Special AT Rich Sequence Binding Protein1Regulates Hedgehog Signaling Pathways Of Breast Cancer And Impacts The Self-renewal Of Breast Cancer Stem Cells

Background:It was recognized publicly that the reason why breast cancer occurs relapse andmetastasis after treating is the resistance to treatment itself. The tumor stem cellstheory believes that the tumor is composed of a group of functional heterogeneitycells. Only a small part of cancer cells has the capacity of stem cells to self-renewaland differentiate into different tumor cells, which play a decisive role in maintainingmalignant proliferation of tumor, called cancer stem cells. The cancer stem cells areconsidered as the root of breast cancer occurring,diverting and relapsing aftertreatment. SATB1(special AT rich sequence binding protein1) is the one whichinvolved in chromatin remodeling, DNA methylation, histone acetylation, andregulates the expression of many genes. SATB1is a tissue-specific expression ofnuclear matrix binding protein, which can be combined with a very high affinity fornuclear matrix attachment region (MAR) ATC sequence and plays an important rolein maintaining the pluripotent nature of embryonic stem cells and other aspects, isregarded as a "gene organizer". Breast cancer stem cells is in a series of complexregulatory networks of signaling pathways, include the Hedgehog signaling pathwaywhich related closely with breast development, incidence of breast cancer andself-renewal of breast cancer stem cells. Gli-1、Gli-2and Ptch1, the key gene ofHedgehog signaling pathway is highly expressed in breast cancer stem cells, can leadto the number and diameter of breast cancer microspheres increase significantly whentheir function are activated abnormally.Objective：To explore the role of nuclear matrix protein SATB1in regulating Hedgehogsignaling pathways of breast cancer and breast cancer stem cell self-renewal.Methods:1、Establishing a model of MCF-7cancer stem cells by serum-free suspensionculture in vitro. 2、Designing the RNAi lentiviral vector of SATB1, RNAi the expression ofSATB1in MCF-7cells by retroviruses transfected to observe the change of breastcancer microspheres number and diameter.3、Establishing a model of MCF-7cancer stem cells by serum-free suspensionculture in vitro.To detect the number and size of diameter of the microspheres withthe same number cells of MCF-7,MCF-7/siControl and MCF-7/siSATB1to comparethe the self-renewal capability of three group cells.4、To detect the changes of proliferation dynamic in MCF-7,MCF-7/siControland MCF-7/siSATB1cells by CCK-8.5、Detecting and sterile sorting the proportion of breast cancer stem cells withphenotype CD44+CD24-in MCF-7,MCF-7/siControl and MCF-7/siSATB1cells byflow cytometry.6、Detecting the mRNA expression level of the key gene in Hedgehog signalingpathway of Gli-1,Gli-2and Ptch1in MCF-7,MCF-7/siControl and MCF-7/siSATB1cells by QRT-PCR.Results:1、Constructing and packaging the RNAi lentiviral vector of SATB1successfully, and in accordance with the different display of green fluorescence undera fluorescence microscope we know when MOI=20and add ENi.s the infectionefficiency is the best. Under MOI=20and add ENi.S instead of medium cultureconditions, which transfect MCF-7cells and screen out the best efficiency of RNAigroup for cell culture(QRT-PCR showes that the the mRNA inhibition rate is63%, P＜0.01;western-blot display the protein inhibition rate is47.78%，P＜0.01).2、Detecting the proliferation activity of MCF-7, MCF-7/siControl and MCF-7/siSATB1cells by CCK-8, the results show that the average OD value of the threegroup cells are1.749±0.104,1.699±0.086and1.154±0.075.Compare with the MCF-7cells, the absorbance value of MCF-7/siSATB1cells is decreased by40.26%(P＜0.01), however the absorbance value of MCF-7/siControl compared undifferentiatedto MCF-7cells.3、The microspheres enriched the MCF-7stem cells formed significantly inserum-free suspension culture of cancer stem cells in vitro model. MCF-7, MCF-7/siControl and MCF-7/siSATB1cells were observed under an invertedfluorescence microscope, There are127numbers of microspheres in MCF-7cells, therate of microspheres is12.7%and the average diameter of the microspheres is158.00±43.34um. There are78numbers of microspheres in MCF-7/siSATB1cells,the rate of microspheres is7.8%and the average diameter of the microspheres is90.99±44.23um. Comparing to the MCF-7cells, the rate of microspheres in MCF-7/siSATB1decrease38.58%and42.41%for the average diameter of themicrospheres.Both of their P value is＜0.014、Detecting the proportion of breast cancer stem cells with phenotype CD44+CD24-in MCF-7,MCF-7/siControl and MCF-7/siSATB1cells by flow cytometry. Theresults show that AS-ACI=3.127±0.235%of MCF-7cells,2.587±0.163%forMCF-7/siControl cells and1.203±0.425%for MCF-7/siSATB1cells.Comparing tothe MCF-7cells, the proportion of breast cancer stem cells is decreased by61.53%inMCF-7/siSATB1cells（P＜0.001）.5、Detecting the mRNA expression level of the key gene in Hedgehog signalingpathway of Gli-1,Gli-2and Ptch1in MCF-7,MCF-7/siControl and MCF-7/siSATB1cells by QRT-PCR. The results show that the average CT values of Gli-1in MCF-7,MCF-7/siControl and MCF-7/siSATB1cells are28.24±0.68,28.12±0.73and29.51±0.55,comparing to the MCF-7cells, the expression of Gli-1is decreased by47.25%in MCF-7/siSATB1cells (P＜0.01). The average CT values of Gli-2in MCF-7,MCF-7/siControl and MCF-7/siSATB1cells are27.91±0.46,26.96±0.63and28.25±0.55,comparing to the MCF-7cells, the expression of Gli-2is decreased by39.45%in MCF-7/siSATB1cells (P＜0.01).The average CT values of Ptch1in MCF-7,MCF-7/siControl and MCF-7/siSATB1cells are25.58±0.55,25.36±0.61and26.72±0.52, comparing to the MCF-7cells, the expression of Ptch1is decreased by43.17%in MCF-7/siSATB1cells (P＜0.01).Conclusion:The expression of SATB1in MCF-7cells is inhibited obviously by lentiviralvector transfection of RNAi,and the same result to the proliferation activity of MCF-7cells.With the depression of SATB1, the proportion of breast cancer stem cells withphenotype CD44+CD24-is decreased, which leads to diminish the forming capacity of microspheres enriched the MCF-7stem cells and reduce the self-renewal capacity ofbreast cancer stem cells. Lower expression of SATB1in MCF-7cell cause themRNA expression of Gli-1,Gli-2and Ptch1in Hedgehog pathway which related tobreast cancer stem cell self-renewal closely decreased. In one word, The expressionlevel of SATB1in MCF-7cells is closely related with the MCF-7stem cells, and itprobably become a new target for the treatment of breast cancer.