| Objective To investigate the effect of silencing CTNNB1 to inhibit Wnt/β-catenin Pathway on the proliferation,migration,invasion and self-renewal ability of thyroid cancer stem cells and its related mechanism.Methods A magnetic bead sorting technique was used to screen and isolate CD133-positive cell subsets in anaplastic thyroid carcinoma(ATC)ARO cell line.Western-blot method,CCK-8 method and tumor cell sphere formation experiments were adopted to identify the characteristics of Cancer stem cell.The subgroups of CD133-positive ARO cells with good growth status were randomly divided into blank control group,negative control group and experimental group.The cells in the blank group were not subjected to any treatment except conventional culture.The negative control group was transfected with the no-load lentivirus carrying only the fluorescently labeled GFP gene.The experimental group was transfected with the recombinant lentivirus carrying the sh RNA and GFP targeted to silence the target gene.After 72 h,the transfection efficiency of Experimental cells was observed under an inverted fluorescence microscope.Cells with transfection efficiency greater than 80% were selected for further experiments.Western-blot method was used to detect the expression of target protein β-catenin and epithelial-mesenchymal transition marker proteins E-cadherin and Vimentin in three groups of cells.CCK-8 method was used to detect changes in cell proliferation activity after transfection.Tumor cell spheroid formation experiments was used to detect changes in cell self-renewal capabilities after transfection.Transwell experiments was used to detect cell proliferation and migration capabilities after transfection.Results The relative expression level of Oct-4 protein(0.98±0.01)in CD133-positive subgroup of ARO cells was significantly higher than that of CD133-negative subgroup(0.59±0.01).The difference is statistically significant(t=81.04,P<0.01).CD133-positive cells grow significantly faster than CD133-negative cells when both of them were cultured for 4～6 days(F=177.18,P<0.01).Which proves that CD133-positive cells have stronger proliferation capacity.After continuous culture for 1 week under serum-free conditions,CD133-positive cells were able to form tumor cell spheres in suspension,while CD133-negative cells were not.Which proves that CD133-positive cells are more capable of selfrenewal.After 72 hours of lentivirus transfection,the cells were observed under an inverted fluorescence microscope.When the multiplicity of infection(MOI)was about 50,the transfection efficiency was above 80%.Western-blot results showed that the relative expression of β-catenin protein in the experimental group was(0.85±0.02),which was significantly lower than that in the blank group(1.64±0.04)and the negative control group(1.57±0.05).The difference is statistically significant(F=408.45,P<0.01).There was no significant difference between the blank group and the negative control group(P>0.05).Which proves that a cell model that stably inhibits CTNNB1 gene expression was successfully constructed.CCK-8 results showed that the cell proliferation ability of the experimental groups cultured 2d and 3d were lower than that of the blank group and the negative control group.The difference was statistically significant(all P<0.01).However,there was no significant difference in OD between the blank group and the negative control group(P>0.05).Tumor cell spheroid formation experiment results show that the experimental group has a downward trend in terms of cell spheroid formation rate and cell sphere volume compared with the blank group.The difference was statistically significant(P<0.05).The results of the Transwell experiment showed that the number of transmembrane cells in the experimental group was(94.80±5.11)/field in the migration experiment,which was significantly less than that in the blank group(308.80±12.13)/ field.The difference is significant(t=-36.339,P<0.01).In the invasion experiment,the number of cells passing through the membrane in the experimental group was(85.00±6.70)/ field,which was significantly less than the blank group(267.00±9.08)/field.The difference was significant(t=-36.041,P<0.01).Western-blot detection of the expression of epithelial phenotype molecule E-cadherin and interstitial phenotype molecule Vimentin indicated that the relative expression of interstitial phenotype molecule Vimentin protein in the experimental group was(0.85±0.05),which was significantly reduced(P<0.01);compared with the blank group(1.28±0.04)and the negative control group(1.27±0.06).The relative expression of E-cadherin protein in the experimental group was(1.09±0.08),which was significantly higher than that in the blank group(0.72±0.03)and the negative control group(0.72±0.02).Conclusion Inhibition of Wnt/β-catenin signaling pathway can reduce the proliferation,migration,invasion and self-renewal ability of thyroid cancer stem cells.Figure 10;Table 4;Reference 166... |
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