| BackgroundBreast cancer is a common disease of global women. In recent years, the incidence ofthe disease in China showed a rising trend, the incidence rate among the femalemalignancy is first or second. It’s a serious threat to the health of the female population.The disease is often occurrence of distant metastases, as a result, patients not effectivelycure and patients with high mortality, poor quality of life. Studies suggest that breastcancer stem cells is the chief culprit in breast cancer metastasis and recurrence. Stem cellbiological characteristics due to breast cancer: breast cancer stem cell membraneexpression of the drug transporter protein ABCG2/Bcrp1, ordinary chemotherapy drugs isdifficult to enter the cell; conventional chemotherapy drugs and radiation are resisted;breast cancer stem cells will be able to reach other parts of the body and result inmetastasis or micrometastasis in early breast cancer. Therefore,the key targets to breastcancer stem cells is thought out by more and more experts and scholars. Targets to causingmetastasis and recurrence of breast cancer, studing the biology characteristics of breastcancer stem cell, finding the effects of drugs to breast cancer stem cells will be a betteroption to control the development of breast cancer.ObjectivesNotch1abnormal activation plays an extremely important role in maintaining thebiological characteristics of breast cancer stem cells. Silence Notch1gene from the breastcancer stem cells which cultured on suspended microspheres, detecting Notch1,E-cadherin, VEGF, CXCR4expression differences, and exploring Notch1molecules playrole in process of breast cancer stem cells convert to migrating cancer stem cells andassociated mechanisms, which will be provide a theoretical basis for the treatment of breast cancer.Methods1. human breast cancer MCF-7cell line is cultured in adherent cell culture and themicrosphere suspension culture, detection of CD44+/CD24-/lowcell ratio is checking themicrosphere suspension culture efficient enrichment of breast cancer stem cells.2. Using RT-PCR and Western-blot technique, detect Notch1, E-cadherin, VEGF-A,,VEGF-C, CXCR4expression differences among breast cancer cells in ordinary adherentculture and suspended microspheres culture.3. Using of the liposome,targets to Notch1sequence and nonsense sequence smallRNA was transfected into breast cancer cells in the suspension culture and adherent culture,using RT-PCR and Western-blot detection Notch1, E-cadherin, VEGF, CXCR4expressiondifferences respectively.Results1. Microsphere suspension the microsphere suspension culture method effectiveenrichment of breast cancer stem cells, enriched CD44+/CD24-/lowcell concentrationreached87.0±2.5%. MCF-7suspension culture were of varying sizes, spaced,irregularballoon.Balloon cells digested into a single cell, through the suspension culture canproliferate formed balloon. Adherent culture can differentiate into adherent cells.2. Ordinary adherent breast cancer cells compared to the suspended microspherescells of Notch1, VEGF-A, VEGF-C, CXCR4expression levels were significantlyincreased E-cadherin expression level was significantly lower(P<0.05).3. Notch1gene expression after silencing suspension cells, VEGF-A, VEGF-C,CXCR4, and E-cadherin were significantly improved (P<0.05). And CD44+/CD24-/lowphenotypic cell count decreased significantly(P<0.05). Notch1gene expression ofadherent cells after the silence, the VEGF-A, VEGF-C, CXCR4and E-cadherin withoutany change in the the CD44+/CD24-/lowtable no significant change in the number of cells.Conclusions1. Suspended microspheres culture method effective enrichment of breast cancer stemcells, the enrichment of CD44+/CD24-/lowcell concentration reached87.0±2.5%. Build agood model in vitro for the next silent breast cancer stem cells Notch1gene.2. Breast cancer stem cell suspension cultures of Notch1, VEGF-A, VEGF-C, CXCR4expression improved significantly decreased expression of E-cadherin. Suspension culturesof breast cancer stem cells or CD44+/CD24-/lowthe phenotypic cell transfer activity wassignificantly enhanced. 3. Silent breast cancer stem cells Notch1gene, in suspension cultures of breast cancerCD44+/CD24-/lowphenotype cells (cancer stem cells), E-cadherin, VEGF-A, VEGF-C,CXCR4expression was significantly increased. Notch1molecules to increase their transferactivity by reducing the expression of E-cadherin, rather than acting on the VEGF-A,VEGF-C, CXCR4increased transfer activity. Silent breast cancer stem cells Notch1geneCD44+/CD24-/lowphenotype cells decreased, Notch1may play an important role in themaintenance of breast cancer stem cell phenotype CD44+/CD24-/lowphenotype. But silenceadherent cells Notch1gene, VEGF-A, VEGF-C, CXCR4, and E-cadherin none expressionchanges, most of the cells in the adherent cells differentiated cells, Notch1play role only inbreast cancer of CD44+/CD24-/lowphenotypic cell(or breast cancer stem cells). |
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