Cxcr4 Expression Effect And Mechanism Of The Pediatric Neuroblastoma Organ-specific Metastasis

Objective:Established Neuroblastoma cell sublines with different metastasis derived from neuroblastoma cell line of the same patient and to explore the expression and effects of the chemokine receptor CXCR4 in human neuroblastoma cell sublines with differently vitro metastatic potentialities.Methods:Neuroblastoma cell sublines were selected from human neuroblastoma cell line (QDDQ-NM) cultured by ourselves by the unicell cloning technique, the expressions of protein of CXCR4 and CXCR4 mRNA were detected by immunohistochemistry and RT-PCR, the pulmonary metastatic potential was detected by the transwell vitro invasion assay.Results:The two neuroblastoma cell sublines (QDDQ-NM a, QDDQ-NM e) were obtained from human neuroblastoma cell line, and their expression of protein of CXCR4 and CXCR4 mRNA were significantly different, P< 0.01.The vitro metastatic potentialities in QDDQ-NM a was significantly higher than its in QDDQ-NM e, P<0.01, which had high correlation with the expression of CXCR4.Conclusion:Neuroblastoma cell sublines with different metastasis derived from neuroblastoma cell line of the same patient, the high expression of'CXCR4 is assiociated to high vitro metastatic potential of human neuroblastoma cell sublines intimately. Objective:To explore the effect of silencing CXCR4 by siRNA on the invasion capability of neuroblastoma cell line SH-SY5Y in vitro.Method:Three siRNAs targeting CXCR4 were chemically synthesized and transfected into SH-SY5Y cells. The transfection efficiency was observed under fluorescence microscope, CXCR4 mRNA and protein levels were detected by semi-quantitative RT-PCR,Immunohistochemistry,Western blot and the invasion capability of the cells in vitro was evaluated by Boyden Chamber.Result:The three sequence-specific siRNAs targeting CXCR4 efficiently suppressed the expression of CXCR4 at mRNA level in transfected cells are 0.32±0.09,0.35±0.13and0.33±0.11 compared with control groups 0.58±0.13 (P<0.05); the three sequence-specific siRNAs targeting CXCR4 efficiently suppressed the expression of CXCR4 at protein level in transfected cells detected by Immunohistochemistry are75.98±4.81,75.52±3.95and76.35±6.51 compared with control groups 92.196±3.89 (P<0.01); the three sequence-specific siRNAs targeting CXCR4 efficiently suppressed the expression of CXCR4 at protein level in transfected cells detected by Western blot are0.1103±0.0023,0.1203±0.0015and0.1308±0.0018 compared with control groups 0.4832±0.0012 (P<0.01).The results of Boyden Chamber experiment showed that the number of cells under micro-membrane decreased in SH-SY5Y cells transfected with CXCR4 three sequence-specific siRNAs after 48h are 25.48±2.81,30.89±2.77 and 18.83±1.79compared with those in the control group53.11±3.72 (P<0.05).Conclusion:Silencing CXCR4 by siRNA decreases the expression of CXCR4 and the invasion capability of SH-SY5Y cells. CXCR4 gene may be a potentially valuable therapeutic target in the treatment of neuroblastoma.