Aim This study is aimed to determine whether SPK2 (Sphingosine kinase-2) is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons. We also examined whether SPK2 pro- tects n...Aim This study is aimed to determine whether SPK2 (Sphingosine kinase-2) is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons. We also examined whether SPK2 pro- tects neurons from ischemic injury by activating autophagy, and explored the molecular mechanism of SPK2 contrib- uting to the autophagy activation in neurons. Methods Isoflurane preconditioning (ISO) and oxygen glucose dep- rivation (OGD) model was established in primary cultured murine cortical neurons. Neurons were transfected by siRNA to interfere SPK2 and Beclin 1, or lentivirus to overexpress SPK2. Protein expression of SPK2, LC3, and Beclinl were detected with immunofluorescence and Western blot analysis. The neurons were treated with lysosomal inhibitor ammonium chloride (NH4 C1) to test the autophagy flux. The protection of SPK2 on OGD/R induced neu- ronal death was detected with CCK-8 (cell counting kit-8) and LDH cytotoxicity assay kit. Autophagy inhibitor 3- MA (3-Methyladenine) was used to detect the protection of autophagy on SPK2 induced isehemie tolerance. Co-im- munoprecipitation was used to detect the interaction between Beclin 1 and Bel-2. Results In primary cultured neu- ISO enhanced SPK2 and LC3 immunofluorescenee. SPK2 siRNA inhibits LC3II upregulation induced by rons, ISO. Beclin 1 siRNA also inhibits LC3II upregulation induced by ISO. Lentivirus-indueed SPK2 overexpression in- creased LC3II/LC3I ratio and enhanced the autophagy flux in neurons. SPK2 overexpression also exerted neuropro- teetion against OGD model in cortical neurons, as evidenced by improvement of neuronal morphology, increased cellular viability and reduced LDH leakage, while 3-MA partly abolished the SPK2-induced neuroprotection. After SPK2 overexpression, Beclin 1 siRNA inhibited SPK2 induced LC3II upregulation, and the coimmunoprecipitation of Beclin 1 and Bcl-2 was reduced.. Conclusion ISO increases SPK2 and activates autophagy in neurons. SPK2 or Beclin 1 interference cancels ISO induced autophagy activation. SPK2 overexpression activates autophagy, and protects the neurons against ischemic injury. SPK2 may induce autophagy by disrupting Beclin 1/Bcl-2 interaction.展开更多
Intraocular pressure elevation can induce retinal ganglion cell death and is a clinically reversible risk factor for glaucoma,the leading cause of irreversible blindness.We previously demonstrated that casein kinase-2...Intraocular pressure elevation can induce retinal ganglion cell death and is a clinically reversible risk factor for glaucoma,the leading cause of irreversible blindness.We previously demonstrated that casein kinase-2 inhibition can promote retinal ganglion cell survival and axonal regeneration in rats after optic nerve injury.To investigate the underlying mechanism,in the current study we increased the intraocular pressure of adult rats to 75 mmHg for 2 hours and then administered a casein kinase-2 inhibitor(4,5,6,7-tetrabromo-2-azabenzimidazole or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole)by intravitreal injection.We found that intravitreal injection of 4,5,6,7-tetrabromo-2-azabenzimidazole or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole promoted retinal ganglion cell survival and reduced the number of infiltrating macrophages.Transcriptomic analysis showed that the mitogen activated protein kinase signaling pathway was involved in the response to intraocular pressure elevation but was not modulated by the casein kinase-2 inhibitors.Furthermore,casein kinase-2 inhibition downregulated the expression of genes(Cck,Htrsa,Nef1,Htrlb,Prph,Chat,Slc18a3,Slc5a7,Scn1b,Crybb2,Tsga10ip,and Vstm21)involved in intraocular pressure elevation.Our data indicate that inhibition of casein kinase-2 can enhance retinal ganglion cell survival in rats after acute intraocular pressure elevation via macrophage inactivation.展开更多
BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC...BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC).AIM To investigate the role and molecular mechanism of miRNA-145-5p(miR145-5p)in the progression of GC.METHODS Real-time polymerase chain reaction(RT-PCR)was used to detect miRNA expression in human GC tissues and cells.The ability of cancer cells to migrate and invade was assessed using wound-healing and transwell assays,respectively.Cell proliferation was measured using cell counting kit-8 and colony formation assays,and apoptosis was evaluated using flow cytometry.Expression of the epithelial-mesenchymal transition(EMT)-associated protein was determined by Western blot.Targets of miR-145-5p were predicated using bioinformatics analysis and verified using a dual-luciferase reporter system.Serpin family E member 1(SERPINE1)expression in GC tissues and cells was evaluated using RT-PCR and immunohistochemical staining.The correlation between SERPINE1 expression and overall patient survival was determined using Kaplan-Meier plot analysis.The association between SERPINE1 and GC progression was also tested.A rescue experiment of SERPINE1 overexpression was conducted to verify the relationship between this protein and miR-145-5p.The mechanism by which miR-145-5p influences GC progression was further explored by assessing tumor formation in nude mice.RESULTS GC tissues and cells had reduced miR-145-5p expression and SERPINE1 was identified as a direct target of this miRNA.Overexpression of miR-145-5p was associated with decreased GC cell proliferation,invasion,migration,and EMT,and these effects were reversed by forcing SERPINE1 expression.Kaplan-Meier plot analysis revealed that patients with higher SERPINE1 expression had a shorter survival rate than those with lower SERPINE1 expression.Nude mouse tumorigenesis experiments confirmed that miR-145-5p targets SERPINE1 to regulate extracellular signal-regulated kinase-1/2(ERK1/2).CONCLUSION This study found that miR-145-5p inhibits tumor progression and is expressed in lower amounts in patients with GC.MiR-145-5p was found to affect GC cell proliferation,migration,and invasion by negatively regulating SERPINE1 levels and controlling the ERK1/2 pathway.展开更多
AIM: To examine the expression of Sph K1, an oncogenic kinase that produces sphingosine 1-phosphate(S1P), and its correlation with the expression of LPAR2, a major lysophosphatidic acid(LPA) receptor overexpressed in ...AIM: To examine the expression of Sph K1, an oncogenic kinase that produces sphingosine 1-phosphate(S1P), and its correlation with the expression of LPAR2, a major lysophosphatidic acid(LPA) receptor overexpressed in various cancers, in human colorectal cancer.METHODS: Real-time reverse-transcription polymerase chain reaction was used to measure the m RNA expression of Sph K1, LPAR2, and the three major S1 P receptors in 27 colorectal cancer samples and corresponding normal tissue samples. We also examined the correlation between the expression of Sph K1 and LPAR2.RESULTS: C o l o r e c t a l c a n c e r t i s s u e i n 2 2 o f 2 7 patients had higher levels of Sph K1 m RNA than in normal tissue. In two-thirds of the samples, Sph K1 m RNA expression was more than two-fold higher than in normal tissue. Consistent with previous reports, LPAR2 m RNA expression in 20 of 27 colorectal cancer tissue samples was higher compared to normal tissue samples. Expression profiles of all three major S1 P receptors, S1PR1, S1PR2, and S1PR3, varied without any trend, with no significant difference in expression between cancer and normal tissues. A highly significant positive correlation was found between Sph K1 and LPAR2 expression [Pearson's correlation coefficient(r) = 0.784 and P < 0.01]. The m RNA levels of Sph K1 and LPAR2 did not correlate with TNM stage.CONCLUSION: Our findings suggest that S1 P andLPA may play important roles in the development ofcolorectal cancer via the upregulation of Sph K1 andLPAR2, both of which could serve as new therapeutictargets in the treatment of colorectal cancer.展开更多
Objective To investigate the role of extracellular signal-regulated kinase (ERK) in apoptosis of human colon cancer (HCT116) cells. Methods After the HCT116 cells were pretreated with specific ERK inhibitor (U012...Objective To investigate the role of extracellular signal-regulated kinase (ERK) in apoptosis of human colon cancer (HCT116) cells. Methods After the HCT116 cells were pretreated with specific ERK inhibitor (U0126) or specific siRNA and exposed to 10 mmol/L sodium butyrate (NaBT) for 24 h, their apoptosis was detected by flow cytometry, levels of SphK2 and ERK protein were measured by Western blot, and translocation of SphK2 was assayed by immunofluorescence microscopy. Results The U0126 and siRNAs specific for SphK2 blocked the export of SphK2 from nuclei to cytoplasm and increased the apoptosis of HCT116 cells following NaBT exposure. Over-expression of PKD decreased NaBT-induced apoptosis of HCT116 cells, which was reversed by U0126. Furthermore, transfection of HCT116 cells with constitutively activated PKD plasmids recovered the UO126-blocked export of SphK2. Conclusion ERK regulates the export of SphK2 and apoptosis of HCT116 cells by modulating PKD. Modulation of these molecules may help increase the sensitivity of colon cancer cells to the physiologic anti-colon cancer agent, NaBT.展开更多
目的应用生物信息学方法探讨鞘氨醇-1-磷酸转运体2(sphingosine-1-phosphate transporter 2,SPNS2)在乳腺癌中的表达情况,并分析SPNS2对乳腺癌预后、诊断或免疫浸润的影响。方法癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库检...目的应用生物信息学方法探讨鞘氨醇-1-磷酸转运体2(sphingosine-1-phosphate transporter 2,SPNS2)在乳腺癌中的表达情况,并分析SPNS2对乳腺癌预后、诊断或免疫浸润的影响。方法癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库检索乳腺癌组织和非乳腺癌样本中的SPNS2 mRNA差异表达数据,并分析SPNS2与乳腺癌之间的关系。用Cox单因素及多因素模型分析SPNS2 mRNA表达对乳腺癌预后的影响。使用Kaplan-Meier曲线评估SPNS2基因的表达与存活率之间的相关性,分析SPNS2对乳腺癌患者生存预后的影响。使用受试者工作特征曲线(receiver operating characteristic,ROC)分析SPNS2对乳腺癌的诊断效能。使用肿瘤免疫估算资源(Tumor Immune Estimation Resource,TIMER)数据库分析SPNS2表达与乳腺癌免疫微环境中不同类型免疫细胞的相关性。搜索相互作用基因检索的工具(Search Tool for the Retrieval of Interacting Genes,STRING)数据库分析乳腺癌中SPNS2与相关蛋白质之间的相互作用。分析京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)数据库中得到的差异基因富集的信号通路。提取正常乳腺上皮细胞和乳腺癌细胞系RNA,通过RT-qPCR实验比较SPNS2的表达水平。结果在TCGA乳腺癌数据库中,SPNS2在乳腺癌组织中表达水平显著低于癌旁组织(P<0.001),SPNS2 mRNA的表达与T、M分期、病理分期、PAM50分型、年龄和组织学类型等因素相关(P<0.05);Cox分析表明年龄>60岁、T4期、M1期等因素是乳腺癌发生预后不良的风险因素(P<0.01);Kaplan-Meier分析显示低表达SPNS2乳腺癌患者具有更长的疾病特异生存期(disease-specific survival,DSS)和无进展间隔期(progression-free interval,PFI)(P<0.05);ROC曲线提示SPNS2诊断具有较好的敏感性和特异性。TIMER数据库分析显示在Luminal型乳腺癌中,SPNS2与CD4+T细胞,巨噬细胞、中性粒细胞等免疫细胞呈正相关(P<0.05)。STRING和KEGG数据库分析表明SPNS2相关蛋白富集于细胞周期和PPAR信号传导等途径(P<0.05)。RT-qPCR实验结果显示与正常乳腺上皮细胞相比,SPNS2在乳腺癌细胞系中低表达(P<0.001)。结论SPNS2是一种潜在的诊断乳腺癌和评价预后的生物学标志物。展开更多
Objective:Cardiac remodeling is a common pathological change in various cardiovascular diseases and can ultimately result in heart failure.Thus,there is an urgent need for more effective strategies to aid in cardiac p...Objective:Cardiac remodeling is a common pathological change in various cardiovascular diseases and can ultimately result in heart failure.Thus,there is an urgent need for more effective strategies to aid in cardiac protection.Our previous work found that sphingosine-1-phosphate(S1P)could ameliorate cardiac hypertrophy.In this study,we aimed to investigate whether S1P could prevent cardiac fibrosis and the associated mechanisms in cardiac remodeling.Methods:Eight-week-old male C57BL/6 mice were randomly divided into a sham,transverse aortic constriction(TAC)or a TAC+S1P treatment group.Results:We found that S1P treatment improved cardiac function in TAC mice and that the cardiac fibrosis ratio in the TAC+S1P group was significantly lower and was accompanied by a decrease inα-smooth muscle actin(α-SMA)and collagen type I(COL I)expression compared with the TAC group.We also found that one of the key S1P enzymes,sphingosine kinase 2(SphK2),which was mainly distributed in cytoblasts,was downregulated in the cardiac remodeling case and recovered after S1P treatment in vivo and in vitro.In addition,our in vitro results showed that S1P treatment activated extracellular regulated protein kinases(ERK)phosphorylation mainly through the S1P receptor 2(S1PR2)and spurred p-ERK transposition from the cytoplasm to cytoblast in H9c2 cells exposed to phenylephrine.Conclusion:These findings suggest that SphK2 and the S1PR2/ERK pathway may participate in the anti-remodeling effect of S1P on the heart.This work therefore uncovers a novel potential therapy for the prevention of cardiac remodeling.展开更多
AIM: To elucidate the mechanisms of mesenteric vasodilation in portal hypertension (PHT), with a focus on endothelin signaling. METHODS: PHT was induced in rats by common bile duct ligation (CBDL). Portal pressure (PP...AIM: To elucidate the mechanisms of mesenteric vasodilation in portal hypertension (PHT), with a focus on endothelin signaling. METHODS: PHT was induced in rats by common bile duct ligation (CBDL). Portal pressure (PP) was measured directly via catheters placed in the portal vein tract. The level of endothelin-1 (ET-1) in the mesenteric circulation was determined by radioimmunoassay, and the expression of the endothelin A receptor (ETAR) and endothelin B receptor (ETBR) was assessed by immunofluorescence and Western blot. Additionally, expression of G protein coupled kinase-2 (GRK2) and β-arrestin 2, which influence endothelin receptor sensitivity, were also studied by Western blot. RESULTS: PP of CBDL rats increased significantly (11.89 ± 1.38 mmHg vs 16.34 ± 1.63 mmHg). ET-1 expression decreased in the mesenteric circulation 2 and 4 wk after CBDL. ET-1 levels in the systemic circulation of CBDL rats were increased at 2 wk and decreased at 4 wk. There was no change in ETAR expression in response to CBDL; however, increased expression of ETBR in the endothelial cells of mesenteric arterioles and capillaries was observed. In sham-operated rats, ETBR was mainly expressed in the CD31+ endothelial cells of the arterioles. With development of PHT, in addition to the endothelial cells, ETBR expression was noticeably detectable in the SMA+ smooth muscle cells of arterioles and in the CD31+ capillaries. Following CBDL, increased expression of GRK2 was also found in mesenteric tissue, though there was no change in the level of β-arrestin 2. CONCLUSION: Decreased levels of ET-1 and increased ETBR expression in the mesenteric circulation following CBDL in rats may underlie mesenteric vasodilation in individuals with PHT. Mechanistically, increased GRK2 expression may lead to desensitization of ETAR, as well as other vasoconstrictors, promoting this vasodilatory effect.展开更多
文摘Aim This study is aimed to determine whether SPK2 (Sphingosine kinase-2) is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons. We also examined whether SPK2 pro- tects neurons from ischemic injury by activating autophagy, and explored the molecular mechanism of SPK2 contrib- uting to the autophagy activation in neurons. Methods Isoflurane preconditioning (ISO) and oxygen glucose dep- rivation (OGD) model was established in primary cultured murine cortical neurons. Neurons were transfected by siRNA to interfere SPK2 and Beclin 1, or lentivirus to overexpress SPK2. Protein expression of SPK2, LC3, and Beclinl were detected with immunofluorescence and Western blot analysis. The neurons were treated with lysosomal inhibitor ammonium chloride (NH4 C1) to test the autophagy flux. The protection of SPK2 on OGD/R induced neu- ronal death was detected with CCK-8 (cell counting kit-8) and LDH cytotoxicity assay kit. Autophagy inhibitor 3- MA (3-Methyladenine) was used to detect the protection of autophagy on SPK2 induced isehemie tolerance. Co-im- munoprecipitation was used to detect the interaction between Beclin 1 and Bel-2. Results In primary cultured neu- ISO enhanced SPK2 and LC3 immunofluorescenee. SPK2 siRNA inhibits LC3II upregulation induced by rons, ISO. Beclin 1 siRNA also inhibits LC3II upregulation induced by ISO. Lentivirus-indueed SPK2 overexpression in- creased LC3II/LC3I ratio and enhanced the autophagy flux in neurons. SPK2 overexpression also exerted neuropro- teetion against OGD model in cortical neurons, as evidenced by improvement of neuronal morphology, increased cellular viability and reduced LDH leakage, while 3-MA partly abolished the SPK2-induced neuroprotection. After SPK2 overexpression, Beclin 1 siRNA inhibited SPK2 induced LC3II upregulation, and the coimmunoprecipitation of Beclin 1 and Bcl-2 was reduced.. Conclusion ISO increases SPK2 and activates autophagy in neurons. SPK2 or Beclin 1 interference cancels ISO induced autophagy activation. SPK2 overexpression activates autophagy, and protects the neurons against ischemic injury. SPK2 may induce autophagy by disrupting Beclin 1/Bcl-2 interaction.
基金supported by the National Natural Science Foundation of China,Nos.81570849,81100931the Natural Science Foundation of Guangdong Province of China,Nos.2015A030313446,2020A1515011413(all to LPC).
文摘Intraocular pressure elevation can induce retinal ganglion cell death and is a clinically reversible risk factor for glaucoma,the leading cause of irreversible blindness.We previously demonstrated that casein kinase-2 inhibition can promote retinal ganglion cell survival and axonal regeneration in rats after optic nerve injury.To investigate the underlying mechanism,in the current study we increased the intraocular pressure of adult rats to 75 mmHg for 2 hours and then administered a casein kinase-2 inhibitor(4,5,6,7-tetrabromo-2-azabenzimidazole or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole)by intravitreal injection.We found that intravitreal injection of 4,5,6,7-tetrabromo-2-azabenzimidazole or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole promoted retinal ganglion cell survival and reduced the number of infiltrating macrophages.Transcriptomic analysis showed that the mitogen activated protein kinase signaling pathway was involved in the response to intraocular pressure elevation but was not modulated by the casein kinase-2 inhibitors.Furthermore,casein kinase-2 inhibition downregulated the expression of genes(Cck,Htrsa,Nef1,Htrlb,Prph,Chat,Slc18a3,Slc5a7,Scn1b,Crybb2,Tsga10ip,and Vstm21)involved in intraocular pressure elevation.Our data indicate that inhibition of casein kinase-2 can enhance retinal ganglion cell survival in rats after acute intraocular pressure elevation via macrophage inactivation.
文摘BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC).AIM To investigate the role and molecular mechanism of miRNA-145-5p(miR145-5p)in the progression of GC.METHODS Real-time polymerase chain reaction(RT-PCR)was used to detect miRNA expression in human GC tissues and cells.The ability of cancer cells to migrate and invade was assessed using wound-healing and transwell assays,respectively.Cell proliferation was measured using cell counting kit-8 and colony formation assays,and apoptosis was evaluated using flow cytometry.Expression of the epithelial-mesenchymal transition(EMT)-associated protein was determined by Western blot.Targets of miR-145-5p were predicated using bioinformatics analysis and verified using a dual-luciferase reporter system.Serpin family E member 1(SERPINE1)expression in GC tissues and cells was evaluated using RT-PCR and immunohistochemical staining.The correlation between SERPINE1 expression and overall patient survival was determined using Kaplan-Meier plot analysis.The association between SERPINE1 and GC progression was also tested.A rescue experiment of SERPINE1 overexpression was conducted to verify the relationship between this protein and miR-145-5p.The mechanism by which miR-145-5p influences GC progression was further explored by assessing tumor formation in nude mice.RESULTS GC tissues and cells had reduced miR-145-5p expression and SERPINE1 was identified as a direct target of this miRNA.Overexpression of miR-145-5p was associated with decreased GC cell proliferation,invasion,migration,and EMT,and these effects were reversed by forcing SERPINE1 expression.Kaplan-Meier plot analysis revealed that patients with higher SERPINE1 expression had a shorter survival rate than those with lower SERPINE1 expression.Nude mouse tumorigenesis experiments confirmed that miR-145-5p targets SERPINE1 to regulate extracellular signal-regulated kinase-1/2(ERK1/2).CONCLUSION This study found that miR-145-5p inhibits tumor progression and is expressed in lower amounts in patients with GC.MiR-145-5p was found to affect GC cell proliferation,migration,and invasion by negatively regulating SERPINE1 levels and controlling the ERK1/2 pathway.
基金Supported by Grant 2010 from Tokyo MetropolisJapan
文摘AIM: To examine the expression of Sph K1, an oncogenic kinase that produces sphingosine 1-phosphate(S1P), and its correlation with the expression of LPAR2, a major lysophosphatidic acid(LPA) receptor overexpressed in various cancers, in human colorectal cancer.METHODS: Real-time reverse-transcription polymerase chain reaction was used to measure the m RNA expression of Sph K1, LPAR2, and the three major S1 P receptors in 27 colorectal cancer samples and corresponding normal tissue samples. We also examined the correlation between the expression of Sph K1 and LPAR2.RESULTS: C o l o r e c t a l c a n c e r t i s s u e i n 2 2 o f 2 7 patients had higher levels of Sph K1 m RNA than in normal tissue. In two-thirds of the samples, Sph K1 m RNA expression was more than two-fold higher than in normal tissue. Consistent with previous reports, LPAR2 m RNA expression in 20 of 27 colorectal cancer tissue samples was higher compared to normal tissue samples. Expression profiles of all three major S1 P receptors, S1PR1, S1PR2, and S1PR3, varied without any trend, with no significant difference in expression between cancer and normal tissues. A highly significant positive correlation was found between Sph K1 and LPAR2 expression [Pearson's correlation coefficient(r) = 0.784 and P < 0.01]. The m RNA levels of Sph K1 and LPAR2 did not correlate with TNM stage.CONCLUSION: Our findings suggest that S1 P andLPA may play important roles in the development ofcolorectal cancer via the upregulation of Sph K1 andLPAR2, both of which could serve as new therapeutictargets in the treatment of colorectal cancer.
基金supported by National Natural Science Foundation of China(81272180)National Basic Research Program of China(2012CB518200)
文摘Objective To investigate the role of extracellular signal-regulated kinase (ERK) in apoptosis of human colon cancer (HCT116) cells. Methods After the HCT116 cells were pretreated with specific ERK inhibitor (U0126) or specific siRNA and exposed to 10 mmol/L sodium butyrate (NaBT) for 24 h, their apoptosis was detected by flow cytometry, levels of SphK2 and ERK protein were measured by Western blot, and translocation of SphK2 was assayed by immunofluorescence microscopy. Results The U0126 and siRNAs specific for SphK2 blocked the export of SphK2 from nuclei to cytoplasm and increased the apoptosis of HCT116 cells following NaBT exposure. Over-expression of PKD decreased NaBT-induced apoptosis of HCT116 cells, which was reversed by U0126. Furthermore, transfection of HCT116 cells with constitutively activated PKD plasmids recovered the UO126-blocked export of SphK2. Conclusion ERK regulates the export of SphK2 and apoptosis of HCT116 cells by modulating PKD. Modulation of these molecules may help increase the sensitivity of colon cancer cells to the physiologic anti-colon cancer agent, NaBT.
文摘目的应用生物信息学方法探讨鞘氨醇-1-磷酸转运体2(sphingosine-1-phosphate transporter 2,SPNS2)在乳腺癌中的表达情况,并分析SPNS2对乳腺癌预后、诊断或免疫浸润的影响。方法癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库检索乳腺癌组织和非乳腺癌样本中的SPNS2 mRNA差异表达数据,并分析SPNS2与乳腺癌之间的关系。用Cox单因素及多因素模型分析SPNS2 mRNA表达对乳腺癌预后的影响。使用Kaplan-Meier曲线评估SPNS2基因的表达与存活率之间的相关性,分析SPNS2对乳腺癌患者生存预后的影响。使用受试者工作特征曲线(receiver operating characteristic,ROC)分析SPNS2对乳腺癌的诊断效能。使用肿瘤免疫估算资源(Tumor Immune Estimation Resource,TIMER)数据库分析SPNS2表达与乳腺癌免疫微环境中不同类型免疫细胞的相关性。搜索相互作用基因检索的工具(Search Tool for the Retrieval of Interacting Genes,STRING)数据库分析乳腺癌中SPNS2与相关蛋白质之间的相互作用。分析京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)数据库中得到的差异基因富集的信号通路。提取正常乳腺上皮细胞和乳腺癌细胞系RNA,通过RT-qPCR实验比较SPNS2的表达水平。结果在TCGA乳腺癌数据库中,SPNS2在乳腺癌组织中表达水平显著低于癌旁组织(P<0.001),SPNS2 mRNA的表达与T、M分期、病理分期、PAM50分型、年龄和组织学类型等因素相关(P<0.05);Cox分析表明年龄>60岁、T4期、M1期等因素是乳腺癌发生预后不良的风险因素(P<0.01);Kaplan-Meier分析显示低表达SPNS2乳腺癌患者具有更长的疾病特异生存期(disease-specific survival,DSS)和无进展间隔期(progression-free interval,PFI)(P<0.05);ROC曲线提示SPNS2诊断具有较好的敏感性和特异性。TIMER数据库分析显示在Luminal型乳腺癌中,SPNS2与CD4+T细胞,巨噬细胞、中性粒细胞等免疫细胞呈正相关(P<0.05)。STRING和KEGG数据库分析表明SPNS2相关蛋白富集于细胞周期和PPAR信号传导等途径(P<0.05)。RT-qPCR实验结果显示与正常乳腺上皮细胞相比,SPNS2在乳腺癌细胞系中低表达(P<0.001)。结论SPNS2是一种潜在的诊断乳腺癌和评价预后的生物学标志物。
基金supported by the National Natural Science Foundation of China(No.81873505).
文摘Objective:Cardiac remodeling is a common pathological change in various cardiovascular diseases and can ultimately result in heart failure.Thus,there is an urgent need for more effective strategies to aid in cardiac protection.Our previous work found that sphingosine-1-phosphate(S1P)could ameliorate cardiac hypertrophy.In this study,we aimed to investigate whether S1P could prevent cardiac fibrosis and the associated mechanisms in cardiac remodeling.Methods:Eight-week-old male C57BL/6 mice were randomly divided into a sham,transverse aortic constriction(TAC)or a TAC+S1P treatment group.Results:We found that S1P treatment improved cardiac function in TAC mice and that the cardiac fibrosis ratio in the TAC+S1P group was significantly lower and was accompanied by a decrease inα-smooth muscle actin(α-SMA)and collagen type I(COL I)expression compared with the TAC group.We also found that one of the key S1P enzymes,sphingosine kinase 2(SphK2),which was mainly distributed in cytoblasts,was downregulated in the cardiac remodeling case and recovered after S1P treatment in vivo and in vitro.In addition,our in vitro results showed that S1P treatment activated extracellular regulated protein kinases(ERK)phosphorylation mainly through the S1P receptor 2(S1PR2)and spurred p-ERK transposition from the cytoplasm to cytoblast in H9c2 cells exposed to phenylephrine.Conclusion:These findings suggest that SphK2 and the S1PR2/ERK pathway may participate in the anti-remodeling effect of S1P on the heart.This work therefore uncovers a novel potential therapy for the prevention of cardiac remodeling.
基金Supported by Grant from National Key New Drug Creation Project of China, No. 2009ZX09102
文摘AIM: To elucidate the mechanisms of mesenteric vasodilation in portal hypertension (PHT), with a focus on endothelin signaling. METHODS: PHT was induced in rats by common bile duct ligation (CBDL). Portal pressure (PP) was measured directly via catheters placed in the portal vein tract. The level of endothelin-1 (ET-1) in the mesenteric circulation was determined by radioimmunoassay, and the expression of the endothelin A receptor (ETAR) and endothelin B receptor (ETBR) was assessed by immunofluorescence and Western blot. Additionally, expression of G protein coupled kinase-2 (GRK2) and β-arrestin 2, which influence endothelin receptor sensitivity, were also studied by Western blot. RESULTS: PP of CBDL rats increased significantly (11.89 ± 1.38 mmHg vs 16.34 ± 1.63 mmHg). ET-1 expression decreased in the mesenteric circulation 2 and 4 wk after CBDL. ET-1 levels in the systemic circulation of CBDL rats were increased at 2 wk and decreased at 4 wk. There was no change in ETAR expression in response to CBDL; however, increased expression of ETBR in the endothelial cells of mesenteric arterioles and capillaries was observed. In sham-operated rats, ETBR was mainly expressed in the CD31+ endothelial cells of the arterioles. With development of PHT, in addition to the endothelial cells, ETBR expression was noticeably detectable in the SMA+ smooth muscle cells of arterioles and in the CD31+ capillaries. Following CBDL, increased expression of GRK2 was also found in mesenteric tissue, though there was no change in the level of β-arrestin 2. CONCLUSION: Decreased levels of ET-1 and increased ETBR expression in the mesenteric circulation following CBDL in rats may underlie mesenteric vasodilation in individuals with PHT. Mechanistically, increased GRK2 expression may lead to desensitization of ETAR, as well as other vasoconstrictors, promoting this vasodilatory effect.