Preliminary Study On Influence Of Expression And Blocking Of CXCR4 On Invasiveness Of Bladder Carcinoma Cells

Objective:To study the expression of chemokine receptor CXCR4 in human bladder carcinoma cell EJ and EJ-M₃ and its effect on vitro metastatic potential of two cell lines.Constructed eukaryotic expression vectors of small interfering RNA targeting human CXCR4 gene and observed the expression of CXCR4 gene and effect of siRNA on the EJ-M₃ vitro metastatic potential after the vector transfected into cells.Methods:Indirect immunofluorescent technique and flow-cytometric measurement were performed to detect the expression of CXCR4 in EJ and EJ-M₃. The recombinant plasmid pshRNA-CXCR4-1 and pshRNA-CXCR4-2 were constructed and transfected into EJ-M₃ cell line.The expression level of CXCR4 was determined by Indirect immunofluorescent technique and the transcription of CXCR4 gene was detected by semi-quantitative RT-PCR.Through knockdown CXCR4 by siRNA,the vitro metastatic potential of EJ-M₃ cells was examined in vitro experiments.Analyzed the correlation of data from siRNA experiment with data from invasion experiment.Results:The expression of CXCR4 protein was shown in cytoplasm of the two bladder carcinoma cell lines.The expression rates of CXCR4 protein in EJ-M₃(65.7±10.35%) was higher than that in EJ(25.4±4.69%) and the difference was significant(p＜0.05).The recombinant plasmid pshRNA-CXCR4-1 and pshRNA-CXCR4-2 were constructed and transfected into EJ-M₃ cell and expression level of CXCR4 in pshRNA-CXCR4-1 group was significantly lower than that in pshRNA-CXCR4-2 group,negative control group and blank group according to results of Indirect immunofluorescent technique.Semi-quantitative RT-PCR showed that the.expression of CXCR4 mRNA in pshRNA-CXCR4-1 group(0.153±0.02) was lower than that in in pshRNA-CXCR4-2 group(0.46±0.03 ),negative control group(0.41±0.05 ) and blank group(0.43±0.07 ),the differences were significant(p＜0.05).The exosomatic invasive capability of in pshRNA-CXCR4-1 group(39.67±8.45 ) was lower than that in negative control group(123.63±6.36) and blank group(135.33±9.28) and the differences were sigrfificant(p＜0.05).The exosomatic invasive capability had positive correlation with the expression of CXCR4(p＜0.05) and the exosomatice invasive capability has positive correlation with the expression of CXCR4 mRNA(p＜0.05).Conelusion:The results demonstrate that the small interfering RNA-1 targeting CXCR4 gene shows dramastic inhibition on protein expression and RNA transcription and it can also inhibit the exosomatice invasive capability of EJ-M₃.The study suggests that CXCR4 has an important influence on the exosomatice invasive capability of EJ-M₃ and plays a significant role in regulating the invasive and metastatic behavior in vitro of EJ-M₃.It also can supply theory proof for research of intraluminal implantation associated with bladder carcinoma and CXCR4 siRNA may be potentially usefully in the next research of tumor therapy.