Effect Of The Short Hairpin RNA Of Fragile Histidine Triad On Proliferation And Apoptosis Of Gastric Cancer Cell Line BGC-823

Background and objective:Gastric carcinoma is one of the most common malignant tumors of mankind, which is closely related environmental carcinogens. Gastric carcinoma has high incidence and death rate in China, so it's a serious threaten to human health. Furthermore, its early detection rate is about 10%. Most cases are at midst or advanced stage until received a final diagnosis. The therapeutic efficacy is unsatisfactory to severe patients. The survival rate of five years is 20%-30%. Early diagnosis and comprehensive treatment is the key point to improve the therapeutic effect of gastric cancer. Gastric carcinogenesis experience a feature of multiphase and multifactor, such genetics alterations as mutation, activation of oncogene, lost of anti-oncogene, chaos of apoptosis regulatory mechanism are involved in the process of carcinogenesis. Fragile histidine triad (FHIT) gene located in chromosomal region 3p14.2 has been cloned in 1996 by Ohta et al. It spans not only the t(3:8) (p14.2; q24) translocation breakpoint found in familial renal cell carcinoma but also the most common human fragile site, FRA3B. It is subordinated to histidine triad (HIT) protein superfamily. FHIT is the first anti-oncogene that connects fragile site with tumors. It is found that FHIT has a close correlation with cancers (such as gastric cancer) which are associated with environment carcinogenic agents, and its expression deletion and abnormality rate are high. All of these suggest that FHIT gene may play a key role in tumor development and treatment. The mechanism of this anti-oncogene is not very clear. Several pathways may be associated with its anti-cancer effect. The further study on the role and mechanism of FHIT may provide new idea for diagnosis and therapy of gastric carcinoma. Our previous studies have shown that FHIT gene could enhance the sensitivity of MGC-803 cells to DDP and ADM, while reduce sensitivity to 5-FU. Moreover, FHIT expression and ADM could promote the apoptosis and G0/G1 phase block of cell cycle of gastric cancer cell synergistically. So, it will have clinical significance to do research on the correlation of expression of FHIT and chemosensitivity in gastric cancer patients. RNA interference (RNAi) is a new powerful tool to inhibit the target gene expression by post-transcriptional gene silencing, which has been utilized widely in the research of functional genome and tumor gene therapy. Based on the above-mentioned studies, our research is designed to construct two eukaryotic plasmids expressing short hairpin RNA (shRNA) of fragile histidine triad (FHIT) and transfect them into gastric cancer cell line BGC-823, then observe the effect on proliferation and apoptosis of these cells, in order to provide basis for the further study of the effect of FHIT on chemosensitivity of gastric cancr.Methods:Specific shRNA plasmids to FHIT were constructed, and then transfected into BGC-823 cells by lipofectamine methods. Cells were divided into four groups:control group, PGPU6/GFP/Neo-shNC transfected group as negative group and PGPU6/GFP/Neo-shRNA transfected groups.After selection with G418, the stable cell clones were attained. The expression of FHIT mRNA was determined by real-time quantitative 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:1. shRNA-FHIT plasmids were successfully transfected into gastric cancer cell line BGC-823. After 3 months'selection with G418, Stable clones with shRNA-FHIT plasmids were obtained.2. Compared with the control group and negative control group, the expression of FHIT mRNA was down-regulated in the shRNA-FHIT plasmid transfected cells(P< 0.05). 3. Compared with the control group and negative control group, proliferation were promoted, whereas cell apoptosis rate were decreased, while the cells at G0/G1 cell stage decreased, the cells at S and G2 cell stage increased in the shRNA-FHIT plasmid transfected cells, these differences between shRNA-FHIT transfected cells and the two control groups of gastric cancer cells had statistical significance(P<0.05).Conclusions:1. ShRNA-FHIT plasmids were successfully transfected into BGC-823 cells, and the cells which express FHIT in a stable lower level were obtained.2. FHIT-targeted shRNA can obviously decrease the FHIT expression at stable lower level in BGC-823 cells, promote their proliferation, suppress their apoptosis and weaken the G0/G1 phase block of cell cycle.