The Inhibition Mechanism Of Choriocarcinoma Invasion By Silencing Upa With Lentivirus Carried Short Hairpin RNA

Objective:As we know,uPA is overexpressed in choriocarcinoma.To observe invasive mechanism of uPA and determine whether a relationship exists among uPA、VEGF-B、MMP-2in choriocarcinoma via Lentivirus carried short hairpin RNA (shRNA).Methods:1.construct a lentivral vector-mediated shRNA against uPA, and package lentivirus particles.2.Lentiviral vector mediated shRNA targeting uPA gene (LV-uPA-shRNA)was transfected into human high invasive choriocarcinoma cell line JEG-3cells,the empty vector and culture media as control.3. the uPA、VEGF-B、MMP-2mRNA expression were detected by quantitative Real-Time PCR(qRT-PCR).4.the invasion of JEG-3cells were detected by Matrigel assay.Result:1.The shRNA sequence was successfully inserted into the lentiviral vector confirmed by PCR analysis and DNA sequencing.The titer of concentrated virus was7.36×106Tu/ml.2.qRT-PCR showed that uPA mRNA expression in non-transfected group,empty vector group and LV-uPA-shRNA group were as follows: 1.025±0.267、0.800±0.099、0.066±0.021,the mRNA expression in LV-uPA-shRNA group was significantly lower than empty vector group and non-transfected group,and the difference was significant at P<0.05.There was no difference between empty vector group and non-transfected group,P>0.05.3.qRT-PCR showed that VEGF-B mRNA expression in non-transfected group, empty vector group and LV-uPA-shRNA group were as follows:1.061±0.410、0.789±0.223、0.061±0.036,the mRNA expression in LV-uPA-shRNA group was significantly lower than empty vector group and non-transfected group,and the difference was significant at P<0.05.There was no difference between empty vector group and non-transfected group,P>0.05.4.qRT-PCR showed that MMP-2mRNA expression in non-transfected group,empty vector group and LV-uPA-shRNA group were as follows:1.009±0.161、0.717±0.222、0.071±0.021,the mRNA expression in LV-uPA-shRNA group was significantly lower than empty vector group and non-transfected group,and the difference was significant at P<0.05.There was no difference between empty vector group and non-transfected group,P>0.05.5.Matrigel assay showed that the number of penetrating cells in non-transfected group,empty vector group and LV-uPA-shRNA group were as follows:200.00±11.13、205.00±14.52、61.33±2.08, the number of penetrating cells in LV-uPA-shRNA group was significantly lower than empty vector group and non-transfected group,and the difference was significant at P<0.05.There was no difference between empty vector group and non-transfected group,P>0.05.Conclusion:1.The recombinant lentiviral vector mediated shRNA targeting uPA gene can invade JEG-3cells effectively and silence the expression of uPA significantly.This study showed a effective way of gene silencing.2. silencing the expression of uPA resulted in significant inhibition of JEG-3invasion.It revealed that uPA played a key role in JEG-3invasion.3.Silencing the expression of uPA can down-regulate the expression of VEGF-B、MMP-2. The down-regulation may enhance the effect of inhibiting JEG-3invasion.