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. 2021 Oct 19;11:738144. doi: 10.3389/fonc.2021.738144

Figure 2.

Figure 2

Autophagy positively regulates miR-449a expression at the transcriptional level through FoxO1 binding at region 1 of the promoter in CRC cells. (A) SW480 and SW620 cells were treated with amiodarone (10 μM) for 48 h. Anti-LC3 antibody and Hoechst stain were used to label the treated cells for LC3 puncta and nuclei, respectively. Cells were investigated under a confocal microscope. Quantification of LC3 puncta was conducted by randomly counting 30 cells. The data represent means ± SEM. (B) The expression of miR-449a was determined by real-time RT–PCR under amiodarone (10 μM) treatment for 48h in colorectal (SW480 and SW620), prostate (PC3), liver (Huh7 and Hep3B), breast (MCF-7), lung (A549), and gastric (AGS) cells. U54 was used as an endogenous control to normalize miR-449a expression. (C) SW480 cells were treated with amiodarone (10 μM) for 48 h or treated with the inhibitor 3MA (10 mM) for 24 h followed by immunoblotting to determine LC3 protein level. The levels of pri-miR-449a and miR-449a in SW480 cells were measured by real-time PCR. (D) Schematic diagram shows the miR-449a reporter plasmids constructed. The cells were transiently transfected with the constructed plasmid pGL3-basic-500, 1000 or 2000 bp. The relative luciferase activities were measured after normalization with renilla luciferase activity with or without amiodarone (10 μM) treatment for 48 h. (E) Localization of FoxO1 and nucleus was shown using anti-FoxO1 antibody and Hoechst staining, respectively. The cells were investigated under a multi-photon confocal microscope and quantified by randomly counting 50 cells. (F) SW480 cells were transiently transfected with FoxO1 plasmid (4 μg, pcDNA3/FLAG-FoxO1) by Lipofectamine™ 2000 for 48 h. The expression of FoxO1 was measured using anti-FoxO1 antibody by immunoblotting. The activity of miR-449 reporter plasmid was measured by luciferase assay. (G) The expression of miR-449a was measured after the cells were transiently transfected with FoxO1 plasmid DNA (8 μg) for 48 h by real-time PCR. (H) The cells were treated with amiodarone (10 μM) in the presence or absence of FoxO1 inhibitor AS1842856 (40 nM) for 48 h. The expression of miR-449a was determined by real-time PCR. (I) SW480 cells after amiodarone (10 μM) treatment for 24 h were fixed with formaldehyde followed by immunoprecipitation with anti-FoxO1 antibody. The DNA in the immunoprecipitate was amplified using the primer sets encompassing region 1 and 2 of miR-449a promoter region. Student’s t-test analysis was conducted and p-value ≤ 0.05 represents significance.