Abstract:

Objective  To construct two eukaryotic plasmids expressing short hairpin RNA (shRNA) of fragile histidine triad (FHIT) and to study their effects on proliferation and apoptosis of the gastric cancer cell line BGC-823. Methods  Specific shRNA plasmids to FHIT were constructed, and then transfected into the BGC-823 cells with lipofectamine methods. Cells were divided into four groups：the control group，the PGPU6/GFP/Neo-shNC transfected group as the negative group and the PGPU6/GFP/Neo-shRNA transfected groups .After selection with G418, the stable cell clones were attained. Expression of FHIT mRNA was determined by quantitive real-time PCR. The effect of FHIT on the growth characteristics of gastric cancer cells was observed by methyl thiazolyl tetrazolium (MTT) and flow cytomery (FCM). Results  Stable clones with shRNA-FHIT plasmids were obtained. Compared with the negative control cells, expression of FHIT mRNA was down-regulated in the shRNA-FHIT plasmid transfected cells(P＜0.05). Proliferation was promoted, whereas the cell apoptosis rate was decreased. Cells at the G0/G1 cell stage decreased, while the cells at S and G2 cell stages increased. All these differences between shRNA-FHIT transfected cells and the two control groups of gastric cancer cells had statistical significances (P＜0.05). Conclusions  shRNA-FHIT plasmids were successfully transfected into BGC-823 cells, and the cells which express FHIT in a stable lower level were obtained. FHIT-tageted shRNA could obviously decrease the FHIT expression in BGC-823 cells at a stable lower level, promote the proliferation of BGC-823 cells, suppress their apoptosis and weaken the G0/G1 phase block of cell cycle.

Key words: Fragile histidine traid gene；Stomach neoplasm；RNA interference；Proliferation；Apoptosis