Effect Of C-jun Gene Silencing On The Growth And Angiogenesis Of Implanted Human Nasopharyngeal Carcinoma In The Nude Mice

Objective:to investigate the effect of c-jun gene silencing on the growth and the angiogenesis of implanted radioresistant human nasopharyngeal carcinoma CNE-2R cells in the nude mice,as well as investigated whether c-jun could influence the angiogenesis of nasopharyngeal carcinoma by regulating VEGF.Methods:using shRNA c-jun-shRNA-expressing lentivirus carrier cells CNE-2R to silence c-jun,build the c-jun-shRNA-CNE-2R cell group(c-jun-shRNA group)and NC-shRNA-CNE-2R cell group(NC group),with CNE-2R cells as blank Control group(Control group).Western blot analysis were used to detect the expression of vascular endothelial growth factor(VEGF)protein levels in cells of the three groups.Positive clones were selected and transplanted in nude mice to produce tumor animal mode.Growth curves and volumes of tumors were observed.After 6 weeks,the weight of transplanted tumor tissues were detected and graded,and CD34,VEGF were tested with immunochemistry method.The change of microvessel density(MVD)was detected according to the expression of CD34.Data were presented as the mean±standard deviation(SD).Statistical analyses were performed using SPSS 17.0(SPSS,IL,USA)or the GraphPad Prism 5.0 software.Statistical differences were determined by a two-tailed t test.The ANOVA test was used to assess the significance of the differences among the experimental groups,tumor volume growth was analyzed by repeated measurement of variance.All the cellular experiments were performed three or four times.A P value less than 0.05indicated statistically significant difference and indicated by*.Results:(1)The virus transfection of human nasopharyngeal carcinoma CNE-2R cells were successful,and the c-jun gene was silenced.(2)The expression of VEGF protein in c-jun-shRNA group were significantly lower than that of NC group and Control group both in vitro and in vivo.(3)After the model was established,nude mice had no death during the observation period;after a week of inoculation,the tumor reached a tumor rate of 100%at the transplant site.(4)In the six weeks of continuous observation,there was no obvious difference in the growth volume of the transplanted tumor in the first two weeks.The tumor volume of the c-jun-shRNA group was slower than that of the NC group and Control group.In the third week,the fourth,the fifth weeks,the tumor volume was significantly smaller than that of the NC group and Control group(all P<0.05).In the 6th weekend,the tumor volume of the c-jun-shRNA group was(720.85±72.10)mm~3,the volume of NC group(1196.81±90.69)mm~3,and Control group(1203.76±100.42)mm~3.Repetitive measure analysis of variance showed that the group effect,time effect and grouped by time effect of statistic respectively grouping group F=52.276,P=0.001,F=89.437,P=89.437,F group=25.187,P group=0.001,the group effect,time effect and grouped by time effect have statistical significance.Grouping effect that nude mouse transplantation tumor c-jun-shRNA group was obviously less than Control and NC groups,time effect description of three groups of transplanted tumor volume increased with the extension of time,grouped by time effect statistically difference and time with a cross in different groups;It can be found from the growth rate of transplanted tumor volume that the increase in the growth of the tumor volume in the c-jun-shRNA group over time was significantly lower than that in the Control and NC groups.(5)The quality of the three groups of nude mice transplanted tumor:c-jun-shRNA group was(0.423±0.040)g,the NC group was(0.798±0.081)g,and the Control group quality was(0.825±0.053)g,and the c-jun-shRNA group transplanted tumor was lower than the NC group and Control group(all P<0.05).(6)The results of immunohistochemical staining of transplanted tumor showed that MVD of c-jun-shRNA group(11.69±3.30)was graded 1-2.MVD of the NC group was(32.56±7.33),the Control group MVD was(35.45±4.87),and the classification was 3-4,and the MVD count and VEGF expression in the c-jun-shRNA group were significantly lower than that of the NC group and Control group(all P<0.05).Conclusion:(1)c-jun gene silencing can down-regulate the expression of VEGF in CNE-2R of human nasopharyngeal carcinoma.(2)c-jun gene silencing inhibits the growth and microangiogenesis of nasopharyngeal carcinoma.(3)c-jun gene silencing inhibits the growth and microangiogenesis of radioimmunoassay via down-regulation of VEGF.