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OBJECTIVE To investigate the effect and mechanism of picroside Ⅱ on the malignant progression of non-small cell lung cancer （NSCLC）. METHODS A549 cells were divided into the control group， picroside Ⅱ low-， medium- and high- concentration groups， K6PC-5 [sphingosine kinase 1 （SPHK1） activator] group， and picroside Ⅱ high-dose+K6PC-5 group. Cell proliferation， migration and invasion were detected. Besides， the expression of proliferating cell nuclear antigen （PCNA）， matrix metalloproteinase-2 （MMP-2）， MMP-9， SPHK1， sphingosine-1-phosphate receptor 3 （S1PR3） and extracellular signal-regulated kinase 1/2 （ERK1/2） protein in the cells were also observed. BALB/c nude mice were subcutaneously inoculated with A549 cell suspension to establish NSCLC xenograft models. Then they were assigned to the nude mouse-control group， nude mouse-picroside Ⅱ low-， medium- and high-dose groups， nude mouse-K6PC-5 group， and nude mouse-picroside Ⅱ high-dose+K6PC-5 group （with 5 mice in each group） to investigate the effect of picroside Ⅱ on their tumor mass and volume. RESULTS Compared with the control group， the OD450 values， EdU-positive cell rates， scratch healing rates， cell invasion number， and the relative expression levels of PCNA， MMP-2， MMP-9， SPHK1， S1PR3 and ERK1/2 protein in the low-， medium- and high-concentration groups of picroside Ⅱ were significantly decreased. Compared with the nude mouse-control group， the tumor mass and volume in the nude mouse-low-， medium- and high-dose groups of picroside Ⅱ were significantly decreased or shrunk. The changes of above indicators were concentration/dose-dependent （P＜0.05）. The changing trend of the corresponding indicators in the K6PC-5 ZYTS181） group and the nude mouse-K6PC-5 group was opposite （P＜0.05）. Compared with the picroside Ⅱ high-concentration group or the nude mice-picroside Ⅱ high-dose group， the above quantitative indicators in the picroside Ⅱ high- concentration+K6PC-5 group cells and the nude mouse-picroside Ⅱ high-dose+K6PC-5 group nude mice were significantly increased or enlarged （P＜0.05）. CONCLUSIONS Picroside Ⅱ may inhibit the malignant progression of NSCLC by inhibiting SPHK1/sphingosine-1-phosphate/S1PR3 signaling pathway.
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OBJECTIVE@#To elucidate the renoprotective effect of resveratrol (RSV) on sphingosine kinase 1 (SphK1) signaling pathway and expression of its downstream molecules including activator protein 1 (AP-1) and transformation growth factor-β1 (TGF-β1) in lipopolysaccharide (LPS)-induced glomerular mesangial cells (GMCs).@*METHODS@#The rat GMCs line (HBZY-1) were cultured and randomly divided into 5 groups, including control, LPS (100 ng/mL), and 5, 10, 20 µmol/L RSV-treated groups. In addition, SphK1 inhibitor (SK-II) was used as positive control. GMCs were pretreated with RSV for 2 h and treated with LPS for another 24 h. GMCs proliferation was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The proteins expression of SphK1, p-c-Jun and TGF-β1 in GMCs were detected by Western blot, and DNA-binding activity of AP-1 was performed by electrophoretic mobility shift assay (EMSA). The binding activity between RSV and SphK1 protein was detected by AutoDock Vina and visualized by Discovery Studio 2016.@*RESULTS@#LPS could obviously stimulate GMCs proliferation, elevate SphK1, p-c-Jun and TGF-β1 expression levels and increase the DNA-binding activity of AP-1 (P<0.05 or P<0.01), whereas these effects were significantly blocked by RSV pretreatment. It was also suggested that the effect of RSV was similar to SK-II (P>0.05). Moreover, RSV exhibited good binding affinity towards SphK1, with docking scores of -8.1 kcal/moL and formed hydrogen bonds with ASP-178 and LEU-268 in SphK1.@*CONCLUSION@#RSV inhibited LPS-induced GMCs proliferation and TGF-β1 expression, which may be independent of its hypoglycemic effect on preventing the development of mesangial cell fibrosis and closely related to the direct inhibition of SphK1 pathway.
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Animals , Rats , Lipopolysaccharides/pharmacology , Mesangial Cells , Resveratrol/pharmacology , Transcription Factor AP-1 , Transforming Growth Factor beta1 , Intercellular Signaling Peptides and Proteins , Cell Proliferation , DNA , Cells, CulturedABSTRACT
ObjectiveTo investigate the effects of hydroxycamptothecin liposomes （LHCPT） on myocardial fibrosis in rats with heart failure by regulating the sphingosine kinase 1 （SphK1）/sphingosine-1-phosphate （S1P） signaling pathway. MethodsSD rats were divided into control group， model group， hydroxycamptothecin （HCPT） group， LHCPT group， captopril group， and LHCPT+K6PC-5 （SphK1 activator） group， with 12 rats in each group. The heart failure rat models in all groups except the control group were established by intraperitoneal injection of doxorubicin and then the corresponding drugs were given once a day. After four weeks， we applied color Doppler ultrasound to detect left ventricular end systolic diameter （LVESD）， left ventricular end diastolic diameter （LVEDD）， and left ventricular ejection fraction （LVEF） in rats； HE and Masson staining for myocardial pathological damage and myocardial fibrosis in rats， respectively； ELISA method for the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in rat myocardial tissues； qRT-PCR for the expression of transforming growth factor-β1 （TGF-β1）， type I collagen （Collagen I）， and type Ⅲ collagen （Collagen Ⅲ） in rat myocardial tissues； Western blot for the expression of SphK1 and S1P proteins in rat myocardial tissues. ResultsCompared with the control group， the model group showed severe myocardial pathological damage and myocardial fibrosis， increased LVESD， LVEDD， levels of TNF-α and IL-6， expression of TGF-β1， Collagen I， Collagen Ⅲ， SphK1， S1P and decreased LVEF （P<0.05）. Compared with the model group， the HCPT group， LHCPT group and captopril group showed alleviated myocardial pathological damage and myocardial fibrosis， decreased LVESD， LVEDD， levels of TNF-α and IL-6， expression of TGF-β1， Collagen I， Collagen Ⅲ， SphK1， S1P and increased LVEF （P<0.05）. Compared with the LHCPT group， the LHCPT+K6PC-5 group showed aggravated myocardial pathological damage and myocardial fibrosis， increased LVESD， LVEDD， levels of TNF-α and IL-6， expression of TGF-β1， Collagen I， Collagen Ⅲ， SphK1， S1P and decreased LVEF （P<0.05）. ConclusionLHCPT may inhibit myocardial fibrosis in heart failure rats by inhibiting the SphK1/S1P signaling pathway.
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Sphingosine kinase (SphK), sphingosine-1-phosphate (S1P) and S1P receptor (S1PR) are involved in the tumor biological processes such as tumor cell proliferation and migration, and play an important role in the development of cancer. In recent years, researchers have increasingly focused on the interaction between cancer cells and the tumor microenvironment. The tumor microenvironment is genetically stable and can be induced to an antitumor phenotype, which has significant therapeutic advantages. Studies have shown that SphK/S1P/S1PR can regulate multiple aspects of the tumor microenvironment. This review summarizes the effects of SphK and S1P/S1PR signaling on the tumor microenvironment from four perspectives: tumor immune microenvironment, cancer associated fibroblasts, tumor angiogenesis and tumor hypoxic microenvironment, and also outlines potential drug research related to these signal molecules, aiming to elucidate the role of SphK/S1P/S1PR in tumor occurrence and development and provide new ideas for the research of anti-tumor drugs.
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@#Sphingosine kinase 1 (SphK1) is an important protein that regulates the lipid microenvironment of cell membranes, and plays an important role in the dynamic equilibrium of ceramide, sphingosine and sphingosine-1-phosphate.The overexpression of SphK1 is closely related to the occurrence, development and migration of tumors as well as the generation of drug resistance.SphK1 inhibitors can induce apoptosis of various tumor cells and reverse drug resistance, which has a good prospect for drug development.In this article, the structural biology of SphK1, the structural types and structure-activity relationships of SphK1 inhibitors are reviewed.
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Objective To investigate the therapeutic effect of SPK1 gene transfected adipose derived mesenchymal stem cells(ADMSC)on experimental autoimmune encephalomyelitis mice and the effect on T helper cell 17(Th17)/regulatory T(Treg) cells balance. Methods EAE was induced by myelin oligodendrocyte glycoprotein 35-55 in mice.Totally 44 mice were randomly divided into four groups:normal control group(NC group),model group(EAE group),ADMSC group,and ADMSC-SPK1 group.Forty days after injection,the pathological changes of brain and spinal cord,Th17/Treg-related inflammatory markers in brain tissue,expressions of interleukin-17A(IL-17A)and forkhead box protein p3(Foxp3)in brain and spinal cord tissue,and flow cytometric results of spleen immune cells were detected. Results Forty days after the injection,serious inflammatory cell infiltration and demyelination occurred in the brain and spinal cord of EAE group,whereas demyelination and axonal injury were improved in ADMSC group and ADMSC-SPK1 group.Compared with EAE group,the ADMSC group and ADMSC-SPK1 group had significantly improved levels of IL-17A(
Subject(s)
Animals , Mice , Adipose Tissue/cytology , Cytokines , Encephalomyelitis, Autoimmune, Experimental/therapy , Interleukin-17 , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Mice, Inbred C57BL , Phosphotransferases (Alcohol Group Acceptor)/genetics , T-Lymphocytes, Regulatory/cytology , Th17 Cells/cytology , TransfectionABSTRACT
In recent years the role of sphingosine kinase 2 (SphK2), a key enzyme in the sphingolipid pathway, in the process of tumorigenesis has gradually been elucidated. Recent research has shown that SphK2 inhibitors can be used as anticancer drugs alone or in combination with existing drugs to increase the therapeutic sensitivity of drug-resistant tumors. Among them, one selective SphK2 inhibitor, ABC294640, shows excellent oral bioavailability and biodistribution in vivo and has now entered Phase II clinical research. Therefore, developing innovative drugs based on SphK2 is of great interest. Herein, we discuss progress in understanding the role of SphK2 in tumorigenesis and review the recent development of inhibitors of SphK2.
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Ischemic stroke is one of the most important causes of death and disability in humans,but the effective methods for treating brain injury after stroke are quite limited.Sphingosine 1-phosphate (S1P) is a pleiotropic lipid.There is certain interdependence between its metabolism and regulation and the molecular mechanisms involved in important biological events following cerebral ischemia.Membrane lipid therapy with S1P as the core may be an effective neuroprotective strategy of ischemic stroke.
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Objective: To evaluate the inhibitory role of a novel oncolytic adenovirus (OA), GP73-SphK1sR-Ad5, on the growth of hepatocellular carcinoma (HCC). Methods: GP73-SphK1sR-Ad5 was constructed by integrating Golgi protein 73 (GP73) promoter and sphingosine kinase 1 (SphK1)-short hairpin RNA (shRNA) into adenovirus serotype 5 (Ad5), and transfecting into HCC Huh7 cells and normal human liver HL-7702 cells. The expression of SphK1 and adenovirus early region 1 (E1A) was detected by quantitative real-time PCR (qRT-PCR) and western blot, respectively. Cell viability was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, and apoptotic rate was determined by flow cytometry. An Huh7 xenograft model was established in mice injected intratumorally with GP73-SphK1sR-Ad5. Twenty days after injection, the tumor volume and weight, and the survival time of the mice were recorded. The histopathological changes in tumor tissues were observed by hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM). Results: Transfection of GP73-SphK1sR-Ad5 significantly upregulated E1A and downregulated SphK1 in Huh7 cells, but not in HL7702 cells. GP73-SphK1sR-Ad5 transfection significantly decreased the viability and increased the apoptotic rate of Huh7 cells, but had no effect on HL7702 cells. Intratumoral injection of GP73-SphK1sR-Ad5 into the Huh7 xenograft mouse model significantly decreased tumor volume and weight, and prolonged survival time. It also significantly decreased the tumor infiltration area and blood vessel density, and increased the percentages of cells with nucleus deformation and cells with condensed chromatin in tumor tissues. Conclusions: GP73-SphK1sR-Ad5 serves as a novel OA and can inhibit HCC progression with high specificity and efficacy.
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Sphingosine 1-phosphate(SIP) is an important signal molecule produced by the metabolism of cell membrane sphingomyelin. SIP can be transported to the extracellular domain, and plays a role in activating a series of downstream signals, such as Ras/RafyERK, PI3K/AKT, by acting on SIP receptors. It can also act as a second messenger directly on intracellular targets such as HDAC, TRAF2. Its mediated signaling pathway plays a role in many physiological and pathological processes such as cell proliferation and migration, inflammatory cytokine infiltra tion, angiogenesis and autoimmunity. This article explores the role of SIP and its signaling pathways in inflammation-related diseases from the extracellular and intracellular roles of SIP, and reviews the research progress of SI P signaling pathway related drugs.
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OBJECTIVE@#To evaluate the inhibitory role of a novel oncolytic adenovirus (OA), GP73-SphK1sR-Ad5, on the growth of hepatocellular carcinoma (HCC).@*METHODS@#GP73-SphK1sR-Ad5 was constructed by integrating Golgi protein 73 (GP73) promoter and sphingosine kinase 1 (SphK1)-short hairpin RNA (shRNA) into adenovirus serotype 5 (Ad5), and transfecting into HCC Huh7 cells and normal human liver HL-7702 cells. The expression of SphK1 and adenovirus early region 1 (E1A) was detected by quantitative real-time PCR (qRT-PCR) and western blot, respectively. Cell viability was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, and apoptotic rate was determined by flow cytometry. An Huh7 xenograft model was established in mice injected intratumorally with GP73-SphK1sR-Ad5. Twenty days after injection, the tumor volume and weight, and the survival time of the mice were recorded. The histopathological changes in tumor tissues were observed by hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM).@*RESULTS@#Transfection of GP73-SphK1sR-Ad5 significantly upregulated E1A and downregulated SphK1 in Huh7 cells, but not in HL7702 cells. GP73-SphK1sR-Ad5 transfection significantly decreased the viability and increased the apoptotic rate of Huh7 cells, but had no effect on HL7702 cells. Intratumoral injection of GP73-SphK1sR-Ad5 into the Huh7 xenograft mouse model significantly decreased tumor volume and weight, and prolonged survival time. It also significantly decreased the tumor infiltration area and blood vessel density, and increased the percentages of cells with nucleus deformation and cells with condensed chromatin in tumor tissues.@*CONCLUSIONS@#GP73-SphK1sR-Ad5 serves as a novel OA and can inhibit HCC progression with high specificity and efficacy.
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Animals , Female , Mice , Adenoviridae , Apoptosis , Carcinoma, Hepatocellular/therapy , Cell Line, Tumor , Liver Neoplasms/therapy , Membrane Proteins/genetics , Mice, Inbred BALB C , Oncolytic Virotherapy/methods , Phosphotransferases (Alcohol Group Acceptor)/genetics , Promoter Regions, GeneticABSTRACT
BACKGROUND: Emerging evidence suggests that sphingolipids may be involved in type 2 diabetes. However, the exact signaling defect through which disordered sphingolipid metabolism induces β-cell dysfunction remains unknown. The current study demonstrated that sphingosine-1-phosphate (S1P), the product of sphingosine kinase (SphK), is an essential factor for maintaining β-cell function and survival via regulation of mitochondrial action, as mediated by prohibitin (PHB). METHODS: We examined β-cell function and viability, as measured by mitochondrial function, in mouse insulinoma 6 (MIN6) cells in response to manipulation of cellular S1P and PHB levels. RESULTS: Lack of S1P induced by sphingosine kinase inhibitor (SphKi) treatment caused β-cell dysfunction and apoptosis, with repression of mitochondrial function shown by decreases in cellular adenosine triphosphate content, the oxygen consumption rate, the expression of oxidative phosphorylation complexes, the mitochondrial membrane potential, and the expression of key regulators of mitochondrial dynamics (mitochondrial dynamin-like GTPase [OPA1] and mitofusin 1 [MFN1]). Supplementation of S1P led to the recovery of mitochondrial function and greatly improved β-cell function and viability. Knockdown of SphK2 using small interfering RNA induced mitochondrial dysfunction, decreased glucose-stimulated insulin secretion (GSIS), and reduced the expression of PHB, an essential regulator of mitochondrial metabolism. PHB deficiency significantly reduced GSIS and induced mitochondrial dysfunction, and co-treatment with S1P did not reverse these trends. CONCLUSION: Altogether, these data suggest that S1P is an essential factor in the maintenance of β-cell function and survival through its regulation of mitochondrial action and PHB expression.
Subject(s)
Animals , Mice , Adenosine Triphosphate , Apoptosis , GTP Phosphohydrolases , Insulin , Insulin-Secreting Cells , Insulinoma , Membrane Potential, Mitochondrial , Metabolism , Mitochondria , Mitochondrial Dynamics , Oxidative Phosphorylation , Oxygen Consumption , Phosphotransferases , Repression, Psychology , RNA, Small Interfering , Sphingolipids , SphingosineABSTRACT
@#[Abstract] Objective: To investigate the effect of sphingosine kinase 1 (SphK1) knockdown on the proliferation and migration of colon cancer RKO cells induced by mesenchymal stem cells (MSCs). Methods: RKO cells were treated with MSCs conditioned medium (MSC-CM) or control medium (Control-CM), respectively. Cell proliferation was detected by CCK-8 assay. Cell migration ability was tested by Transwell chamber assay. The proteins expression of Ki-67, MMP-2/9, CD44 and CD133 was detected by Western blotting. Then, the expression of SphK1 in RKO cells was suppressed by targeted gene lentivirus shRNA vector transfection. The effects of SphK1 knockdown on the proliferation, migration and protein expressions of Ki-67, MMP-2/9, CD44 and CD133 of RKO cells induced by MSC-CM were observed. Results: The RKO cells proliferation was promoted by MSC-CM in a time-dependent manner; moreover (P<0.05), the migration ability of cells was significantly enhanced after being treated with MSC-CM(P<0.01). In addition, MSC-CM significantly increased the protein expressions of Ki-67, MMP-2/9, CD44 and CD133(all P<0.05 or P<0.01). Lentiviral ShRNA vector transfection could significantly inhibit the expression of SphK1. Down-regulation of SphK1 significantly inhibited the proliferation, migration and protein expressions of Ki-67, MMP-2/9, CD44 and CD133 of RKO cells induced by MSC-CM(all P<0.05 or P<0.01). Conclusion: MSC-CM promotes the proliferation and migration of colon cancer RKO cells. Down-regulation of SphK1 reverses the cell proliferation and migration induced by MSC-CM via inhibiting the expression of MMP-2/9, CD44 and CD133.
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Objective To investigate the effect of moxifloxacin(MXF)on inflammatory reaction of mice abdominal peritoneal macrophage induced by LPS.Methods The level of TLR4、SPHK1 and NF-κB mRNA was determined by realtime-PCR;The relative levels of TLR4、SPHK1 and NF-κB were determined by Western blot.TNF-αand IL-1 se-creted by macrophages were detected by ELISA.Results At low and medium concentration(8,16 mg/L)of the MXF have inhibitory effect on the increasing of TLR 4,SPHK1,NF-κB expression level and cell supernatant of TNF-αand IL-1 in LPS stimulation of mice peritoneal macrophages.High concentration(64 mg/L)of the MXF promoted the role of inflammation.Conclusions The MXF of low and medium concentrations can inhibit the inflammatory response in LPS stimulation of mice peritoneal macrophages,and high concentration of the MXF showed contrary effects.
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Formation of liver fibrosis mainly involves activation of hepatic stellate cell and imbalance between synthesis and degradation of extracellular matrix.The sphingosine kinase (SphK)/sphingosine 1-phosphate (S1P)/sphingosine 1-phosphate receptors (S1PRs) signaling pathway plays an important role in the regulation of cell life activities including proliferation,migration,and inflammatory response.This article introduces the distribution and biological functions of SphK,S1 P,and S1PRs and elaborates on the research advances in mechanism of action of the SphK/S1P/and S1PRs signaling pathway in liver fibrosis.Many studies have confirmed the important role of the SphK/S1P/and S1PRs signaling pathway in liver fibrosis,and in-depth exploration helps to provide new thoughts for clinical treatment of liver fibrosis and development of new drug targets.
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Objective To examine the role of SPHK-1/S1P and NFκB p65 signal pathway in non-small cell lung cancer (NSCLC)drug-resistant cells.Methods The drug-resistant cell line of lung cancer H460/DDP was constructed and its biological characteristics were identified successfully.The expression of SPHK-l was tested by RT-PCR and Western blot methods.S1 P and some proteins related to NFκB pathway were studied by Western blot. Results The drug-resistant lung cancer cell line H460/DDP was constructed and its drug-resistant ability was evaluated (IC50H460/DDP = 50.62μg/mL, RIH460/DDP = 2.95 ). Cisplatin at a concentration of 10 - 80 μg/mL significantly decreased cell death of drug-resistant cell line (P<0 .01 ).Western blot assay analysis showed that overexpressions of SPHK-l,S1 P and NFκB p65 were significantly higher in drug-resistant cell line than in their parent cell line H460 (P=0.0415,P=0.0465,P=0.0218).RT-PCR method revealed that SPHK-1 mRNA was overexpressed in drug-resistant cell line compared with that in their parent cell line H460 (P<0.05).More NFκB p65 protein in cell nucleus was expressed in drug-resistant cells than in parent cells.Conclusion SPHK-1/S1P and NFκB p65 signal pathway may play an improtant role in the drug-resistant H460 cell line in non-small cell lung cancer.
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Aim To investigate the effect of sphingo-sine kinase 1 (SphK1 )on unilateral ureteral obstruc-tion(UUO)-induced tubulointerstitial fibrosis and ex-plore the possible mechanism.Methods The CD-1 mice were randomly divided into four groups:sham-op-eration group(Sham),PF-543 treatment control group (Sham +PF-543),model group(UUO)and PF-543 treatment group(UUO +PF-543).On 1 ,3,7 and 1 4 d after operation,eight mice were selected randomly from each group and sacrificed.The protein expressions of SphK1 ,mature TGF-β1 ,FN,ColⅠ,LC3,Beclin1 ,Atg5 and Atg1 2 were observed by Western blot.The histo-logical changes were examined by Masson′s trichrome stain.Immunhistochemistry was performed to measure the levels of expression of SphK1 ,FN and Col Ⅰ. Transmission electron microscope was used to observe the autophagic body.Results SphK1 expression and autophagy were both upregulated in a mouse model of kidney fibrosis induced by UUO. Meanwhile, in-creased mature TGF-β1 and deposition of extracellular matrix(ECM)were observed in tubulointerstitial areas compared with sham-operated mice.After intraperito-neal injection with the SphK1 specific inhibitor PF-543 in UUO mice,enhanced expression of SphK1 and acti-vated autophagy were significantly abrogated.Howev-er,aggravation of renal fibrosis was detected when SphK1 inhibitor PF-543 was applied to suppress SphK1 expression in UUO mice.Conclusion SphK1 activa-tion is renoprotective through the induction of autoph-agy in the pathogenesis of kidney fibrosis.
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Formation of liver fibrosis mainly involves activation of hepatic stellate cell and imbalance between synthesis and degradation of extracellular matrix.The sphingosine kinase (SphK)/sphingosine 1-phosphate (S1P)/sphingosine 1-phosphate receptors (S1PRs) signaling pathway plays an important role in the regulation of cell life activities including proliferation,migration,and inflammatory response.This article introduces the distribution and biological functions of SphK,S1 P,and S1PRs and elaborates on the research advances in mechanism of action of the SphK/S1P/and S1PRs signaling pathway in liver fibrosis.Many studies have confirmed the important role of the SphK/S1P/and S1PRs signaling pathway in liver fibrosis,and in-depth exploration helps to provide new thoughts for clinical treatment of liver fibrosis and development of new drug targets.
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AIM: To observe the effects of miR-542-5p on the proliferation of rat small intestine crypt epithe-lial IEC-6 cells induced by sphingosine-1-phosphate (S1P).METHODS: Two IEC-6 cell lines (SphK1-IEC-C1 and SphK1-IEC-C2) were established, which expressed sphingosine kinase-1 (SphK1) stably.Radioactive tracer was used to detect SphK1 activity and S1P secretion.The cell proliferation was observed by cell counting and described by drawing growth curve, and the cell cycle analysis was carried out by flow cytometry.The level of miR-542-5p was evaluated by RT-qPCR.RESULTS: Compared with control vector cells without SphK1 cDNA, both SphK1-IEC-C1 and SphK1-IEC-C2 cell lines showed that Sphk1 was elevated, both intracellular and extracellular S1P increased dramatically, the rate of cell growth was faster, the percentage of the cells in S phase increased, and miR-542-5p expression decreased.S1P (0.5~10 μmol/L) led to the decrease in miR-542-5p expression.On the contrary, SphK1 silencing resulted in the increase in miR-542-5p expression in the IEC-6 cells.The miR-542-5p was elevated in SphK1-IEC-C1 cells and SphK1-IEC-C2 cells, which caused the decrease in the percentage of the cells in S phase.The cell growth rate in the above-mentioned 2 cell lines decreased compared with negative control group.CONCLUSION: In IEC-6 cells, S1P promotes proliferation by inhibiting miR-542-5p expression, which induces the cell cycle transferring from G1 phase to S phase.
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Objective To investigate the impact of sphingosine kinase 1 (SPK1) modified adipose tissue-derived stromal cells(ADSC)on tissue engineered bone osteogenesis.Methods ADSC cells isolated from SD rat fat cells were divided into CON group and SPK1 group,and then the cells were respectively infected with 10 MOI CON and SPK1 lentivirus for 48 h.The infection efficiency was confirmed by using flow cytometry.Alizarin red and oil red O was used to stain the cells 14 days after ADSC infection,and osteogenic and adipogenic ability was evaluated by detecting A595nm and A490 nm.In the meantime,the activity change of alkaline phosphatase(ALP)was detected.The SD rat femoral defect model was created,and then after combining ADSC with β-TCP,the tissue engineered bone was pressed to the defect site.The repairment of bone defect was detected by X-ray in 4,6 weeks.After infection of CON and SPK1 virus,bone morphogenetic protein(BMP7)expression in ADSC of these two groups was detected.Results The infection efficiency of CON and SPK1 lentivirus was 94.4％ vs.94.9％ respectively by flow cytometry.The SPK1 protein expression level in CON group and SPK1 group was (0.73±0.10) vs.(1.29±0.17)(P＜0.05).The A value of CON and SPK1 group at 595 nm was (0.20±0.02) vs.(0.41±0.01) (P＜0.05),respectively.The A value of CON and SPK1 group at 490 nm was (0.72±0.01) vs.(0.51±0.02)(P＜0.05),respectively.The expression level of ALP in CON and SPK1 group was (1.42±-0.09) vs.(2.68±0.09) (P＜0.01),respectively.In the repairment of bone defect,high density tissue at rat bone defect was significantly larger in SPK1 group than in CON group in 4 weeks,and in 6 weeks,bone defect of SPK1 group was healed,but CON group still had defect.The expression level of BMP7 in CON and SPK1 group was (1.13±0.16) vs.(4.46±0.23)(P＜0.05),respectively,48 h after infection.Conclusion SPK1 modified ADSC has an enhanced osteogenesis ability in vitro and in vivo,which was related to the activation of BMP7.