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目的 :研究大鼠增殖抑制基因(rat hyperplasia suppressor gene,r HSG)过表达对胶质瘤C6细胞侵袭的影响,并探讨其可能的作用机制。方法 :用携带r HSG基因的重组腺病毒Adv-r HSG-GFP感染胶质瘤C6细胞,同时设置Adv-GFP感染的阴性对照组以及仅PBS处理的空白对照组。采用实时荧光定量PCR和蛋白质印迹法检测各组细胞中r HSG的表达变化。然后,采用细胞划痕愈合实验和Transwell迁移及侵袭实验分别检测r HSG过表达对胶质瘤C6细胞迁移和侵袭的影响,细胞免疫荧光法和细胞黏附实验分别分析r HSG过表达后C6细胞骨架形成和黏附能力的变化,实时荧光定量PCR和蛋白质印迹法测定各组细胞中Rho/Rock信号通路关键分子Ras同源基因家族成员A(Ras homolog gene family,member A,RhoA)、Ras相关的C3肉毒底物1(Ras-related C3 botulinum toxin substrate 1,Rac1)、细胞分裂周期蛋白42(cell division cycle 42,Cdc42)、Rho相关卷曲螺旋形成蛋白激酶1(Rho associated coiled-coil forming protein kinase 1,Rock1)和Rock2的表达水平。结果 :重组腺病毒Adv-r HSG-GFP感染48和72 h后,胶质瘤C6细胞中r HSG的表达水平明显升高(P值均<0.01)。与2个对照组相比,Advr HSG-GFP组细胞的划痕愈合率明显降低(P值均<0.01),迁移、侵袭和黏附细胞数均明显减少(P值均<0.05)。r HSG过表达后C6细胞形态发生变化,丝状伪足缩短,部分呈片状;而且细胞中Rho A、Rac1、Cdc42、Rock1和Rock2的m RNA及蛋白表达水平均明显降低(P值均<0.05)。结论 :r HSG过表达可以抑制胶质瘤C6细胞的侵袭,其作用机制可能与阻滞Rho/Rock信号通路有关。
OBJECTIVE: To study the effect of r HSG over-expression on the invasion of glioma C6 cells and to explore its possible mechanism. Methods: C6 glioma cells were infected with recombinant adenovirus Adv-r HSG-GFP carrying r HSG gene, and negative control group with Adv-GFP infection and blank control group only treated with PBS were set up. Real-time fluorescence quantitative PCR and Western blotting were used to detect the expression of r HSG in each group. Then, the effect of r HSG overexpression on the migration and invasion of C6 glioma cells was detected by cell scratch healing assay and Transwell migration and invasion assay. The cytosolic C6 glioma cells were identified by immunofluorescence and cell adhesion assay respectively (RhoA), RhoA (Ras homolog gene family), Ras (C3, C3) related to Rho / Rock signaling pathway in each group were detected by real-time fluorescence quantitative PCR and Western blotting. Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division cycle 42 (Cdc42), Rho associated coiled-coil forming protein kinase 1 , Rock1) and Rock2 expression levels. Results: The expression of r HSG in glioma C6 cells was significantly increased after 48 and 72 h of infection with recombinant adenovirus Adv-r HSG-GFP (all P <0.01). Compared with the two control groups, the wound healing rate of Advr HSG-GFP group was significantly lower (P <0.01), migration, invasion and adhesion cells were significantly reduced (P <0.05). r HSG over-expression of C6 cells morphological changes, filopodia shorten, some flaky; and the cells Rho A, Rac1, Cdc42, Rock1 and Rock2 m RNA and protein expression levels were significantly lower (P values < 0.05). Conclusion: r HSG overexpression can inhibit the invasion of glioma C6 cells, its mechanism may be related to block Rho / Rock signaling pathway.