Inhibition Of S1PR3 Promotes The Apoptosis Of T-cellacute Lymphoblastic Leukemia Cells In Children

PART Ⅰ GPCRS EXPRESSION IS DISRUPTED IN CHILDREN WITH T-CELLACUTE LYMPHOBLASTIC LEUKEMIAObjective: To explore the differentially expressed G protein coupled receptors in children with T cell acute lymphocyte leukemia by High throughput RNA sequencing.Methods: The T-ALL bone marrow samples were collected from 13 children diagnosed with acute T lymphocyte leukemia in Childrens Hospital of Chongqing Medial University,and tumor cells were isolated by Ficoll lymphocyte separation solution;4 healthy children’s peripheral blood samples were collected,and normal T cells were isolated by human T cell extraction kit;The RNA of Tumor cells and normal T cells were extracted and then subjected to RNA-sequencing to detect genes mRNA expression levels;Differentially expressed genes between T-ALL tumor cells and normal T cell were analysed by R-language.Heat maps and volcano maps about the differentially expressed genes were drew in R language.Differentially expressed gene were submitted to Gene Ontology for GO analysis.Results: The results of RNA-seq analysis showed that there were a large number of abnormal expressions of tumor-associated pathway genes in bone marrow tumor cells of 13 children with T-ALL compared with normal T cells of healthy children.The expression of a large number of GPCRs was abnormal;Differentially expressed GPCRs were submitted for GO analysis.Among them,the sphingosine-1-phosphate(S1P)signaling pathway-related genes are enriched.Conclusion: There are a large number of abnormal expression of GPCRs gene in bone marrow cells of children with T cell acute lymphocyte leukemia,in which S1 P signaling pathway-related genes are abnormally enriched.PART Ⅱ INCREASED EXPRESSION OF S1PR3 IN CHILDREN WITH T-CELLACUTE LYMPHOBLASTIC LEUKEMIAObjective: To analyse the expression of S1 P signaling pathway-related genes in RNA-seq data.To analyse the expression of S1 P signaling pathway related genes in multiple T-ALL studies in GEO database.To verify the expression of S1 P signaling pathway related genes in T-ALL cell lines and T-ALL patients.Methods: The expression of S1 P signaling pathway related genes in RNA-seq data was analyzed.The expression of S1 P signaling pathway related genes in T-ALL was analyzed by searching GEO database.The expression of S1 P and its carrier apolipoprotein M(Apo M)in the bone marrow supernatant of children with T-ALL and non-tumor patients were detected by ELISA.The expression of S1 P signaling pathway related genes in T-ALL cells was analyzed by GEO database.Normal T cells were extracted by human T cell extraction kit,thymus tissues of healthy children were collected,ground and filtered to obtain thymocytes.Bone marrow of non-tumor patients was collected to collect CD34+ bone marrow cells.Bone marrow samples were collected from newly diagnosed T-ALL children.T-ALL cell lines were cultured.The expression levels of SPHK1,SPHK2 and SGPL1 in normal T cells,CD34+ bone marrow cells and T-ALL cell lines were detected by QPCR.The expression of S1PR3 in normal T cells,normal thymocytes,CD34 bone marrow cells and T-ALL cell lines was detected by Q-PCR.The expression of S1PR3 in the cell membrane of T-ALL cell line was detected by flow cytometry.Q-PCR was used to detect the expression of S1PR3 in normal T cells and T-ALL tumor cells.Results: Compared with normal T cells,RNA-seq showed that the expression of S1 P synthase SPHK2 was increased in T-ALL tumor cells,the expression of S1 P degrading enzyme SGPL1 was decreased,and the expression of S1PR3 was significantly increased in T-ALL tumor cells,S1PR1,S1PR2,S1PR4 and S1PR5 expression is relatively reduced.The data of multiple T-ALLs in the GEO database showed that the expression of S1PR3 in T-ALL tumor cells was significantly increased,the expression of SPHK1 and SPHK2 was relatively increased,the expression of S1PR1,S1PR2,S1PR4 and S1PR5 was relatively decreased,and the expression of SGPL1 was relatively decreased.The results of ELISA showed no significant difference in the expression of S1 P and Apo M in T-ALL bone marrow supernatant and normal bone marrow supernatant.The results of GEO database analysis and Q-PCR showed that compared with peripheral blood T cells,thymocytes and CD34+ bone marrow cells,the expression of S1PR3 in T-ALL cells was significantly increased,the expression of SPHK2 was increased,and the expression of SGPL1 was decreased.The results of flow cytometry showed that the expression of S1PR3 on the surface of JK cells was the highest in T-ALL cell line,followed by CEM,MOLT4 and CUTLL1.The Q-PCR results of tumor cells in clinical patients showed that the expression of S1PR3 in T-ALL was significantly increased.Conclusion: Analysis of T-ALL related studies in GEO database,and verification in T-ALL cell lines and patient samples,We found that S1 P signaling pathway related genes were differentially expressed in T-ALL,and S1PR3 expression was significantly increased.PART Ⅲ INHIBITION OF S1PR3 PROMOTES THE APOPTOSIS OF ACUTE T CELL LYMPHOBLASTIC LEUKEMIA CELLS IN CHILDRENObjective: To analysis the effect of S1PR3 on the apoptosis of T-ALL cells in vitro.Methods: The S1PR3 specific small molecule inhibitor TY52156(0,1,3,7,10,15 u M),CAY10444(0,3,5,10,20,30 u M)were respectively applied to the T-ALL cell lines,by Annexin V-APC/7-AAD staining,flow cytometry was used to detect the apoptosis of T-ALL cell lines.The tumor cells of primary T-ALL patients were collected and cultured.CAY10444(0,3,5,10,20,30 u M)was applied to the primary T-ALL cells,a nnexin V-APC/7-AAD staining was performed,then flow cytometry was used to detect the apoptosis of primary T-ALL cells.At the same time,Laser scanning confocal microscope was used to observe the proportion of Cleaved-caspase3+ cells.After TY52156(0,7,15 u M),CAY10444(0,10,30 u M)applied to CD34+bone marrow cells,by annexin V-APC/7-AAD staining,apoptosis of CD34+bone marrow cells was detected by flow cytometry.Results: Flow cytometry results showed that S1PR3 specific small molecule inhibitor could significantly promote the apoptosis of T-ALL cells,and the apoptosis ratio was drug concentration dependent.The IC50 of TY52156 on CEM,MOLT4,JK and CUTLL1 was 5.23 u M,5.78 u M,5.93 u M and 6u M,respectively.CAY10444 acted on CEM,MOLT4,JK and CUTLL1 with IC50 of 10.12 u M,13.17 u M,21.59 u M and 27.22 u M,respectively.CAY10444,a S1PR3 specific small-molecule inhibitor,acted on tumor cells of a patient with primary t-all and caused the apoptosis of 88% leukemia cells at 30 u M.S1PR3 specific small molecule inhibitor CAY10444 acted on tumor cells of a primary T-ALL patient,and as the drug concentration gradually increased,the proportion of Cleaved caspase-3+ cells increased.Flow cytometry showed that S1PR3 specific small-molecule inhibitors TY52156 and CAY10444 had no significant effect on the apoptosis of CD34+ bone marrow cells.Conclusion: Specific inhibition of S1PR3 promoted the apoptosis of TALL cells.