关键词: [关键词]结直肠癌, Grb2, RNA干扰, 慢病毒, 信号通路

Abstract: Objective To analyze the effect of Grb2 inhibition by shRNA transfection on the proliferation of colon cancer cells HT29,and to estimate the feasibility of Grb2 as a target for the treatment of colorectal cancer. Method The Grb2 shRNA was transfected to 293T cells by shRNA lentiviral vector system, 5 strains of different sequence Grb2 shRNA lentiviral particles was obtained and infected colon cancer HT29 cells respectively. 5 colon cancer HT29 cell lines infected with the Grb2 shRNA lentiviral particles for the experimental group (i.e. infected the Grb2 shRNA cells HT29/shGrb2-69,HT29/shGrb2-70,HT29/shGrb2-71, HT29/shGrb2-72,HT29/shGr-b2-73),eGFP shRNA slow virus infection colon cancer HT29 cell for the negative control group. MTT method was used to determine the cell proliferation situation, the expression of Grb2 mRNA was detected by RT-PCR and Grb2、P42/44 ERK、phosphorylation P42/44 ERK、serine/threonine protein (Akt)、phosphorylated Akt (P-Akt)、STAT5 and other molecules signaling pathway detected by Western Blot. Results 72h after infection，HT29/shGrb2-69, HT29/shGrb2-73 cell were suppression in the mRNA and protein levels. After 24h infection ,HT29/shGrb2-69、HT29/shGrb2-73 cell A values were 0.176±0.045、0.186±0.013,and 48h for 0.347±0.048,0.382±0.041 (Significantly lower than the blank control, negative control, P<0.001),and 72 hours after infection were 0.934±0.038、0.983±0.205 (compare with the blank control, negative control, P<0.01); 72h after infection，Phospho-P42/44 ERK, Akt, Phospho-Akt were decreased, while the ERK,STAT5 expression were not affected. Conclusion The application of a lentiviral vector system transfected of Grb2 shRNA to HT29 cells inhibited the levels of Grb2 mRNA and protein significantly, and induced HT29 cell proliferation and down-regulated signal transduction molecules expression.

Key words: colorectal cancer, Grb2, RNAi, lentivirus, signaling pathway