The Research Of Inhibitory Effect Of Baicalin On Hypoxia Three Negative Breast Cancer Stem Cells Based On HIF-1a/IGF-? Signaling Pathway

Part 1 Effects of hypoxia on the enrichment and gene expression of three negative breast cancer stem cellsObjective:To study the effect of hypoxia on the ball ratio of human breast cancer stem cells and its effect on gene expression.Methods:Human breast cancer cells MDA-MB-231,respectively under normoxic and hypoxic conditions in suspension culture and enrichment of breast cancer stem cells(breast CancerStem cells,BCSCs MDA-MB-231)microspheres,and evaluate the ball rate.Flow cytometry was used to detect the proportion of breast cancer stem cells inCD44+/CD24/low phenotype.Immunomagnetic cell sorting and CD44+/CD24/lowphenotype of breast cancer stem cells.Detection of gene expression between normoxia and hypoxia breast cancer stem cells by cytokine antibody microarray.Results:1.In the hypoxia group,BCSCs microspheres had a ball formation rate of 80.22±0.05%,which was superior than that of the normal group 60.21±0.07%(P<0.05).2.The BCSCs ratio of CD44+/CD24/low phenotype in hypoxia group was 78.63±0.03%detected by flow cytometry,which was higher than that of normoxia group 55.32±0.06%(P<0.05).3.The detection of cytokine antibody microarray showed that the expression of the 20 genes in hypoxia BCSCs group was significantly higher than that in normoxia group.Conclusion:Hypoxia can increase the rate of BCSCs microspheres formation and increase the ratio of CD44+/CD24/low phenotype to BCSCs.Compared with hypoxia and normoxia BCSCs,20 genes expression were up-regulated,such as IGF-II,L-Selectin,etc.Part 2 The effect of Baicalin on the inhibition of hypoxia three negative breast cancer stem cellsObjective:To study the inhibitory effect of Baicalin on hypoxia three negative breast cancer stem cells(MDA-MB-231)in vitro and in vivo.Methods:MTT was used to detect the effect of Baicalin on the proliferation of breast cancer cell line MDA-MB-231 in vitro.In vivo,a breast cancer tumor model in nude mice,to observe the general condition and body weight of nude mice,tumor volume and weight changes,pathological changes of transplanted tumor detection,immunohistochemical detection of tumor ER,PR,Her-2 expression and breast cancer stem cell markers CD44,CD24 expression.Detection of BCSCs markers CD44,CD24 and CD133 expression by tissue microarray.Results:1.MTT assay showed that baicalin could inhibit the proliferation of breast cancer stem cells(MDA-MB-231),which was concentration dependent,and the inhibitory effect of 200mg/L was the best in high dose group(P<0.05).2.In vivo experiments,we successfully established a nude mouse model of three negative breast cancer.3.Baicalin in medium dose(100mg/L)and high dose group(200mg/L)can improve the general state of mice,reduce the size and weight of transplanted tumor(P<0.05).The expression of BCSCs markers CD44,CD24 and CD133 in the middle and high dose group could be reduced(P<0.05).Conclusion:Baicalin can inhibit the proliferation and transformation of breast cancer stem cells MDA-MB-231 in vitro and in vivo.Part 3 The study of the mechanism of baicalin inhibiting hypoxia threenegative breast cancer stem cellsObjective:To investigate the inhibitory mechanism of baicalin on hypoxic breast cancer stem cells(MDA-MB-231)by targeting the HIF-1/?GF-? signal pathway.Methods:The Real-time PCR showed that hypoxia breast cancer stem cells MDA-MB-231 after baicalin affected,the HIF-1a,?GF-?,L-Selectin expression of mRNA were reduced.Western-blot showed hat hypoxia breast cancer stem cells MDA-MB-231 after baicalin affected,the HIF-1a,?GF-?,L-Selectin expression of protein were reduced.Results:1.After the intervention of baicalin,the expression of mRNA and protein of HIF-1a,?GF-?,L-Selectin of hypoxia breast cancer stem cells MDA-MB-231 decreased(P<0.05).2.After the intervention of baicalin,the expression of HIF-1a,?GF-? and L-Selectin in the transplanted tumor of nude mice was decreased(P<0.05).Conclusion:Baicalin can downregulate the level of HIF-la,?GF-?I,L-Selectin expression in hypoxia breast cancer stem cells MDA-MB-231 in vitro.In vivo,the expression of HIF-la,?GF-?,L-Selectin of tumor can be reduced by baicalin.The mechanism of baicalin inhibitory effect of hypoxia three negative breast cancer stem cells may be related to regulating the expression of HIF-1a/?GF-? signaling pathway and L-Selectin.