The Effects Of TPX2 Silencing On Esophageal Carcinoma Cell Growth And The Expression Of TPX2

In the worldwide, esophageal carcinoma is one of the most popular malignant tumors in digestive system. And China is the country which has the highest incidence and mortality of Esophageal Carcinoma in the world. Squamous cell carcinoma is the most popular histological type, which accounts for 90%. Malignant transformation of cells is not the result of a single genetic mutation; it is caused by several genes and multistage changes. It has important significance to search the changes for mechanism of esophageal carcinoma.RNA interference (RNAi) technique, it inhibits the production of the target gene mRNA through the processing of transcription, and it results in the reduction of special proteins. After all, RNAi induce the phenomenon of gene expression's decrease, so it is called posttranscriptional gene silencing (PTGS). The fundamental principle of RNAi is that dsRNA was splitted by nucleicacidase into small ribonucleotide with the length of 21-25bp, and the small ribonucleotide as the mediator can effectively revoke the degradation of homologous sequences special mRNA. The cross-correlation technology of RNA interference could silence the special genes, it also can replace the technology of gene knockout, and it is a important method to research the function of gene regulation. RNAi may be used to inhibit the expression of abnormal gene in tumor cell, then the cells resume the normal physiological function of the mutational gene, meanwhile, it enhanced the sensitivity of the drugs to humor cells, at the same time, according the research of the functions of all kinds of cytokines, we may could screen the effective targeting gene of the gene therapy to cure the tumors. Recently, TPX2 is considered as a new candidate oncogene by many researchers. The results of multiple experiments which were published in domestic and abroad journals showed that overexpression of TPX2 could induce the amplification of centrosome which lies in cells, we may found heteroploid even polyploids DNA in the cells, and it interferes with the nucleolus's normal disruption, and inhibits the self-activated physiological pathway. In many abroad documents, it is over-expressed in many cancer tissues, for examples:lung squamous cell carcinoma, salivary-gland carcinoma, pancreatic cancer, ovarial cancer, but it is low-expression in the respective normal tissues. And we found that the report of the TPX2 expression of esophageal carcinoma cells is rarely founded.So our research was covered with those aspects.1. We designed and synthesised three kinds of TPX2 siRNA (TPX2 siRNA988, TPX2 siRNA1042 and TPX2 siRNA1725), then we transfected them into EC9706 cells in which TPX2 was highly expressed, and we investigated the effective transfection site and density to inhibit the expression of TPX2; 2. The method of cellar proliferation was used to observe the effect of siRNA inhibition of EC9706. RT-PCR and Western blot were used to detect the expressive influence of TPX2 mRNA and protein; it could tell us the distinction in different time points of transfection.Materials and Methods1 The culture of the esophageal carcinoma EC9706 cells:The EC9706 cells were adherent-cultured with 1640culture fluid of 10% fetal bovine serum under the condition of 37℃and 5%CO2.2 Design and synthesis.of three kinds of TPX2 siRNA:three nucleotide sequence of siRNA (TPX2 siRNA-988, TPX2 siRNA-1042, TPX2 siRNA-1725) corresponding to different sites of TPX2 gene were designed and synthesized by Shanghai GenePharma Co. Ltd.3 Transfecion:TPX2 siRNA was transfected by LipofectamineTM2000Transfection Reagent into esophageal carcinoma EC9706 cells which were in logarithmic growth phase. 4 Screening of optimum TPX2siRNA:the experiment was designed in 300 nM transfecting concentration after 48 h transfection. Blank control and negative control were set. The technique of RT-PCR was used for detection of TPX2mRNA in different groups.5 Screening of the optimum dosage TPX2siRNA-1725:the experiment was designed in 100 nM,200 nM and 300 nM transfecting concentrations with TPX2 siRNA-1725 after 48 h transfection. Blank control and negative control were set. The technique of RT-PCR was used for detection of TPX2mRNA in different groups.6 Screening of the optimum time point of TPX2siRNA-1725:300 nM of TPX2 siRNA were transfected into EC9706 cells. Then they were cultured 24 h,48 h,72 h and 96 h, we set blank control and negative control. RT-PCR and Western blot were used to detect the expression of TPX2mRNA and protein, and cellar proliferation was detected by MTT assay.7 Statistical analyses:The SPSS 15.0 statistical software package was used for all analyses. The data were expressed by mean±standard deviation(X±S)and analyzed using the ANVOA. The level of significant difference isα=0.05Results1 The screening results of optimum TPX2siRNA:three kinds of TPX2siRNA were transfected into EC9706cells for 48 h, the expression level of TPX2 mRNA in TPX2 siRNA-1725 group decreased, and it has the statistical significance(P< 0.05). So we got the result that TPX2 siRNA-1725 is the optimum TPX2siRNA.2 The screening results of optimum dosage of TPX2 siRNA-1725:TPX2siRNA-1725 were transfected into EC9706 cells with the density of 100 nM,200 nM and 300 nM for 48 h, the expression of TPX2 mRNA in 300 nM groups were suppressed evidently compared with control groups, and it has the statistical significance(P<0.05). So we got the result that the optimum dosage of TPX2 siRNA-1725 is 300 nM.3 The screening results of optimum transfection time point:EC9706 cells were transfected at the density of 300 nM TPX2siRNA for 24 h,48 h,72 h and 96 h, the expression quality of TPX2mRNA and protein were lower in experimental groups compared with control groups after transfecting for 48 h,72 h, especially 72 h. There was significant difference at different transfecting times (P<0.05). And the cellar proliferation was inhibited after 48 h, the inhibitory effect was mostly obvious after 72 h, and the rate of growth suppression reached 35.28%.4 There was a correlation between the expression of TPX2mRNA and the expression of protein after the transient transfection of TPX2siRNA at 24 h,48 h,72 h and 96 h.Conclusions1 The foundation of TPX2siRNA could research the functions of TPX2 conveniently; TPX2 siRNA can specially inhibit the expression of TPX2.2 Specific siRNA1725 of TPX2 can inhibit the expression of TPX2 gene and TPX2 Protein in the esophageal carcinoma EC9706 cells. It indicates that TPX2 siRNA can inhibit the proliferation of esophageal carcinoma by inhibiting expression of TPX2 gene.