Effect of Girdin on apoptosis of human glioblastoma cells

Abstract

Objective To investigate the effects of Girdin on apoptosis of LN229 glioblastoma cells by Girdin small RNA interference (RNAi) technology and the molecular mechanisms. Methods Girdin expression was knocked down in LN229 glioblastoma cells by Girdin small interfering RNA (siRNA). The expression levels of Girdin, cytochrome C (Cyt-C) and Bad proteins in glioblastoma LN229 cells were detected by Western blotting. Stable clones were used to measure cells survival ratio by proliferation assay and methyl thiazol tetrazolium ( MTT) assay. Flow Cytometry was used to examine the content of Cyt-C in siGirdin/LN229 and scr/LN229. 20 male athymic Nu/Nu mice were subcutaneously injected with siGirdin/LN229 or scr/LN229 cells respectively to study the growth of tumor in each group. Results Western blotting showed that the expression of Girdin was decreased significantly. The relative intensity of Cyt-C and Bad in siGirdin/LN229 cells was 3. 57 ±0.43 and 4. 78 ± 1. 03 respectively, and the relative intensity of Cyt-C and Bad in scr/LN229 was 1. 13 ±0. 11,1. 78 ±0.20 respectively (P＜0. 05). The mean fluorescence density of Cyt-C in siGirdin/LN229 and scr/LN229 by flow cytometry was 12 531.00 ±1412. 98 and 183. 67 ±41. 55 respectively. Meanwhile, the number of siGirdin/LN229 cells was obviously decreased as compared with control group in proliferation assay. The number of siGirdin/LN229 cells on the 6th day was (15. 08 ± 1.17) x 104, and that of scr/LN229 cells was (53. 93 ± 6. 26) x 104. MTT assay revealed the survival rate of siGirdin/LN229 on the 5th day was (323. 15 ± 57. 01) % , and that of scr/LN229 was (640.67 ±59.66)%, P＜0.05. Additionally, the volume of xenograft tumors in siGirdin/LN229 group was decreased significantly as compared with control group. The volume of xenograft tumors in siGirdin/LN229 group was (36.42 ±2. 00) mm3, and that in scr/LN229 group was (262. 42 ±24. 12) mm3 at the 7.5th week. Conclusion Girdin plays a critical role in apoptosis of glioblastoma cells. Inhibition of Girdin by siRNA could promote the apoptosis of glioblastoma LN229 cells through the release of Cyt-C from mitochondria and up-regulation of Bad. Key words: Glioma; Girdin; Apoptosis