The Study For Functions Of Nanog In Human Breast Cancer Cells

Objectives:Nanog is a homeodomain-containing transcription factor that functions to maintain self-renewal and proliferation of embryonic stem cells (ESCs). Nanog maintain the pluripotent and replicate ability of embryonic stem cells through the signal way with cells or outside. At first people think Nanog can express only in embryonic stem cells (ESC), embryonic germ (EG) cells and embryos tumor (EC) cells, and there is none expression in adult organization. However, a recent study found that Nanog can also express in some cancer cells(seminoma, breast cancer, ovarian cancer, colon cancer, testosterone and germ cell tumors, etc.). In view of the cancer and ES cells are infinite in proliferation and differentiation ability, we speculate that Nanog may be the key factor to adjust the versatility of cancer cells. In this study we have used RNA interference technology to reduce the expression of Nanog in breast cancer cells, to investigate the influence that Nanog with the proliferation ability in breast cancer cells. And we have also discuss if there are relationship among Nanog and the proliferation related factors (cyclinD1/c-myc/cyclinE/STAT3), to further explore the mechanism how Nanog regulate the proliferation of breast cancer cells.Methods:1. We used Real-time PCR and Immunohistochemistry to detect the relationship between the expression of Nanog and clinical and pathological features.2. We used RNA interference technology to konckdown the expression of Nanog in huamn breast cancer cell lines and the expression of Nanog was detected by Western blotting.3. Using MTT to detect the effects of Nanog down-regulation on proliferaton of human breast cancer cells.4. Flow cytometry anaylisis was used to detect the change of cell cycle.5. Colony formation assay was used to observe the influence of Nanog on colony formation rate of MCF-7cells.6. We used soft agar assay to detect the influence of Nanog down regulation on the ability of human breast cancer MCF-7cells’proliferation and tumor formation.7. Migration assay was performed to detect the influence of Nanog on human breast cancer MCF-7migration ability in vitro.8. Real-time PCR and werstern blotting was used to examine the expression of c-myc, cyclinD1, cyclinE and STAT3to explore the mechanism of Nanog regulating the proliferation, migration of human breast cancer.9. Western bloting detect that if the expression of cyclinDl could be recoveried following the Normal expression of Nanog.10. Chromatin immunoprecipitation assay was used to detect that if Nanog can regulate the expression of cyclinDl in a direct way.Results:1. Real-time PCR and Immunohistochemistry detect that the expression of Nanog in breast cancer tissues were increasing following the increase of malignant degree, which closely related to the size of the tumor tissue, lymph node metastasis and to the clinical stage (P<0.05).2. RNAi technology was used to transfect human breast cancer cells and3monoclonal cell strains with low expression of Nanog were screened in MCF-7cells.3. The proliferation ablity of MCF-7and MDA-MB-231was inhibited by the knockdown of Nanog.4. Flow cytometry detect the cell cycle were blocked in G0/G1after Nanog were knock down.5. The colony formation rate of MCF-7was decreased by the knockdown of Nanog.6. Soft agar assay shows that the ability of human breast cancer MCF-7cells" proliferation and Tumor formation were reduced following the knock down of Nanog.7. The migration of MCF-7cells were decreased with the down-regulation of Nanog.8. The expression of cyclinDl and c-myc were decreased significantly after Nanog down regulation, while the expression STAT3and cyclinE were not influenced.9. Rescue experiment detect that the expression of cyclinDl could be recoveried following the normal expression of Nanog.10. Chromatin immunoprecipitation assay was used to detect that Nanog can bind to the promoter of cyclinDl and promote the transcription of cyclinD1.Conclusions:1. The expression level of Nanog are higher in the high malignant degrees of breast cancer tissues than low malignant degrees, and the expressiomn of Nanog are closely related with the size of the tumor tissue, lymph node metastasis and the clinical stages(P<0.05).2. The down expression of Nanog inhibited the proliferation ablity and the percentage of G0/G1was increased while which of G2/M+S was decreased in MCF-7and MDA-MB-231cells.3. The down expression of Nanog induced the low expression of C-myc and CyclinDl, and the further research approved that Nanog by interacting with the promoter of cyclinDl to affect the expression of cyclinDl and to influence the proliferation/cell cycle of breat cancer cells.