| Objective:To investgate the role of IL-6/STAT3 inflammatory signalling pathway in regulating the expression of SATB1 gene and breast cancer stem cell self-renewal.Methods:(1)The MCF-7 cells were routinely cultured and passaged.Pre-experiments of transfection was done to verify that infection efficiency of the lentiviral vectors of STAT3 and RNAi lentiviral vectors of STAT3(provided by Shanghai Genechem Co.,Ltd.),and to screen out the most efficient STAT3 siRNA.(2)The lentiviral vectors of STAT3 and the RNAi lentiviral vectors of STAT3were transfected into MCF-7 cells,respectively,while a control group and two negative virus control groups were set up..(3)Establishing a model of the breast cancer stem cells by serum-free suspension culture in vitro,To detect the number and size of diameter of the microspheres with the same number cells of the control group(MCF-7),MCF-7/STAT3 siRNA group(26100),MCF-7/negative virus control groups(CON077 and CON235)and MCF-7/STAT3 overexpression group(14233)(The number and the size of the microspheres reflect the number of BCSCs and self-renewal capability of cells).(4)Collect the microspheres of five groups and re-digest them into single cell suspensions.To detect the changes of proliferation dynamic in the control group(MCF-7),MCF-7/STAT3 siRNA group(26100),MCF-7/negative virus control groups(CON077 and CON235)and MCF-7/STAT3 overexpression group(14233)cells by CCK-8.(5)The STAT3 and SATB1 at mRNA levels was determined by QRT-PCR assay.The expression of total STAT3,pSTAT3,SATB1 at protein levels was measured by Western blot assay.(6)The proportion of breast cancer stem cells with phenottype CD44~+CD24~-in the five groups cell were tested by flow cytometry.Results:(1)The MCF-7 cell line consisted of polygon-like and spindle-shaped cells into a piece of adherent growth.Pre-experiments of transfection was done to screen out that the most efficient of STAT3 siRNA group is 26100 group:the cells in 18940group,18942 group and 26100 group were expressed lower mRNA levels of SATB1,respectively reduced 28%,38%and 66%,But the 26100 group were significantly lower than that 18940 group and 18942 group(P<0.05,n=3).Using the RNAi lentiviral vectors of STAT3 26100 for iRNA group.(2)Microsphere assay results:the microspheres of five group cells enriched the BCSCs formed significantly after 7 days of serum-free suspension culture and add IL-6 induction.There are more and even greater microspheres in 14233 group,there are fewer and smaller microspheres in 26100 group.,while the microspheres of that in the MCF-7 group,CON077 group and CON235 group were at medium level.(3)The results of proliferation activity determined by CCK-8 showed that the proliferation activity in microspheres of 14233 group higher than control group(P<0.01),the proliferation activity in microspheres of 26100 group lower than control group(P<0.01),while the changes of that in control group?CON077 group and CON235 group were not obvious(P>0.05).(4)The results of several key components of IL-6/STAT3 signaling and SATB1gene determined by RT-PCR demonstrated that compare with the CON235 group the up-regulation mRNA expression of STAT3 and SATB1 in 14233 group(P<0.05);compare with the CON077 group the down-regulation mRNA expression of STAT3and SATB1(P<0.05)in 26100 group;while compare with the MCF-7 group the the changes of mRNA expression of STAT3 and SATB1 that in CON077 group and CON235 group were not obvious(P>0.05).The results of western blot analysis suggested that the 14233 group had higher levels of STAT3,pSTAT3 and SATB1compared to MCF-7 group,CON235 group,CON077 group and 26100 group;the CON235 group,CON077 group and MCF-7 group had higher levels of STAT3,pSTAT3 and SATB1 compared to 26100 group;the changes of protein expression of STAT3,pSTAT3and SATB1 that in MCF-7 group,CON077 group and CON235 group were not obvious(P>0.05).(5)The results of flow cytometry analysis suggested that the 14233 group of cells had higher levels of the proportion of BCSCs with phenotype CD44~+CD24~-compared to MCF-7 group,CON235 group,CON077 group and 26100 group;the CON235 group,CON077 group and MCF-7 group of cells had higher levels of the proportion of BCSCs with phenotype CD44~+CD24~-compared to 26100 group;the changes of the proportion of BCSCs with phenotype CD44~+CD24~-that in MCF-7group,CON077 group and CON235 group of cells were not obvious(P>0.05).Conclusion:(1)Activation the IL-6/STAT3 pathway can induce the mammary microspheres formed increased significantly and the proportion of BCSCs with phenotype CD44~+CD24~-increased significantly.(2)Activation the IL-6/STAT3 pathway can induce the expression of SATB1increased significantly.(3)Activation the IL-6/STAT3 inflammatory signaling pathway plays a postive role in BCSCs self-renewal and proliferation,the mechanism involving expression of SATB1. |
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