The Primary Research Of The Effect And Mechanism Of Pokemon On The Expression Of Smad4 In MCF-7 Cells

Objective To explore the impact of Pokemon on the expression of Smad4 in human breast cancer MCF-7 cells,and to analyze the associated molecular mechanism.Methods The experiments includ two parts. The first part:1) The breast cancer cell MCF-7 in vitro culture was the research object. Transfecting the mock-vehicle as control group,and transfecting Pokemon expression plasmids as experimental group,Q-PCR technology,Western Blot and immunoflurescence were utilited in this study to detect the expression of Smad4 in different ways;2) The breast cancer cell MCF-7 in vitro culture was the research object,and exploring the effect of Pokemon on Smad4 promoter activity.Through transient transfecting the mock-vehicle as control group,and transient transfecting Pokemon expression plasmids as experimental group.Luciferasy assay was used to detect the impact of Pokemon on the promoter activity of Smad4.The second part:1) The breast cancer cell MCF-7 in vitro culture was the research object.Through transient transfecting the mock-vehicle as control group,and transient transfecting Pokemon expression plasmids as experimental group.Beside that,The MCF-7 cell strain steadily expressing Pokemon was used in this test.Flow cytometry was used to detect the breast cancer stem cells in both control group and experimental group;2) The breast cancer cell MDA-MB-231 in vitro culture was the research object.Through transient transfecting the mock-vehicle and Pokemon expression plasmids,Flow cytometry was used to detect the breast cancer stem cells in both control group and experimental group.Results The first part:1.Pokemon inhibited the expression of Smad4 in human breast cancer MCF-7 cells.Transfecting the mock-vehicle and Pokemon expression plasmids in MCF-7 cells:1)Q-PCR results showed that Pokemon inhibited the transcription of m RNA Smad4(p<0.01).2)Immunofluorescence assay showed that Pokemon inhibited the expression of Smad4 protein(p<0.05).3)Western Blot showed that Pokemon inhibited the expression of Smad4 protein(p<0.05). 4)In MCF-7 cells strain steadily expressing Pokemon,Western Blot showed that Pokemon inhibited the expression of Smad4 protein(p<0.05).2. Pokemon inhibited the promoter activity of Smad4 in human breast cancer MCF-7 cells.1)Identify that the three of p GL4.10-Smad4 promoter fragments constructed in our laboratory be of transcriptional activity in MCF-7 Cells.Luciferasy assay revealed:compared with transfected p GL4.10,the ratio of fluorescence were significantly increased in p GL4.10-Smad4 groups(p GL4.10-Smad4pp1 p<0.001,p GL4.10-Smad4pp2 p<0.05,p GL4. 10-Smad4pp3 p<0.05).2)To exploring the effect of Pokemon on Smad4 promoter activity in MCF-7 cells.Luciferasy assay showed:compared with control groups,the Smad4 promoter activity was significantly descread in experimental groups(p GL4.10-Smad4pp1 p<0.05, p GL4.10-Smad4pp2 p<0.001,p GL4.10-Smad4 p<0.05).Showing that Pokemon inhibited the activity of Smad4 promoter in MCF-7 cells.The second part:1.Pokemon down-regulated the rate of BCSCs in human breast cancer MCF-7 cells:Flow cytometry showed:Pokemon down-regulated the rate of CD24-/CD44~+ Cells in both transient expressing(p<0.05)and strain steadily expressing Pokemon(p< 0.05)of MCF-7 cells.2.Pokemon down-regulated the rate of BCSCs in human breast cancer MDA-MB-231 cells:Flow cytometry showed that:Pokemon down-regulated the rate of CD24-/CD44~+cells(p<0.05) and CD49f~+/Ep CAM~+cells(p<0.05) in MDA-MB-231 cells.Conclusion 1)Pokemon could inhibit the expression of Smad4 in human breast cancer MCF-7 cells,by suppressing the promoter activity of Smad4.2)Pokemon down- regulated the rate of breast cancer stem cells in MCF-7 and MDA-MB-231 cells.