m6A RNA demethylase FTO promotes the growth, migration and invasion of pancreatic cancer cells through inhibiting TFPI-2

Abstract

Pancreatic cancer (PC) is one of the most fatal cancers with a very poor prognosis. Here, we found that N⁶-methyladenosine (m⁶A) RNA demethylase fat mass and obesity-related protein (FTO) promote the growth, migration and invasion of PC. FTO expression level is increased in human PC and is associated with poor prognosis of PC patients. Knockdown of FTO increases m⁶A methylation of TFPI-2 mRNA in PC cells, thereby increasing mRNA stability via the m⁶A reader YTHDF1, resulting in up-regulation of TFPI-2 expression, and inhibits PC proliferation, colony formation, sphere formation, migration and invasion in vitro, as well as tumour growth in vivo. Rescue assay further confirms that FTO facilitates cancer progression by reducing the expression of TFPI-2. Mechanistically, FTO promotes the progression of PC at least partially through reducing m⁶A/YTHDF1 mediated TFPI-2 mRNA stability. Our findings reveal that FTO, as an m⁶A demethylase, plays a critical role in promoting PC growth, migration and invasion, suggesting that FTO may be a potential therapeutic target for treating PC.

Keywords: FTO; TFPI-2; m6A; pancreatic cancer; progression.

Figure 1.

Expression of m⁶A demethylase FTO is significantly upregulated in pancreatic cancer (PC). (a) FTO expression in PC tissues and normal tissues based on GEPIA database. (b) Meta-analysis of FTO expression based on Oncomine database in PC. (c) qRT-PCR and (d) western blot analysis of FTO mRNA and protein levels in PC tissues and adjacent normal pancreatic tissues. (e) IHC analysis of FTO protein expression in PC specimens and adjacent normal pancreatic tissue specimens. (f) Kaplan-Meier analysis indicated that higher FTO expression was remarkably correlated with shorter overall survival of PC patients. *P < 0.05, ***P < 0.001.

Figure 2.

FTO significantly facilitated the proliferation, migration and invasion of pancreatic cancer (PC) cells in vitro. (a) qRT-PCR and (b) western blot were used to determine the knockdown efficiency of FTO in PANC-1 and SW1990 cells. (c) Dot blot was used to determine the effect of FTO knockdown on the m⁶A modification level of total mRNA in PANC-1 and SW1990 cells. (d) CCK-8 assay revealed that FTO knockdown significantly inhibited the proliferation of pancreatic cancer cells. (e) Wound healing experiment indicated that silenced FTO significantly inhibited migration of PC cells. (f) Migration and (g) invasion assays revealed that silenced FTO significantly reduced the migration and invasion ability of PC cells. **P < 0.01, ***P < 0.001.

Figure 3.

Silencing FTO significantly inhibited pancreatic cancer (PC) growth in vitro and in vivo. (a, b) Knockdown efficiency of FTO in PANC-1 and SW1990 cells were detected by (a) qRT-PCR and (b) western blot. (c, d) Knockdown of FTO repressed the ability of PC cell (c) colony and (d) sphere formation. (e-g) Knockdown of FTO significantly inhibited subcutaneous graft (e) tumour growth in nude mice, and reduced (f) tumour volume and (g) weight. **P < 0.01, ***P < 0.001.

Figure 4.

TFPI-2 was identified as the downstream target gene of m⁶A demethylase FTO. (a) m⁶A modify sites of TFPI-2 mRNA were detected by SRAMP database. (b) The binding probability of FTO protein to TFPI-2 mRNA predicted by RPISeq. (c) The interaction between FTO protein and TFPI-2 mRNA predicted by PRIdictor. (d) RMBase v2.0 database showed that FTO regulates TFPI-2 mRNA m⁶A motif that was mainly GGACU located at the junction of mRNA CDS and 3’-UTR. (e, f) TFPI-2 mRNA and protein expression levels in silenced FTO PANC-1 and SW1990 cells detected by (e) qRT-PCR and (f) western blot. (g) MeRIP analysis showed that FTO knockdown significantly increased m⁶A levels of TFPI-2 mRNA. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

TFPI-2 and FTO expression were negatively correlated in pancreatic cancer (PC). (a) Protein expression levels and IHC scores of TFPI-2 and FTO by IHC analysis in human PC specimens. (b) Pearson’s correlation analysis of IHC scores of clinical PC specimens. (c) IHC analysis of TFPI-2 and FTO protein expression levels in subcutaneous graft tumour with PANC-1.

Figure 6.

FTO regulated the expression of target gene TFPI-2 by inhibiting the stability of TFPI-2 mRNA mediated through m⁶A/YTHDF1. (a) PRIdictor database predicted the reader proteins of TFPI-2 mRNA m⁶A modification. (b) Knockdown efficiency of siYTHDF1 was evaluated by qRT-PCR analysis. (c) The effect of YTHDF1 on TFPI-2 mRNA stability was detected by RNA stability assay. **P < 0.01, ***P < 0.001.

Figure 7.

TFPI-2 knockdown significantly attenuated the inhibitory effect of silenced FTO on the malignant phenotype of pancreatic cancer (PC). (a) Knockdown efficiency of siTFPI-2 was evaluated qRT-PCR. (b) CCK-8 assay indicated that knockdown of TFPI-2 effectively alleviated the repressive effect of silenced FTO on cell proliferation. (c and d) knockdown of TFPI-2 effectively alleviated the repressive effect of silenced FTO on cell (c) colony and (d) sphere formation. (e-g) Knockdown of TFPI-2 effectively alleviated the repressive effect of silenced FTO on cell migration and invasion determined by (e)wound healing assay, (f) migration assay, and (g) invasion assay. **P < 0.01, ***P < 0.001, ns, no significance.

Figure 8.

FTO promotes the progression of pancreatic cancer by inhibiting the expression of TFPI-2, partially through decreasing the stability of TFPI-2 mRNA mediated via m⁶A /YTHDF1.