Inhibitory Effect Of Rat Hyperplasia Suppressor Geneoverexpression On Invasion Of C6 Glioma Cells And Its Mechanism

Objective:To study the effect of overexpression of rat hyperplasia suppressor gene(rHSG)on invasion of glioma C6 cells,and to investigate its potential mechanism.Methods:The recombinant adenovirus vector Adv-rHSG-GFP carrying rHSG gene sequence was infected into rat glioma C6 cells.At the same time,C6 cells treated by PBS or infected with Adv-GFP were used as the control groups.The expressions of rHSG mRNA and protein in different groups were detected by real-time fluorescent quantitative PCR and Western blotting.Then,the effects of rHSG overexpression on the migration and invasion of glioma C6 cells were analyzed by scratch wound healing assay,Transwell migration and invasion assay.The changes of cytoskeleton formation and adhesion ability of C6 cells after rHSG overexpression were analyzed by immunofluorescence and cell adhesion assay.The expressions of Ras homolog gene family,member A(RhoA),Rasrelated C3 botulinum toxin substrate 1(Rac1),cell division cycle 42(Cdc42),Rho associated coiled-coil forming protein kinase 1(Rock1)and Rock2 in Rho/Rock signaling pathway in each group were detected by real-time fluorescent quantitative PCR and Western blotting.Results:1.Detected by real-time fluorescence quantitative PCR and Western blotting,the expression of rHSG was significantly up-regulated in C6 cells infected by recombinant adenovirus Adv-rHSG-GFP for 48 and 72 h(both P< 0.01)compared with the two control groups(PBS group and Adv-GFP group).However,there is no difference between the PBS group and the Adv-GFP group.2.The scratch wound healing assay indicates thatthe cell wound healing rate of the Adv-rHSG-GFP group was significantly decreased(P < 0.01)compared with the Adv-GFP group and the PBS group after 30 hours,but there is no significant difference(P > 0.05)between the PBS group and the Adv-GFP group(P > 0.05).3.The Transwell tests indicated that the migration and invasion of the Adv-rHSG-GFP group were significantly reduced(P < 0.01)compared with the Adv-GFP group and the PBS group,but the PBS group has no significant difference with the the Adv-GFP group(P> 0.05).4.The cell adhesion testexpounds that the adhesion number of the Adv-rHSG-GFP group were reduced significantly at different time(30,60,90 and 120 min)compared with the Adv-GFP group and the PBS group(P < 0.05),and the difference is narrowing as time goes on;but there is no significant difference between the two groups(P> 0.05).5.The immunofluorescence experimentelucidates that when the C6 cells had been infected by the recombinant adenovirus Adv-rHSG-GFP for 48 hours,their morphology would change significantly,most of them changed form long fusiform,stellate,filopodia axialledand so on to filopodial shortening,disorganized and skeleton adjusted.6.Quantitative Real-time PCR and Western blot experiments indicate thatwhen the C6 cells had been infected by the the recombinant adenovirus Adv-rHSG-GFP for 48 and 72 hours,the expression of the related moleculars in Rho/Rock signal pathway(RhoA,Rac1,Cdc42,Rock1 and Rock2)were significantly lower(P < 0.05)compared with the Adv-GFP group and the PBS group,but there was no significant difference between the two control groups(P > 0.05).Conclusion1.The virus was successfully infected.When the C6 cells had been infected for 48 hours and 72 hours,the expression of rHSG was overexpression.2.When the expression of rHSG was increased in C6 glioma cells,the migration,invasion and adhesion numbers of cells were significantly inhibited.3.The overexpression of rHSG inhibitedthe formation of the framework of C6 glioma cell and promoted cell filopodia shortened and rearranged.4.The overexpression of rHSG could inhibite the expression of the mRNA and protein of the key molecules in Rho/rock signaling pathway in C6 glioma cells,suggesting that rHSG may inhibit the invasion of C6 glioma cells by blocking the Rho/rock signaling pathway.