Inhibiting roles of FOXA2 in liver cancer cell migration and invasion by transcriptionally suppressing microRNA-103a-3p and activating the GREM2/LATS2/YAP axis

Abstract

Forkhead box A2 (FOXA2) has emerged as a tumor inhibitor in several human malignancies. This work focused on the effect of FOXA2 on liver cancer (LC) cell invasion and migration and the involving molecules. FOXA2 expression in LC tissues and cell lines was determined. The potential target microRNA (miRNA) of FOXA2 was predicted via bioinformatic analysis and validated through a ChIP assay. The mRNA target of miRNA-103a-3p was predicted via bioinformatic analysis and confirmed via a luciferase assay. Altered expression of FOXA2, miR-103a-3p and GREM2 was introduced in cells to identify their roles in LC cell migration and invasion. Consequently, FOXA2 and GREM2 were poorly expressed while miR-103a-3p was highly expressed in LC samples. Overexpression of FOXA2 or GREM2 suppressed migration and invasion of LC cells, while up-regulation of miR-103a-3p led to inverse trends. FOXA2 transcriptionally suppressed miR-103a-3p to increase GREM2 expression. Silencing of GREM2 blocked the effects of FOXA2. GREM2 increased LATS2 activity and YAP phosphorylation and degradation. To conclude, this study demonstrated that FOXA2 suppressed miR-103a-3p transcription to induce GREM2 upregulation, which increased LATS2 activity and YAP phosphorylation to inhibit migration and invasion of LC cells.

Keywords: FOXA2; GREM2; LATS2; Liver cancer; MiR-103a-3p; YAP.

Fig. 1

FOXA2 is poorly expressed in LC and inhibits LC cell migration and invasion. a FOXA2 expression in LC tumor tissues and in the paired adjacent normal tissues determined by RT-qPCR (n = 50, paired t test, ***, p < 0.001); b FOXA2 expression in THLE-2, HepG2, SK-HEP-1 cells determined by RT-qPCR and western blot analysis (one-way ANOVA, ***, p < 0.001); c transfection efficacy of pcDNA-FOXA2 determined by RT-qPCR and western blot analysis (one-way ANOVA, ***, p < 0.001); d migration and invasion abilities of cells determined by Transwell assays (one-way ANOVA, **, p < 0.01); e transfection efficacy of siRNA-FOXA2 in THLE-2 cells examined by RT-qPCR and western blot analysis (unpaired t test, *, p < 0.05); f migration and invasion of THLE-2 cells after siRNA-FOXA2 transfection examined by Transwell assays (unpaired t test, *, p < 0.05). Data were presented as the mean ± SD from three independent experiments

Fig. 2

FOXA2 transcriptionally suppresses miR-103a-3p. a miR-103a-3p expression in LC tumor tissues and in the paired adjacent normal tissues determined by RT-qPCR (n = 50, paired t test, **, p < 0.01); b miR-103a-3p expression in THLE-2, HepG2, SK-HEP-1 cells determined by RT-qPCR (one-way ANOVA, *, p < 0.05); c miR-103a-3p expression in cells after miR-103a-3p mimic/inhibitor transfection determined by RT-qPCR; d migration and invasion abilities of cells after miR-103a-3p mimic/inhibitor transfection measured by Transwell assays; e putative binding sequence of miR-103a-3p and FOXA2 predicted on Jaspar (http://jaspar.genereg.net/); f enrichment of miR-103a-3p promoter sequence in the precipitation of FOXA2-DNA compound in the ChIP-qPCR assay (one-way ANOVA, **, p < 0.01); g correlation between miR-103a-3p expression and FOXA2 expression in 50 pairs of LC tissues and the adjacent tissues analyzed by Pearson’s Correlation Analysis (r = -0.632, * p < 0.01); h miR-103a-3p expression in cells detected after pcNDA-FOXA2 transfection (one-way ANOVA, * p < 0.05). Data were presented as the mean ± SD from three independent experiments

Fig. 3

miR-103a-3p directly targets GREM2. a the downstream target mRNAs of miR-103a-3p predicted on StarBase, TargetScan and miRDB; b GREM2 expression in LC predicted on GEPIA; c putative binding sites of miR-103a-3p and GREM2 predicted on Starbase; d GREAM2 expression in LC tumor tissues and in the paired adjacent normal tissues determined by RT-qPCR and immunohistochemical staining (n = 50, paired t test, **, p < 0.01); e mRNA and protein expression of GREM2 in THLE-2, HepG2, SK-HEP-1 cells determined by RT-qPCR and western blot analysis, respectively (one-way ANOVA, **, p < 0.01); f GREM2 expression in LC cell lines after pcDNA-GREM2 or siRNA-GREM2 transfection determined by RT-qPCR and western blot analysis, respectively (one-way ANOVA, **, p < 0.01); g number of migrated and invaded LC cells after pcDNA-GREM2 or siRNA-GREM2 transfection determined by Transwell assays (one-way ANOVA, *, p < 0.05); h correlation between GREM2 and miR-103a-3p expression in 50 pairs of LC tissues and the adjacent tissues analyzed by Pearson’s Correlation Analysis (r = -0.721, **, p < 0.01); i mRNA and protein expression of GREM2 in LC cell lines after miR-103a-3p transfection determined by RT-qPCR and western blot analysis (one-way ANOVA, *, p < 0.05); j binding relationship between miR-103a-3p and GREM2 validated through a dual luciferase reporter gene assay (two-way ANOVA, * p < 0.05); Data were presented as the mean ± SD from three independent experiments

Fig. 4

GREM2 activates LATS2 to promote YAP phosphorylation. a correlation between LATS2 and GREM2 expression in LC in TCGA database; b expression of LATS2 and YAP and phosphorylation of YAP in LC cells after pcDNA-GREM2/siRNA-GREM-2 transfection determined by western blot analysis (one-way ANOVA, *, p < 0.05); Data were presented as the mean ± SD from three independent experiments

Fig. 5

FOXA2 inhibits migration and invasion of LC cells through the miR-103a-3p/GREM2/LATS2 axis. a protein levels of LATS2 and YAP and phosphorylation of YAP in cells determined by western blot analysis (one-way ANOVA, *, p < 0.05); b migration and invasion abilities of cells transfected with pcDNA-FOXA2 and siRNA-GREM2 determined by Transwell assays (one-way ANOVA, *, p < 0.05). Data were presented as the mean ± SD from three independent experiments

Fig. 6

A diagram presentation of the molecular mechanism. In the nuclear of LC cells, FOXA2 binds to the promoter region of miR-103a-3p to repress miR-103a-3p transcription. This triggers the following GREM2 up-regulation and LATS2 activation, leading to further YAP phosphorylation and degradation in cytoplasm, resulting in a decline in cell migration and invasion