| Objective Breast cancer is a common women malicious disease. In Europe and North America, the incidence of breast cancer has been the first place of all the women malicious diseases for many years. As the development of economy, the incidence of breast cancer began to raise quickly in some developed littoral area of China, it has already been the first or second place of all the cancers of women. Breast cancer seriously threatened women health. At present, the major clinical treatments to breast cancer are surgery, chemical therapy and endocrine therapy. Gene therapy has emerged recently, it showed tremendous potential ability to treat breast cancer. So, it is very important to find some new genes which can early diagnose breast cancer, or predict the prognosis of breast cancer, or provide a target for gene therapy. Lately, the hottest spot in molecular oncology is the research of cell cycle, and Cyclins are the most popular gene family in this field. As a oncogene and a gatekeeper gene of G1/s phase in cell cycle, Cyclin E, especially LMW Cyclin E has been noticed for the strong relationship to breast caner prognosis by many researchers. This study measured the expression of Cyclin E in breast cancer, conclude the relationship between Cyclin E expression and breast cancer prognosis. Then we designed siRNA sequences according to RNAi technology to inhibit the expression of Cyclin E in vivo and in vitro, and analysed the influence of Cyclin E to breast cancer's biology behavior, and finally discovered the potential value of Cyclin E as a new taget to gene therapy.Methods Tumor tissues were obtained from the pathology department of Huazhong University of Science and Technology Tongji Medical College Union Hospital. Each patient had received a diagnosis of breast cancer according to the TNM method which is recommended by UICC and AJCC between 2000 and 2003. Among them there are 18 cases of benign tumor and 80 cases of breast cancer. All patients have not accepted any treatment before surgery. We examined the expression of Cyclin E, HER-2/neu, nm23-H1 and actin by immunohistochemical methods. We designed and synthesized two pairs of siRNA according to the Cyclin E cDNA sequence in Genebank, then inserted them into pGENESIL-1 plasmids respectively. The recombinants (named pGENESIL/Cyclin E-1 and pGENESIL /Cyclin E-1) were sequenced and identified. We transfected the vector into MCF-7 cell line, RT-PCR and Western blot were performanced to examine the expression of Cyclin E in mRNA and protein level respectively. Cell cycle was measured by FCM. And after being treated with chemotherapy drugs, the MCF-7 growth and proliferation were examined by CCK-8 assay. The nude mice bearing human breast cancer were prepared with MCF-7 and transfected MCF-7 and Cyclin E siRNA was injected to nude mice bearing human breast cancer. The growth of tumor was observed.Results The over expression rate of Cyclin E in malignant tissues is obviously higher than that in benign tumor tissues, P<0.01. The over expression of Cyclin E in later stage of disease is higher than that in early stage of disease, P<0.05.The expression of Cyclin E in ER positive tissues is lower than that in ER negative tissues, P<0.05. The expression of Cyclin E in PR positive tissues and in PR negative tissues has no significant different, P>0.05. The expression of Cyclin E in HER-2/neu positive tissues is higher than that in HER-2/neu negative tissues, P<0.05. And the expression of Cyclin E in ER, PR and HER-2/neu all positive tissues is much higher, P<0.01. The expression of Cyclin E in nm23-H1 positive tissues and in nm23-H1 negative tissues has no significant different, P>0.05. The expression of Cyclin E in actin positive and continuous distribution tissues is lower than that in actin negative or uncontinuous distribution tissues, P<0.05. Cyclin E siRNA eukaryotic expression vectors were succesfully constructed. Sequence analysis of inserted fragments revealed the same sequences as synthesized siRNA oligonucleotides. RT-PCR and Western blot showed the expression of Cyclin E was significantly lower after MCF-7 was transfected. After being treated by chemical drugs, the growth and proliferation of transfected MCF-7 was lower then that of non-tranfected cells, and there was a significant difference. And its cell cycle was arrested at G1 phase. The tumor forming ability of transfected MCF-7 was lower than that of non-transfected MCF-7. Cyclin E siRNA could inhibit the growth of tumor beared by nude mice.Conclusions To some extend, the over expression of Cyclin E can reflect the difference between benign and malicious breast diseases. The level of Cyclin E expression has a strong relationship with the clinical phases of breast cancer. The expression of Cyclin E can be a division index of breast cancer prognosis. Inhibition of Cyclin E can obviously depress cancer cells' proliferation, raise their sensibility to chemical therapy and restrain their tumor forming ability. Cyclin E siRNA can be used to inhibit the growth of breast cancer cells. Cyclin E has the potential to be a new target for gene therapy of breast cancer.
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