Effect Of Pokemon Expression Silencing On CD44+CD24- Population In Breast Cancer T-47D Cell Lines And Its Mechanism

Objective This work is proposed to observe the effect of silencing pokemon expression on CD44+CD24-population in Breast cancer T-47 D cell lines,and to explore the impact of pokemon on Epithelial-mesenchymal transition and possible targets,and to analyze possible mechanism and clinical correlationship between pokemon and smad4.we explore the effect of pokemon expression silencing on CD44+CD24-population,pokemon expression silencing on EMT,pokemon expression silencing on TGF?/smad4 signaling pathways,which will lay a foundation for the further research of invasion and metastasis mechanism of breast cancer.Methods: Experiment was divided into two parts.Part I: T-47 D breast cancer cells was cultivated,a couple of specific siRNA were designed in accordance with the pokemon gene sequence and chemically synthesized.After pokemon-siRNA transient transfection in T-47 D cells by Cationic liposome method,the more efficient silence sequence was selected by western blot,the variation of the proportion of breast cancer stem cells was examined by Flow cytometry detection.After pokemon-siRNA transient transfection in T-47 D cells,the change of level of protein of EMT related proteins was detected by western blot assay?Part II:The luciferase reporters carrying smad4 promoter was constructed.The luciferase reporters and Pokemon expression plasmid transfection at the same time,Luciferase activity was examined by Luciferase reporter assay.To further validate the relationship between pokemon and smad4,western blot was used to examine the change of Smad4 protein level after pokemon-siRNA transfection.To analyze the relevance of pokemon and smad4 in clinical breast cancer tissues,the expression of pokemon and smad4 in Breast cancer tissue was examined by immunohistochemistry staining method.Results:1)Effect of silencing pokemon expression on promotion of CD44+CD24-population in breast cancer T-47 D cell line.(1)pokemon-siRNA-002 transient transfection can effectively silence the expression of pokemon gene in T-47 D cell.(2)Flow cytometry assay revealed that negative control group and pokemon-siRNA group were(0.57±0.15)% and(1.10±0.20)% respectively.the number and rate of breast cancer stem cells in pokemon-siRNA were largely increased.2)pokemon-siRNA transient transfection in T-47 D cells down-regulated the protein expression level of beta-catenin,ZO-1 and claudin-1,which indicated that silencing the pokemon expression could possibly promote breast cancer epithelial–mesenchymal transition.3)pokemon can down-regulated the protein expression of smad4 by inhibiting smad4 promoter activity.(1)we constructed the three luciferase reporters carrying the smad4 promote,named pGL4.10-Smad4pp1,pGL4.10-Smad4pp2 p GL4.10-Smad4pp3.luciferase report assays validated luciferase activity of pGL4.10-Smad4pp1 was the strongest.(2)luciferase assays indicated that the pokemon expression plasmid caused a significant reduction in luciferase activity compared with the control.(3)Western blot indicated that pokemon-siRNA markedly promoted smad4 protein level compared with the control.4)The correlation between Pokemon and smad4 expression in breast cancer tissues.Taken the 185 cases of human breast cancer tissues including all kinds of different pathological types to immunohistochemical analysis,the expression of Pokemon and smad4 was not remarkable correlation.Conclusion:1)Silencing pokemon gene expression could promote the CD44+CD24-population in Breast cancer T-47 D cell lines.2)pokemon can down-regulated the protein expression of smad4 by inhibiting smad4 promoter activity...