The Effects Of E2F-1 Gene Expression And Celluar Biological Behaviors By Short Interfering RNAs In Gastric Cancer Cell Line MGC803

Objective To investigate the effects of RNA inference(RNAi)targeting E2F-1 on biological behaviors of gastric cancer cell line MGC803.Methods E2F-1-siRNA and negative control-siRNA were transfected into gastric cancer cell line MGC803 by using Lipofectamine^TM2000.48 hours later,the expression of E2F-1 mRNA and protein in gastric cancer cell line MGC803 was detected by Real-time quantitative PCR and western blotting methods respectively,Cell cycle was examined by flow cytometry,The proliferative rate of MGC803 cells was assessed by methyl thiazolyl tetrazolium(MTT)assay.Cell matrigel invasion assay and cloning assay were used for detecting cell invasive power and proliferative ability by E2F-1 gene down regulation.Results Transfection of gastric cancer cell line MGC803 with E2F-1-siRNA and negative control-siRNA could be mediated by Lipofectamine^TM2000 effectively.Compared with negative control-siRNA and control group the expression level of E2F-1 mRNA and protein measured by Real time quantitative PCR and western blotting decreased by 84.8%,85.3%(P＜0.05)and 77.2%,79.6%evidently in gastric cancer cell line MGC803 transfected with E2F-1-siRNA.Compared to negative control-siRNA and control group,the proliferative inhibiting rate of experimental group was 69% and 81.6%(P＜0.05)respectively.More mean DNA content of MGC803 cells accumulated at G₂/M phase,the rates of mean G₂/M content of cells in experimental group,negative control group and control group were(54.98±3.46)%,(43.07±3.82)%and(31.47±2.12)%respectively,with the difference being significant among them(P＜0.05).At the same time,the invasive power and proliferative ability presented a decrease(P＜0.05).Conclusions E2F-1 gene repression by RNAi can decrease the DNA content in G₁ phase of MGC803 cell,keep cell division arresting in G₂/M phase and restrained cell proliferation,suppress its invasive power ability to some extent.E2F-1 may be a novel therapeutic target for gastric cancer. Objective As an important regulatory factor,the transcription factor E2F-1 has an imitate relationship with the development of tumor in cell life cycle.We investigate the effects of small interference RNA(siRNA)expression vectors of transcription factor E2F-1 on the expression of E2F-1 in human gastric cancer cell line MGC803.Methods Two DNA sequences containing small hair pin structure were designed and synthesized.The complement forms were obtained by annealing and being cloned into vector pSilencer4.1-CMV neo digested by restriction enzyme BamHâ… and Hindâ…¢.The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing.After transfecting into the gastric cancer cell line MGC803 by Lipofectamine TM2000,the expression of E2F-1 mRNA and protein was detected by Real-time quantitative PCR and western blotting methods respectively.Results The recombinant expression vectors were successfully constructed,which were confirmed after the enzyme digestion analysis and the DNA sequencing.Compared with negative control group and control group,the expression level of E2F-1 mRNA and protein decreased by 86.1%,86.4%(P＜0.05)and 79.6%,81.5%evidently in gastric cancer cell line MGC803 transfected with the siRNA expression vectors.Conclusions The E2F-1 specific siRNA expression vector has been successfully constructed,which can down-regulate the expression level of E2F-1 in gastric cancer cell line MGC803 transfected with the pSilencer4.1-E2F-1.At the same time,It may provide a basis applicable strategy for function study and gene therapy of gastric cancer. Objective To investigate the genes differently expressed between gastric cancer cell line MGC803 transfected with E2F-1-siRNA(experimental group) and negative control-siRNA(negative control group)by cDNA microarray technique.Methods The total RNA was extracted from gastric cancer cell line MGC803 transfected with E2F-1-siRNA and negative control-siRNA and then purified.The cDNA was obtained by reverse transcription polymerase chain reaction(RT-PCR),and then labeled with Cy5 and Cy3 fluorescence as probes, which were hybridized with gene chip containing 21522 human 22K gene expression profile.Subsequently,the two signal images were scanned by LuxScan 10K/A dual pathways laser scanner(CapitalBio Corp.)and analyzed by LuxScan3.0 image analysis software(CapitalBio Corp.).Results Of the 21522 target genes,18 genes were screened out for differences in gene expression expression level between gastric cancer cell line MGC803 transfected with E2F-1-siRNA and negative control-siRNA.Eight of the 18 genes were up-regulated and 10 were down-regulated,of which one was with unknown function respectively.Conclusion The carcinogenesis of gastric adenocarcinoma involves in the interaction of multiple genes and regulation in signal pathways.The 18 gene differentially expressed in gastric cancer cell line MGC803 by interfering E2F-1 in vitro may take part in the occurrence and development of gastric cancer.