Objective:To explore the effect and molecular mechanism of SPHK1 in the invasion and metastasis process of non-small-cell lung cancer cells(A549).Methods:Recombinant retrovirus was used to mediate the production of A5...Objective:To explore the effect and molecular mechanism of SPHK1 in the invasion and metastasis process of non-small-cell lung cancer cells(A549).Methods:Recombinant retrovirus was used to mediate the production of A549/vector,A549/SPHK1,A549/scramble,and A549/SPHK1/RNAi that stably expressed or silenced SPHK1.The invasion and migration capacities of A549 cells overexpressing or silencing SPHK1 were determined using Transwell invasion assay and scratch wound repair experiment.The protein and mRNA expression levels of E-cadherin,fibronectin,vimentin in A549/vector,A549/SPHK1,A549/scramble,A549/SPHK1/RNAi were detected with Western blot(WB) and quantitative PCR(QPCR) methods,respectively.Results:Transwell invasion assay and scratch wound repair experiments showed that over-expression of SPHK1 obviously enhanced the invasion and migration capacities of A549 cells.WB and QPCR detection results showed that,the expression of E-cadherin(a molecular marker of epithelial cells) and fibronectin,vimentin(molecular markers of mesenchymal cells) in A549 cells was upregulated after overexpression of SPHK1;while SPHK1 silencing significantly reduced the invasion and metastasis capacities of A549 cells,upregulated the expression of molecular marker of epithelial cells,and downregulated the expression of molecular marker of mesenchymal cells.Conclusions:SPHK1 promotes epithelial mesenchymal transition of non-small-cell lung cancer cells and affects the invasion and metastasis capacities of these cells.展开更多
Glioblastoma(GBM)is the most challenging malignant tumor of the central nervous system because of its high morbidity,mortality,and recurrence rate.Currently,mechanisms of GBM are still unclear and there is no effectiv...Glioblastoma(GBM)is the most challenging malignant tumor of the central nervous system because of its high morbidity,mortality,and recurrence rate.Currently,mechanisms of GBM are still unclear and there is no effective drug for GBM in the clinic.Therefore,it is urgent to identify new drug targets and corresponding drugs for GBM.In this study,in silico analyses and experimental data show that sphingosine kinase 1(SPHK1)is up-regulated in GBM patients,and is strongly correlated with poor prognosis and reduced overall survival.Overexpression of SPHK1 promoted the proliferation,invasion,metastasis,and clonogenicity of GBM cells,while silencing SPHK1 had the opposite effect.SPHK1 promoted inflammation through the NF-κB/IL-6/STAT3 signaling pathway and led to the phosphorylation of JNK,activating the JNK-JUN and JNK-ATF3 pathways and promoting inflammation and proliferation of GBM cells by transcriptional activation of PTX3.SPHK1 interacted with PTX3 and formed a positive feedback loop to reciprocally increase expression,promote inflammation and GBM growth.Inhibition of SPHK1 by the inhibitor,PF543,also decreased tumorigenesis in the U87-MG and U251-MG SPHK1 orthotopic mouse models.In summary,we have characterized the role and molecular mechanisms by which SPHK1 promotes GBM,which may provide opportunities for SPHK1-targeted therapy.展开更多
Objective:To elucidate the expression of SPHK1 in gastric cancer tissues and to analyse its clinical value in gastric cancer using bioinformatics.Methods:The expression of SPHK1 in gastric cancer tissues was analysed ...Objective:To elucidate the expression of SPHK1 in gastric cancer tissues and to analyse its clinical value in gastric cancer using bioinformatics.Methods:The expression of SPHK1 in gastric cancer tissues was analysed using HPA and TCGA databases.The association between the expression of both SPHK1 and MKI67 in gastric cancer was analysed using GEPIA database.Based on Kaplan-Meier Plotter database to analyse the relationship between SPHK1 mRNA expression level and survival of gastric cancer patients.Gene enrichment pathway predicted the potential biological function of SPHK1 on gastric cancer.The TIMER database was used to predict the expression of SPHK1 in immune-infiltrating cells.The expression of SPHK1 in gastric cancer tissues was analysed by IHC and Western Blot.Results:Bioinformatics analysis showed that SPHK1 was highly expressed in gastric cancer tissues compared to paracancerous tissues(P<0.001).The analysis from the GEPIA database reveals a significant increase in the expression of SPHK1 and MKI67 in gastric cancer tissues(P<0.05).Furthermore,there is a positive correlation between the two proteins(P=0.00049).Kaplan-Meier Plotter survival analysis showed that high expression of SPHK1 predicted worse overall survival tine(logrankP=0.002)and post-progression generation time(logrankP=0.0019)in gastric cancer.Gene enrichment pathway results showed that SPHK1 may be involved in multiple gastric cancer progression pathways.TIMER database analysis showed that in macrophages,gastric cancer patients with low SPHK1 expression group had a better post-progression period than those with high SPHK1 expression group(P=0.002).IHC showed that the staining intensity and staining positive area of SPHK1 in gastric cancer tissues were higher than that of adjacent non-tumour tissues.And the staining intensity of TNMI and Ⅱ stages was weaker than that of Ⅲ and Ⅳ stages.Western blot analysis revealed higher levels of SPHK1 protein in gastric cancer tissue compared to paired adjacent non-cancerous tissue in 9 cases(P=0.02展开更多
Graft-versus-host disease (GVHD) significantly contributes to patient morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HSCT). Sphingosine-1-phosphate (S1P) signaling is involved in the...Graft-versus-host disease (GVHD) significantly contributes to patient morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HSCT). Sphingosine-1-phosphate (S1P) signaling is involved in the biogenetic processes of different immune cells. In the current study, we demonstrated that recipient sphingosine kinase 1 (Sphk1), but not Sphk2, was required for optimal S1PR1-dependent donor T-cell allogeneic responses by secreting S1P. Using genetic and pharmacologic approaches, we demonstrated that inhibition of Sphk1 or S1PR1 substantially attenuated acute GVHD (aGVHD) while retaining the graft-versus-leukemia (GVL) effect. At the cellular level, the Sphk1/S1P/S1PR1 pathway differentially modulated the alloreactivity of CD4+ and CD8+ T cells;it facilitated T-cell differentiation into Th1/Th17 cells but not Tregs and promoted CD4+ T-cell infiltration into GVHD target organs but was dispensable for the CTL activity of allogeneic CD8+ T cells. At the molecular level, the Sphk1/S1P/S1PR1 pathway augmented mitochondrial fission and increased mitochondrial mass in allogeneic CD4+ but not CD8+ T cells by activating the AMPK/AKT/mTOR/Drp1 pathway, providing a mechanistic basis for GVL maintenance when S1P signaling was inhibited. For translational purposes, we detected the regulatory efficacy of pharmacologic inhibitors of Sphk1 and S1PR1 in GVHD induced by human T cells in a xenograft model. Our study provides novel mechanistic insight into how the Sphk1/S1P/S1PR1 pathway modulates T-cell alloreactivity and validates Sphk1 or S1PR1 as a therapeutic target for the prevention of GVHD and leukemia relapse. This novel strategy may be readily translated into the clinic to benefit patients with hematologic malignancies and disorders.展开更多
基金supported by the Key Projects of Hunan Provincial Edueation Department with the number of 13A103
文摘Objective:To explore the effect and molecular mechanism of SPHK1 in the invasion and metastasis process of non-small-cell lung cancer cells(A549).Methods:Recombinant retrovirus was used to mediate the production of A549/vector,A549/SPHK1,A549/scramble,and A549/SPHK1/RNAi that stably expressed or silenced SPHK1.The invasion and migration capacities of A549 cells overexpressing or silencing SPHK1 were determined using Transwell invasion assay and scratch wound repair experiment.The protein and mRNA expression levels of E-cadherin,fibronectin,vimentin in A549/vector,A549/SPHK1,A549/scramble,A549/SPHK1/RNAi were detected with Western blot(WB) and quantitative PCR(QPCR) methods,respectively.Results:Transwell invasion assay and scratch wound repair experiments showed that over-expression of SPHK1 obviously enhanced the invasion and migration capacities of A549 cells.WB and QPCR detection results showed that,the expression of E-cadherin(a molecular marker of epithelial cells) and fibronectin,vimentin(molecular markers of mesenchymal cells) in A549 cells was upregulated after overexpression of SPHK1;while SPHK1 silencing significantly reduced the invasion and metastasis capacities of A549 cells,upregulated the expression of molecular marker of epithelial cells,and downregulated the expression of molecular marker of mesenchymal cells.Conclusions:SPHK1 promotes epithelial mesenchymal transition of non-small-cell lung cancer cells and affects the invasion and metastasis capacities of these cells.
基金This work was supported by Beijing Natural Science Foundation(7212157,China)This work was also supported by CAMS Innovation Fund for Medical Sciences(2021-I2M-1-029 and 2022-12M-JB-011,China)National Natural Science Foundation of China(81703536,China).
文摘Glioblastoma(GBM)is the most challenging malignant tumor of the central nervous system because of its high morbidity,mortality,and recurrence rate.Currently,mechanisms of GBM are still unclear and there is no effective drug for GBM in the clinic.Therefore,it is urgent to identify new drug targets and corresponding drugs for GBM.In this study,in silico analyses and experimental data show that sphingosine kinase 1(SPHK1)is up-regulated in GBM patients,and is strongly correlated with poor prognosis and reduced overall survival.Overexpression of SPHK1 promoted the proliferation,invasion,metastasis,and clonogenicity of GBM cells,while silencing SPHK1 had the opposite effect.SPHK1 promoted inflammation through the NF-κB/IL-6/STAT3 signaling pathway and led to the phosphorylation of JNK,activating the JNK-JUN and JNK-ATF3 pathways and promoting inflammation and proliferation of GBM cells by transcriptional activation of PTX3.SPHK1 interacted with PTX3 and formed a positive feedback loop to reciprocally increase expression,promote inflammation and GBM growth.Inhibition of SPHK1 by the inhibitor,PF543,also decreased tumorigenesis in the U87-MG and U251-MG SPHK1 orthotopic mouse models.In summary,we have characterized the role and molecular mechanisms by which SPHK1 promotes GBM,which may provide opportunities for SPHK1-targeted therapy.
基金Anhui Province Higher Education Natural Science Research Project(No.KJ2021ZD0090)Bengbu Medical College Graduate Research Innovation Project(No.Byycx22090)。
文摘Objective:To elucidate the expression of SPHK1 in gastric cancer tissues and to analyse its clinical value in gastric cancer using bioinformatics.Methods:The expression of SPHK1 in gastric cancer tissues was analysed using HPA and TCGA databases.The association between the expression of both SPHK1 and MKI67 in gastric cancer was analysed using GEPIA database.Based on Kaplan-Meier Plotter database to analyse the relationship between SPHK1 mRNA expression level and survival of gastric cancer patients.Gene enrichment pathway predicted the potential biological function of SPHK1 on gastric cancer.The TIMER database was used to predict the expression of SPHK1 in immune-infiltrating cells.The expression of SPHK1 in gastric cancer tissues was analysed by IHC and Western Blot.Results:Bioinformatics analysis showed that SPHK1 was highly expressed in gastric cancer tissues compared to paracancerous tissues(P<0.001).The analysis from the GEPIA database reveals a significant increase in the expression of SPHK1 and MKI67 in gastric cancer tissues(P<0.05).Furthermore,there is a positive correlation between the two proteins(P=0.00049).Kaplan-Meier Plotter survival analysis showed that high expression of SPHK1 predicted worse overall survival tine(logrankP=0.002)and post-progression generation time(logrankP=0.0019)in gastric cancer.Gene enrichment pathway results showed that SPHK1 may be involved in multiple gastric cancer progression pathways.TIMER database analysis showed that in macrophages,gastric cancer patients with low SPHK1 expression group had a better post-progression period than those with high SPHK1 expression group(P=0.002).IHC showed that the staining intensity and staining positive area of SPHK1 in gastric cancer tissues were higher than that of adjacent non-tumour tissues.And the staining intensity of TNMI and Ⅱ stages was weaker than that of Ⅲ and Ⅳ stages.Western blot analysis revealed higher levels of SPHK1 protein in gastric cancer tissue compared to paired adjacent non-cancerous tissue in 9 cases(P=0.02
基金This work is supported in part by SmartState Cancer Stem Cell Biology&Therapy Program and by R01 grants from the National Institutes of Health,including AI118305,HL140953 and CA258440(X.-Z.Y.).
文摘Graft-versus-host disease (GVHD) significantly contributes to patient morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HSCT). Sphingosine-1-phosphate (S1P) signaling is involved in the biogenetic processes of different immune cells. In the current study, we demonstrated that recipient sphingosine kinase 1 (Sphk1), but not Sphk2, was required for optimal S1PR1-dependent donor T-cell allogeneic responses by secreting S1P. Using genetic and pharmacologic approaches, we demonstrated that inhibition of Sphk1 or S1PR1 substantially attenuated acute GVHD (aGVHD) while retaining the graft-versus-leukemia (GVL) effect. At the cellular level, the Sphk1/S1P/S1PR1 pathway differentially modulated the alloreactivity of CD4+ and CD8+ T cells;it facilitated T-cell differentiation into Th1/Th17 cells but not Tregs and promoted CD4+ T-cell infiltration into GVHD target organs but was dispensable for the CTL activity of allogeneic CD8+ T cells. At the molecular level, the Sphk1/S1P/S1PR1 pathway augmented mitochondrial fission and increased mitochondrial mass in allogeneic CD4+ but not CD8+ T cells by activating the AMPK/AKT/mTOR/Drp1 pathway, providing a mechanistic basis for GVL maintenance when S1P signaling was inhibited. For translational purposes, we detected the regulatory efficacy of pharmacologic inhibitors of Sphk1 and S1PR1 in GVHD induced by human T cells in a xenograft model. Our study provides novel mechanistic insight into how the Sphk1/S1P/S1PR1 pathway modulates T-cell alloreactivity and validates Sphk1 or S1PR1 as a therapeutic target for the prevention of GVHD and leukemia relapse. This novel strategy may be readily translated into the clinic to benefit patients with hematologic malignancies and disorders.
