The Effect Of RNA Interference On Expression Of EphB4 And Cell Growth In Glioma Cells

[Objective]: The expression and functional significance of the receptor tyrosine kinase EphB4 was studied in U251 glioma cell line in vitro. This study was designed to identify novel targets for diagnostic, prognostic, and therapeutic approaches of glioma.[Methods]: First, evaluate the expression of EphB4 gene and EphA2 gene in two different malignant glioma cell lines U251 and U87 by RT-PCR; Scend, establish the relevant interference vector-shRNA expression vector, which could be used in the glioma gene therapy; Third, EphB4 short interfering RNA was designed and synthesized, and then was transfected into U251 cell by lipofection to observe and detect the inhibition of EphB4 gene expression in the glioma cell line U251 cell; Forth, detect whether the siRNA can reduce the growth and induce apoptosis of the U251 cell; Fifth, to determine the important role of EphB4 gene played in tumorigenesis of glioma cell line U251, and study the effect of siRNA on the ability of the tumorigenicity in nude mice.[Results]: Each cell line showed a different level of EphB4 and EphA2 expression, with the highest level observed in glioma cell line U251. EphB4 siRNA was designed and synthesized in vitro, then was transfected into U251 cell by liopfection. Treatment of malignant glioma U251 cell with 100nm/L siRNA-EphB4 led to a 75.0% reduction in EphB4 mRNA levels by RT-PCR, which was associated with the increased quantity of cells in the sub-G1 phase fractions. Knockdown of EphB4 using small interfering RNA showed reduction in cell migration and invasion. In addition, murine tumor xenograft studies using EphB4 siRNA showed a marked reduction in tumor growth accompanied by reduced cell division.[Conclusions]: EphB4 knockdown using EphB4 short interfering RNA led to a significantly inhibition in cell viability, migration and invasion, and also induced apoptosis in U251 cell. What's more, it indicated we have successfully established the shRNA eukaryotic expression vector which can inhibit the expression of EphB4 mRNA, and this study provides the precondition for the further study of EphB4 in glioma pathogenesis. These results suggest that inhibition of EphB4 is likely to be a promising molecular target for glioma therapy.