Title Page
Contents
Abstract 6
Introduction 8
2. Results 11
2.1. Screening of a natural compound library identified an enantiomer of betamethasone 21-phosphate and dexamethasone 21-phosphate as a potent inducer of TTP in cancer cells. 11
2.2. Betamethasone 21-phosphate, dexamethasone 21-phosphate, and dexamethasone induce the expression of TTP in MDA-MB-231 cells. 11
2.3. RNA-seq transcriptome analysis of betamethasone 21-phosphate and dexamethasone-treated MDA-MB-231 cells 13
2.4. FOXO1 mediates dexamethasone- and betamethasone 21-phosphate-induced TTP expression in MDA-MB-231 cells. 14
2.5. Dexamethasone enhances the binding of FOXO1 to the TTP promoter in a GR-dependent manner. 15
2.6. Dexamethasone-induced TTP down-regulates ARE-containing genes in cancer cells and mediates the anti-proliferative effect of dexamethasone. 17
3. Discussion 18
4. Materials and Methods 21
4.1. Cells and chemicals 21
4.2. Cell viability/proliferation 21
4.3. Plasmids, small interfering RNAs, transfections, and dual-luciferase assay 21
4.4. Screening of the natural product library and luciferase assay 22
4.5. Quantitative real-time PCR and semi-qRT-PCR 22
4.6. SDS-PAGE and immunoblotting 23
4.7. Chromatin immunoprecipitation (ChIP) assay 23
4.8. RNA preparation and RNA-seq 24
4.10. Statistical analysis 25
Conflict of interest 25
Author contributions 25
References 25
Table 1. PCR primers used in this study 30
Figure 1. Dose-dependent induction of TTP in MDA-MB-231 cells by three compounds selected from the 4th screening of the natural compound library. (A) Dose-dependent changes...[이미지참조] 31
Figure 2. Induction of TTP expression by BTM-21-P, DXM-21-P, and DXM in breast cancer cells in a glucocorticoid receptor (GR)-dependent manner. (A and B) Dose-dependent changes... 32
Figure 3. RNA-Seq data analysis and KEGG pathways. (A and B) Volcano plots for the differentially expressed genes (DEGs) in BTM-21-P-treated and DXM-21-P-treated cells... 34
Figure 4. FOXO1 mediates DXM-induced TTP expression in MDA-MB-231 cells. (A-F) MDA-MB-231 cells were incubated with the indicated concentrations of inhibitors against (A)... 35
Figure 5. DXM induces TTP expression without induction of FOXO1 expression. (A) Validation of the DEGs in the FOXO signaling pathway in KEGG analysis by qRT-PCR... 37
Figure 6. DXM enhances FOXO1 binding to the promoter of the TTP gene. (A) The JASPAR prediction (p value=0.001) for the binding positions of FOXO1 on the promoter of the TTP... 39
Figure 7. DXM-induced TTP mediates the anti-proliferative function in MDA-MB-231 cells. (A-C) DXM treatment decreases expression of ARE-containing genes in MDA-MB-231 cells.... 40
Supplement Figure S1. FOXO1 mediates BTM-21-P-induced TTP expression in MDA-MB- 231 cells. MDA-MB-231 cells were incubated with the indicated concentrations of inhibitors... 36
Supplementary Figure S2. The JASPAR prediction (p value=0.001) for the binding positions of FOXO1, FOXO3, FOXO4, FOXO6, and GR (NR3C1) on the promoter of the TTP gene.... 38