% vs 0.68±0.14%(P<0.05,n=3)〕. However, the NK cell number in the bone marrow (BM) and lymphonodes (LN) of the mice with S1PR5 defect increased very significantly 〔BM: 0.97±0.20 % vs 2.6±0.35% (P<0.01, n=3); LN: 0.35±0.16% vs 1.7±0.15% (P<0.01, n=3)〕. The percentages of CD3(+) lymphocyte in peripheral blood, spleen and lymph node were not statistically significantly different between these 2 types of mice 〔PB: 17.3±7.9% vs 17.0±4.6% (P>0.05, n=3); SP: 33.0±6.0% vs 27.4±1.8% (P>0.05, n=3); LN: 42.3±10.7% vs 51.2±2.7% (P>0.05, n=3)〕.

CONCLUSIONS1PR5 defect can significantly influence the NK cell distribution.

Animals ; Bone Marrow ; Cell Count ; Flow Cytometry ; Lymphocytes ; Lysophospholipids ; Mice ; Receptors, Lysosphingolipid ; Sphingosine ; analogs & derivatives

Sphingosine-1-phosphate (S1P) plays an important role in trafficking leukocytes and developing immune disorders including autoimmunity. In the synovium of rheumatoid arthritis (RA) patients, increased expression of S1P was reported, and the interaction between S1P and S1P receptor 1 (S1P1) has been suggested to regulate the expression of inflammatory genes and the proliferation of synovial cells. In this study, we investigated the level of S1P1 mRNA expression in the blood leukocytes of RA patients. In contrast to the previous reports, the expression level of this gene was not correlated to their clinical scores, disease durations and ages. However, S1P1 was transcribed at a significantly lower level in the circulating leukocytes of RA patients when compared to age-, and sex-matched healthy controls. Since these data may suggest the participation of S1P1, further studies are needed to determine the role of this receptor in the pathogenesis of RA.
Arthritis, Rheumatoid* ; Autoimmunity ; Humans ; Immune System Diseases ; Leukocytes* ; Receptors, Lysosphingolipid* ; RNA, Messenger ; Synovial Membrane

Sphingosine-1-phosphate (S1P) has been demonstrated to be a mediator and marker of heart diseases. We hypothesized that the expression of S1P receptors is involved in the S1P-mediated cardioprotection in vivo and may serve as a biomarker of ischemic heart disease. In vivo models of myocardial ischemia (MI) and ischemia-reperfusion (IR) were established by ligation of the left anterior descending artery (LAD) of rat heart, the mRNA expressions of S1PR1-3 were detected using real time PCR at different time intervals after ischemia (LAD for 15 min, 30 min, and 1 h) and IR. The results showed that mRNA expression of S1PR3, but not S1PR1 and S1PR2, increased greatly after IR. No statistical difference was found in any of the three S1P receptors after MI within 1 h. Regarding the studies of lipid concentration changes in myocardiopathy, we conclude that S1P receptors are not early response biomarkers for MI. There are different mechanisms when S1P plays a protection role in heart during MI and IR. The cooperation of lipid content and S1P receptor expression appears to form a regulation network during MI and IR.
Animals ; Lysophospholipids ; physiology ; Myocardial Reperfusion Injury ; physiopathology ; Rats ; Receptors, Lysosphingolipid ; physiology ; Sphingosine ; analogs & derivatives ; physiology

OBJECTIVETo explore the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) mobilization on S1P5 expression in T lymphocyte subsets of allo-HSCT donors.

METHODSThe peripheral blood was collected from 10 allo-hematopoietic stem cell transplantation (allo-HSCT) donors before and after mobilization with rhG-CSF for 4 days. The flow cytometry was used to detect S1P5 expression in T lymphocyte subsets.

RESULTSThere was no S1P5 expression on the surface of T-lymphocytes both before and after rhG-CSF mobilization. After fixation with permeabilization agent, S1P5 expression could be detected in lymphocytes after rhG-CSF mobilization, which indicates S1P5 may be located in cells. Compared with level before rhG-CSF mobilization, S1P5 expression was significantly increased in T lymphocyte subsets after rhG-CSF mobilization, CD3(+)T cells (57.92±2.32)% vs (7.94±1.47)%(P<0.05）, CD4(+)T cells (72.58±1.73)% vs （5.48±0.82）%(P<0.05）, CD8(+)T cells（51.79±3.57）% vs （6.46±1.01）%（P<0.05）,CD3-/CD56(+)NK cells（40.00±1.47）% vs（4.97±0.74）%（P<0.05）. The up-regulated level of S1P5 expression in CD4(+)T cells was most high(P<0.05).

CONCLUSIONS1P5 expression significantly increases in T lymphocyte subsets after rhG-CSF mobilization, and the up-regulated level of S1P5 expression in CD4(+)T cells is the most high.

Flow Cytometry ; Granulocyte Colony-Stimulating Factor ; Hematopoietic Stem Cell Transplantation ; Humans ; Receptors, Lysosphingolipid ; Recombinant Proteins ; T-Lymphocyte Subsets ; Transplantation, Homologous