Fig. 4
ApoA1 downregulates KRT14 transcription by enhancing the expression of the transcription factor FOXO3a. (a) Volcano plots comparing transcription factors between 4T1ApoA1 and 4T1WT cells. (b) 4T1ApoA1 cells were transfected with the indicated siRNAs for 48 h. mRNA levels of KRT14 were determined by qPCR. (c) mRNA levels of FOXO3a in 4T1ApoA1 and 4T1WT cells determined by qPCR assay. (d) Protein levels of KRT14 determined by western blot. (e) Migration of 4T1 cells transfected with FOXO3a siRNA or control siRNA, determined by wound healing assays. (f) Luciferase reporter gene plasmids controlled by truncated promoters of KRT14 were generated. 4T1WT and 4T1ApoA1 cells were transfected with these plasmids, and luciferase activity was measured 48 h later. The lengths of the upstream region of the KRT14 transcription initiation site are indicated (-1~-2000, -1~-1500, -1~-800, and − 1~-500). (g) Luciferase reporter gene plasmids controlled by mutant promoters of KRT14 were generated, and luciferase activity was measured 48 h later. The wide-type promoter of KRT14 is represented by -1~-2000 WT, while the mutation sites are indicated as -879 ~ -872, -1716 ~ -1709, -1950 ~ -1943, and − 1960 ~ -1953. (h) CHIP-seq was performed in 4T1ApoA1 lysates using an anti-FOXO3a antibody. IGV analysis of CHIP-seq coverage is shown, indicating the promoter region of KRT14 with a red arrow. (i) Representative image of immunohistochemical staining of KRT14 and FOXO3a in surgical human breast cancer specimens. (j) Correlation analysis of FOXO3a expression with KRT14 expression. k) Correlation analysis of FOXO3a expression with serum ApoA1 levels in patients with breast cancer. l) Correlation analysis of FOXO3a expression with serum HDL levels in patients with breast cancer. *p < 0.05; **p < 0.01