Study Of Impact On Expression To Breast Cancer Stem Cells Wnt1, β-catenin And MMP-9by Silencing Notch1Molecules

ObjectiveThe current studies have demonstrated that breast cancer stem cells shouldaccount for breast cancer metastasis, and Notch1molecules expedite thismetastasis process by improving the migration activity of breast cancer stemcells. The purpose of this study is as follows:1.To cultivate breast cancer MCF-7cell strain through the use of suspendedmicrosphere culture technique and test the cells CD44and CD24expressions tovalidate the efficient enrichment of breast cancer stem cells and determine theirenrichment efficiency.2.To obtain breast cancer stem cells through the use of suspendedmicrosphere culture technique, and obtain the unclassified cells of breast cancerthrough the use of adherent culture technique. To contrastly test the expressiondifferences of Notch1, Wnt1, β-catenin and MMP-9, and analyze the migrationactivity changes of breast cancer stem cells and the correlation between thismigration activity and Notch1expression level.3.To targetedly silence Notch1gene with siRNA to block the signalpathway so as to observe the expressions of breast cancer stem cells Wnt1,matrix metalloproteinase-9(MMP-9) and β-catenin, analyze the impact ofNotch1molecular expression to breast cancer stem cells Wnt1, β-catenin andMMP-9, and investigate the action of silencing Notch1molecules to Wnt/β-catenin signal pathway and the role of MMP-9in breast cancer occurrenceand development process, in order to provide a reference for the pathogenesis ofsuch disease Method1.Taking the human breast cancer MCF-7cell strain as the research subjectto cultivate the cells by using adherent culture and suspended microsphereculture techniques. Use flow cytometry (FCM) to detect CD44+/CD24-/lowcellproportion and validate that suspended microsphere culture is capable ofenriching breast cancer stem cells efficiently.2.Extract breast cancer stem cells from breast cancer MCF-7cell strain anddivide them into3groups, i.e., the blank group, the negative fluorescent group(including negative interference molecules and mixture transfection of5uLliposomes li2000) and Notch1molecular interference group (including theinterference of targeted Notch1molecules to siRNA and mixture transfection of5uL liposomes li2000). All samples are cultivated in vitro and western-blot andRT-PCR methods are used to determine Notch1expressions of the blank groupand the healthy control group. After a24-hour period, the Wnt1, β-catenin andMMP-9expressions are determined in the blank group, the negative fluorescentgroup and Notch1molecular interference group through the above methods.Results1.Breast cancer stem cells were efficiently enriched through the use of invitro microsphere suspension culture technique and CD44+/CD24-/lowcells tookup87.0%of the total cell numbers. When adherent cells were put intosuspension culture medium, some cells occured apoptosis and the other cellsgradually proliferated and formed microspheres. These microspheres weresaccules with different size and irregular patterns, and saccule cells weredigested into monoplast suspension which might proliferate to form sacculesagain. If the suspended microsphere cells were put into adherent cell culturemedium, they might come into pseudopodia and adhere to the walls again.2.By compared with the common adherent cells, the transcription level ofNotch1and MMP-9m RNA of suspended microsphere cells improvedsignificantly (P <0.05), while the transcription level of Wnt1mRNA lowered (P<0.05), and no change was found in β-catenin.3.When silencing suspended cell Notch1gene, its Wnt1expressionimproved significantly (P <0.05), while no changes were found in β-catenin andMMP-9. Conclusion1.The suspended microsphere culture technique is capable of enrichingbreast cancer stem cells efficiently under in vitro environment, and theconcentration of the enriched breast cancer stem cells is about87.0%, whichmay construct experimental model for silencing breast cancer stem cells Notch1in vitro.2.Compared to those unclassified breast cancer cells, the Notch1andMMP-9transcription improved in enriched breast cancer stem cells bysuspended microsphere culture technique, while Wnt1transcription lowered andno change occurs in β-catenin.3.After silencing the breast cancer stem cell Notch1enriched by suspendedcells, the cell Wnt1indicates a significantly improved transcription, while nochanges occur in β-catenin and MMP-9.