| Nasopharyngeal carcinoma(NPC)is the most important Epstein-Barr Virus(EBV)-related head and neck malignant tumor in southern China,which has obvious tendency of local lymph node invasion and distant metastasis.This malignant tendency is often the main reason for the failure of NPC treatment.However,the specific mechanism of NPC high invasion and metastasis has not been clarified.The invasion and metastasis of tumors are closely related to tumor angiogenesis.From the perspective of tumor microenvironment,the tumor cells recruit vascular endothelial cells and vascular interstitial cells by secreting a large amount of vascular endothelial growth factor(VEGF)into the local microenvironment,which results in abnormal tumor neovascularization.Not only tumor cells but other non-tumor cells in the microenvironment are also actively involved in this process.Therefore,interfering with the interaction between tumor cells and non-tumor cells in the local microenvironment is a new way of antitumor angiogenesis.To communicate with other non-tumor cells,tumor cells firstly perceive the signal molecules released by non-tumor cells,and convert the external stimulation caused by these cell signal molecules into intracellular signals that can be transmitted.Store-operated Ca2+ entry(SOCE)is a unique mode of extracellular and intracellular signaling transmission,and SOCE-mediated abnormal calcium signaling is actively involved in oncogenesis and development.Our previous study confirmed that both SOCE-blocking drug SKF96365 and 2-aminoethoxydiphenyl borate(2-APB)could inhibit the growth and angiogenesis of NPC,suggesting that SOCE can be used as a target to inhibit tumor angiogenesis of NPC.Stromal interaction molecule 1(STIM1)is a key functional protein that regulates SOCE channels.As a receptor of intracellular Ca2+ concentration,STIM1 regulates cell Ca2+ homeostasis.In addition,our previous experiment results suggested that miRNA-185-5p could inhibit the expression of STIM1 by binding its 3’UTR region.However,it is not clear whether miRNA-185 targets STIM1 to regulate SOCE-mediated Ca2+ signaling,thereby affecting EBV-involved NPC tumor angiogenesis.Therefore,through the following four parts,this study will clarify the regulated mechanism of STIM1-dependent Ca2+ signaling on EBV-involved NPC tumor angiogenesis,and provide new therapeutic strategies and methods that can effectively resist the invasion and metastasis of NPC.Objective:To study the effect of EBV infection on Ca2+ signaling of NPC cells,and to explore the mechanism of EBV infection on tumor angiogenesis of NPC.Methods:The EBV-encoded LMP-1 was detected to verify the effectiveness of EBV infection in the EBV-positive NPC cells(CNE2-EBV,HK1-EBV)by western blotting.Then,the amounts of VEGF production in EB V-negative(CNE2,HK1)and EBV-positive NPC cells with or without EGF stimulation were determined by enzyme-linked immunosorbent assay(ELISA),and human umbilical vein endothelial cells(HUVECs)-formed tube networks were quantitatively evaluated as an in vitro angiogenesis assay.In addition,cytosolic Ca2+changes were measured in individual fluorescent Ca2+indicator-loaded cells.Finally,a mouse model concurrently bearing EBV-positive/negative xenografts was utilized to evaluate the tumor growth and angiogenesis through measuring the size of xenografts and testing CD31 by immunohistochemistry in vivo.Results:(1)LMP-1 expression was found in HK1-EBV and CNE2-EBV cells,but not in HK1 and CNE2 cells.(2)In the absence of EGF stimulation,the amounts of spontaneous VEGF production were not significantly different between the EBV-positive and EBV-negative NPC cells.However,VEGF production of the EBV-positive cells in the presence of external EGF stimulation was significantly increased(HK1 vs.HK1-EBV:P<0.0001,CNE2 vs.CNE2EBV:P<0.001).(3)In the absence of EGF stimulation,endothelial tubular formation had no statistically significant difference between the EBV-negative and EBV-positive cells.However,in the presence of external EGF stimulation,the amplification of tubular formation in EBV-positive NPC cells was more significant than that of the EBV-negative NPC cells,and the difference in the two groups was statistically significant(both P<0.001).(4)EBV infection significantly boosted the cytosolic Ca2+elevation found upon external EGF stimulation(HK1 vs.HK1-EBV:0.7942±0.0500 vs.1.4313±0.0211,P<0.01;CNE2 vs.CNE2-EBV:1.1958±0.1551 vs.1.8372±0.0617,P<0.05),and EBV promoted the Ca2+ responses by boosting the SOCE,but had no obvious effect on the Ca2+ release from the intracellular Ca2+store.(5)The EBV-positive xenografts grew faster compared with the EBV-negative xenografts(P<0.05).In addition,the expression of CD31 was more in the xenografts of EBV-positive cells(P<0.05).(6)Compared with the EBV-negative NPC tissues,the expression of CD31 was more in the EBV-positive NPC tissues(P<0.01).Conclusion:EBV infection may increase the VEGF production and promote tumor angiogenesis in NPC cells through facilitating EGF-induced,SOCEmediated Ca2+ signaling.Objective:To investigate the mechanisms of STIM1-dependent Ca2+signaling regulating EBV-promoted tumor angiogenesis in NPC.Methods:The expression of STIM1 was detected in HK1,CNE2 and HK1EBV,CNE2-EBV by western blotting.The expression of STIM1 in HK1-EBV and CNE2-EBV was knocked down by lentiviral transduction,which was confirmed by western-blotting.Cytosolic Ca2+changes were measured in stably transfected individual NPC cells by fluorescence resonance energy transfer(FRET)technique.Then,the amounts of VEGF production in stably transfected NPC cells with or without EGF stimulation were determined by ELISA,and HUVECs-formed tube networks were quantitatively evaluated as an in vitro angiogenesis assay.A mouse model concurrently bearing the xenografts of stably transfected CNE2-EBV cells was utilized to evaluate the tumor growth and angiogenesis through the measurement of the xenograft size and immunohistochemical analysis of CD31.STIM1 and CD31 in paraffin-embedded tissue of NPC were determined by immunofluorescence homologous double-label assay.Results:(1)The expression of STIM1 protein was higher in HK1-EBV and CNE2-EBV compared with HK1 and CNE2.(2)Lentivirus transfection silenced the expression of STIM1 in HK1-EBV and CNE2-EBV cells.Compared with the negative control group(shRNA-NC),knockdown of STIM1(shRNA-STIM1)could significantly reduce EGF-induced Ca2+ influx via SOCE in EBV-positive NPC cells(HK1-EBV shRNA-NC vs.HK1-EBV shRNA-STIM1:1.1563 ±0.6408 vs.0.3132±0.0962,P<0.05;CNE2-EBV shRNA-NC vs.CNE2-EBV shRNA-STIM1:9.7122±0.6520 vs.5.1942±2.6926,P<0.05).(3)In the absence of EGF stimulation,the amounts of spontaneous VEGF production were not significantly different between the shRNA-NC and shRNA-STIM1 NPC cells.However,in the presence of external EGF stimulation,the amplification of VEGF production in the shRNA-STIM1 cells was less significant than that of the shRNA-NC NPC cells(P<0.0001 and P<0.01).(4)In the absence of EGF stimulation,tubular formation had no statistically significant difference between the shRNA-NC and shRNA-STIM1 NPC cells.However,in the presence of external EGF stimulation,the amplification of tubular formation in the shRNASTIM1 NPC cells was less significant than that of the shRNA-NC cells(P<0.001 and P<0.0001).(5)The shRNA-STIMl xenografts grew slower compared with shRNA-NC the xenografts(P<0.05).In addition,the expression of CD31 was less in the xenograft of shRNA-STIM1 cells(P<0.05).(6)The more expression of CD31 was near the higher expression of STIM1 in paraffin-embedded tissue of NPC.Conclusion:Knockdown of STIM1 decreases the VEGF production and inhibit EBV-promoted tumor angiogenesis in NPC cells through disturbing EGFinduced,SOCE-mediated Ca2+signaling.Objective:To investigate the EGFR and ERK1/2 signaling pathways that affected the tumor angiogenesis of NPC.Methods:Using the mRNA expression profiles of head and neck cancer(HNSC)downloaded from The Cancer Genome Atlas(TCGA)database,gene set enrichment analysis(GSEA)were performed to explore the gene pathway most closely related to STIM1 according to STIM1 expression level.The transcriptome sequencing data of NPC cells with or without EBV infection(Accession:SRX3199730)downloaded from NCBI’s SRA database were carried out to find the differential genes(DGs),and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis of the up-regulated DGs was performed using the David online tool.The expression of EGFR and its phosphorylated protein p-EGFR were determined by western blotting in NPC cells(HK1,HK1-EBV,CNE2,CNE2EBV,HK1-EBV shRNA-NC,HK1-EBV shRNA-STIM1,CNE2-EBV shRNANC and CNE2-EBV shRNA-STIM1)with or without EGF stimulation.Under the external EGF stimulation,the expression of ERK1/2 and its phosphorylated protein p-ERK1/2 were also detected in NPC cells above.Results:(1)The mRNA expression profiles of 522 cases of HNSC were obtained from TCGA database.The results of GSEA showed ErbB signaling pathway was positively correlated with gene set in the group of high STIM1 expression(P<0.05).(2)Transcriptome analysis revealed 5412 up-regulated DGs after EBV infection,and the top 10 results of KEGG analysis showed that the upregulated DGs were enriched in Ca2+ and MAPK signaling pathway.(3)Compared with NPC cells without EGF stimulation,the expression of p-EGFR was higher in cells with EGF stimulation.However,EBV infection and knockdown of STIM1 had no significant effect on the expression of EGFR and pEGFR.(4)EBV infection promoted the expression of p-ERK1/2,while the blockage of EBV-facilitated Ca2+ signaling through S TIM 1-knockdown disturbed it in NPC cells.Conclusion:With the results of Part Ⅰ and Part Ⅱ,the study suggests that(1)EGF activates SOCE-mediated Ca2+ signaling through the EGFR pathway in NPC cells.(2)EBV infection activates ERK1/2 signaling pathway by enhancing SOCE-mediated Ca2+ signaling,while the blockage of EBV-facilitated Ca2+signaling through STIM 1-knockdown disturbs it,which further affects the tumor angiogenesis of NPC.Objective:To elucidate the targeted regulation mechanisms of miRNA-1855p and STIM1 in NPC cells.Methods:Potential target genes of miRNA-185-5p were predicted by miRBASE and miRDB database,and the targeted relationship between miRNA185-5p and STIM1 was further verified by double luciferase reporting assay.The expression of miRNA-185 in normal nasopharyngeal epithelial cells NP-69 and NPC cells 6-10B and 5-8F was detected by quantitative reverse transcriptionpolymerase chain reaction(RT-qPCR).Then,the expression of miRNA-185 in 610B and 5-8F cells was up-regulated by lentivirus transfection,and the expression of STIM1 in up-regulated NPC cells above was determined by RT-qPCR and western blotting.In order to further verify the regulated effectiveness of miRNA185 on STIM1 of NPC cells,STIM1 was overexpressed on the basis of miRNA185 up-regulation,and then the expression of STIM1 was evaluated by western blotting.Results:(1)It was found that STIM1 was the direct target of miRNA-185-5p by searching miRBase and miRDB databases.Further,mutation was introduced into the binding site of miRNA-185-5p in the 3’UTR region of STIM1 in the dual luciferase reporter gene system,and the results showed that miRNA-185-5p could inhibit the transcription of wild-type STIM1 gene,but could not inhibit that of the mutant gene.(2)Compared with NP-69 cells,miRNA-185 expression of 6-10B and 5-8F cells were lower.(3)Upregulation of miRNA-185 could reduce the expression level of STIM1 protein in 6-10B and 5-8F cells,while overexpression of STIM1 could reverse the expression level.Conclusion:With the results of the previous three parts,the study suggests miRNA-185-5p may inhibit tumor angiogenesis by negatively modulating the expression of STIM1 in NPC. |
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