Modulation Of The Biological Features Of Breast Cancer Stem Cells By Knockdown Of CD44

[Purpose]1. To explore the role of CD44mocular in progress of breast cancer by detecting the proportion of CD44+CD24-/low phenotype of cells in breast cancer cell line MDA-MB-435.2. To CD44knockdown in breast cancer cell line MDA-MB-435by using short hairpin small interfering RNA (shRNA) and observe if CD44knockdown inhibit the expression of CD44and effect biological behaviour in breast cancer cell line MDA-MB-435.[Methods]1. Detection the expression of CD44and confirmation proportion of CD44+CD24-/low cells in breast cancer cell line MDA-MB-435by flow cytometry.2. The CD44splicing variant which with the highest expression level in the breast cancer cell lines MDA-MB-231, MDA-MB-435and breast cancer tissue samples was used as template, we construct specific shRNA expression vector. With scrambled siRNA and shRNA-GAPDH plasmids expression vector as control.3. shRNA-CD44s recombinant plasmids were transient transferred into human breast cancer MDA-MB-435cells by lipofectamine2000respectively, we detected if shRNA-CD44s effect cell proliferation, adherence, invasion,cell cycle and cell apoptosis by Mammosphere culture, transwell invation assay and flow cytometry.4. On the basis of the transient transfection by G418selection, the establishment of stably transfected MDA-MB-435cells, the untransfected MDA-MB-435cell line as a control group, To detect the CD44s expression in MDA-MB-435cells by real-time fluorescence quantitative PCR, and observe whether the expression of CD44mRNA was inhibited, the application of in vivo experiments to further investigate the effect of tumor CD44knockdown to the tumorigenic ability.[Results]1. Flow cytometry showed.we were obtained with the proportion of CD44+CD24-/low phenotype of cells up to90%sorting by flow cytometry in breast cancer cell line MDA-MB-435.2. shRNA-CD44s significantly reduced the expression of CD44s gene mRNA in MDA-MB-435cells, promoted cell apoptosis and necrosis, regulated cell cycle, inhibited invasiveness, proliferation and self-renewal capacity of tumor cells.3. Tumorigenicity experiments showed that injection of106untransfected shRNA-CD44s of MDA-MB-435cells21days caused large tumors(on the right) in NOD/SCID mice, while the injection of106shRNA-transfected CD44s of MDA-MB-435cells21days observed no obvious tumor formation(on the left). The untransfected shRNA-CD44s had a wide range of tumor tissues was significantly red fluorescence signal Under the fluorescence microscope, the regional distribution of the fluorescent signal, indicating that the untransfected shRNA-CD44s had tumors expressing CD44protein; while tumor tissue can be seen in the weak or almost no red fluorescent signal in the shRNA-transfected CD44s. HE staining of tumor tissue paraffin, the untransfected shRNA-CD44s of tumor tissue showed a cord-like arrangement Under light microscope, no significant catheter differentiation.central saw large areas of necrosis, cell volume big, translucent eosinophilic cytoplasm and nucleus increased significantly, stained pathological mitotic easy to see, most of the cells showed obvious nucleoli. partially visible bizarre giant cells, while the majority of tumor cells had moderate volume and no central necrosis in the shRNA-transfected CD44s.[Conclusion]1. Knockdown of CD44caused breast cancer cells into a ball diminished capacity, suggesting that BCSCs is inhibited dry and differentiate into non-BCSCs, suggesting that CD44is dry tumor stem cells play a key molecule.2. Knockdown of CD44in MDA-MB-435cells inhibited the proliferation, regulated cell cycle of tumor cells, promoted cell apoptosis and necrosis, reduced cell invasion force.3. the results of this project targeted therapy for breast cancer provides new evidence and clues.