Suppressed The Expression Of E2F1 And TS In MCF-7 Cells By Retrovirus-mediated RNAi

Transcription factor E2F1 plays an important role in controlling cell growth during the G1 to S phase transition. A variety of genes encoding proteins important for cell proliferation and growth are activated and controlled by E2F1.Therefore E2F1 modulation is critical in maintaining control of normal cell proliferation. The role of E2F1 in the development of cancer is complex and poorly understood. Present studies show the over-expression of E2F1 gene is probably a key factor in the formation and development of most human cancer. Deregulation of E2F1 gene expression can lead to uncontrolled cell proliferation and promote angiogenesis, invasion and metastasis of cancer. Studies showed the over-expression of E2F1 correlates with invasion of breast cancer and E2F1 is a promising candidate to become a new prognostic/predictive marker of breast cancer.Thymidylate synthase(TS) is a key enzyme in the synthesis of the thymidine and plays an important role in the synthesis and reparation of DNA. TS closely correlates with cellular division and proliferation. TS is also the target enzyme of 5-FU, which exerts its anti-tumor activity through its inhibition of TS and blocking synthesis of DNA. Some proves indicated high expression of TS in tumor tissues can promote the development of tumor and develop the resistance to 5-FU. Studies showed that the expression of TS correlates with E2F1 expression .TS has the identification site ofE2F1 and the expression of TS is regulated by E2F1. Over-expression of E2F1 will promote the expression of TS. It is showed some tumor tissues over-expressing E2F1 and TS have low sensitivity to traditional chemical therapy and leads to recurrence and metastasis of tumor.Breast cancer is one of the most common malignant tumors, which greatly affects human health and life. The characteristic of epidemiology of breast cancer is the annual rate gradually increases and the age of the disease turns on a younger tendency in China. With the development of treatments, chemotherapy has become one of the most important parts of multidisciplinary therapies in breast cancer. However, resistance to chemotherapy leads to poor therapeutic effects and become one of the great difficulties in tumor therapy. Since the development of gene therapy, the object of gene therapy turns to malignant tumors and RNA interference (RNAi) will cure malignant tumor and become a hot spot in genetic-based therapies in future.RNAi is a new genetic blocking techniques in transcriptional level. It can specially suppress the expression of target gene and cure tumor, especially in gene over-expressing diseases, such as cancers. Though the applications of RNAi techniques still stay in a preliminary period in mammalian animals, We can predict it will be a critical therapeutic method in diseases, especially in tumor.Materials and methods1. Two complementary oligonucleotides designed with hairpin loop for E2F1 siRNA according to the design principle.2. Construction of pSIREN-E2Fl vector and control vector of pSIREN-C.3. Transfection , cloning and screening of MCF-7 cell.4. Detection of E2F1mRNA TSmRNA expression by RT-PCR techniques.5. Detection of E2F1 protein expression by Western blot techniques.6. Statistical analyses, including multivariant analysis and sample correlation were performed with the software package SPSS 11.0(SPSS Inc., Chicago, USA). Data are presented as mean ± s ( x ± s ) . Statistics between groups of samples were made by ANOVA with the LSD test. Differences were considered to be statistically significant when P <0.05. Pearson's correlation coefficent analysiswas used to evaluate the relation. All P-values presented are two-tailed.Results1. The pSIREN-E2Fl vector was confirmed by restriction endonuclease digestion and the sequence analysis of E2F1 siRNA is correct.2. Compared with cells transfected with control vector and untransfected cells, the mRNA expression of E2F1 and TS were greatly decreased in cells transfected the pSIREN-E2Fl vector by RT-PCR techniques (P <0.05). And the rates of suppression both E2F1 and TS are 100% (7/7).3. The mRNA expression of E2F1 and TS are closely related, and the Pearson'scorrelation coefficent r=0.951 (P =0.000) .4. Compared with cells transfected with control vector and untransfected cells, the expression of E2F1 protein were greatly decreased in cells transfected pSIREN-E2Fl vector by Western blot techniques (P <0.05).Conclusions1. The selected E2F1 siRNA sequence, which has been packaged in pSIREN and transfected it into MCF-7cell works well.2. The expression of E2F1 gene is successfully suppressed by the retrovirus-mediated RNAi techniques, indicating that this may provide a new solution for E2F1-targeting therapeutic intervention of breast cancer.3. The expression of E2F1 and TS is closely related in mRNA level and the expression of TS mRNA is successfully suppressed by the retrovirus-mediated RNAi techniques, indicating that this may provide a new solution for TS-targeting therapeutic intervention of tumor.