Upregulation of METTL14 mediates the elevation of PERP mRNA N6 adenosine methylation promoting the growth and metastasis of pancreatic cancer

Abstract

Background: Pancreatic cancer is one of the most lethal human cancers. N⁶-methyladenosine (m⁶A), a common eukaryotic mRNA modification, plays critical roles in both physiological and pathological processes. However, its role in pancreatic cancer remains elusive.

Methods: LC/MS was used to profile m⁶A levels in pancreatic cancer and normal tissues. Bioinformatics analysis, real-time PCR, immunohistochemistry, and western blotting were used to identify the role of m⁶A regulators in pancreatic cancer. The biological effects of methyltransferase-like 14 (METTL14), an mRNA methylase, were investigated using in vitro and in vivo models. MeRIP-Seq and RNA-Seq were used to assess the downstream targets of METTL14.

Results: We found that the m⁶A levels were elevated in approximately 70% of the pancreatic cancer samples. Furthermore, we demonstrated that METTL14 is the major enzyme that modulates m⁶A methylation (frequency and site of methylation). METTL14 overexpression markedly promoted pancreatic cancer cell proliferation and migration both in vitro and in vivo, via direct targeting of the downstream PERP mRNA (p53 effector related to PMP-22) in an m⁶A-dependent manner. Methylation of the target adenosine lead to increased PERP mRNA turnover, thus decreasing PERP (mRNA and protein) levels in pancreatic cancer cells.

Conclusions: Our data suggest that the upregulation of METTL14 leads to the decrease of PERP levels via m⁶A modification, promoting the growth and metastasis of pancreatic cancer; therefore METTL14 is a potential therapeutic target for its treatment.

Keywords: METTL14; N6-methyladenosine; PERP; Pancreatic cancer; m6A.

Fig. 1

m⁶A modification levels and profiles in pancreatic cancer. a, b HPLC quantification of m⁶A levels in mRNA extracted from pancreatic cancer cell lines and human pancreatic cancer tissues, as indicated. * p < 0.05. c Kaplan-Meier analysis of the correlation between the m⁶A levels and the overall survival of pancreatic cancer patients. * p < 0.05. d Quantification of m⁶A levels in 21 pancreatic cancer tissues with (LN Pos) or without (LN Neg) lymphatic metastasis. * p < 0.05

Fig. 2

Aberrant expression of METTL 3-METTL14 complex in pancreatic cancer. a Real-time PCR analysis of the relative mRNA levels of METTL3, METTL14, and WTAP in 20 paired pancreatic cancer tissues. * p < 0.05. b Quantification of western blot analyses of METTL3, METTL14, and WTAP levels in pancreatic cancer and adjacent tissues. * p < 0.05; ** p < 0.01. c Immunohistochemical analysis of METTL3/METTL14/WTAP expression in pancreatic cancer tissues; Kaplan-Meier analysis of the correlation between METTL3/METTL14/WTAP expression and the overall survival of pancreatic cancer patients; and relative METTL3/METTL14/WTAP expression scores in pancreatic cancer tissues per clinical and pathological stage. * p < 0.05; ***p < 0.001; n.s., no significance. d Colorimetric quantification of m⁶A in PANC-1 cells after METTL3 and METTL14 knockdown or overexpression. ** p < 0.01; ***p < 0.001

Fig. 3

Upregulation of METTL14 enhances the growth and metastasis of pancreatic cancer. a Viability of PANC-1 cells expressing shCtrl, shMETTL14, vector or exogenous METTL14 detected by the CCK8 assay. ***p < 0.001. b Representative images from the colony-forming assay (lower panel) and colony number analysis (upper panel). All experiments were performed in triplicate and data are presented as the mean ± SD. **p < 0.01; ***p < 0.001. c Images (left panel; scale bar: 1 cm) and weight analysis (right panel) of subcutaneous tumors from the indicated groups. * p < 0.05; ** p < 0.01. d Images of the orthotopic transplantation mouse model (shCtrl, shMETTL14, vector or METTL14 groups; lower panel), and analysis of the orthotopic tumor diameter (upper panel). * p < 0.05. PANC-1 cells expressing shCtrl, shMETTL14, vector or METTL14 were subjected to a transwell assay with or without Matrigel (Scale bar: 200 μm) (e), and to a wound-healing assay (Scale bar: 200 μm) (f). All experiments were performed in triplicate and data are presented as the mean ± SD. ** p < 0.01; ***p < 0.001. g Images of armpit lymph node metastasis in the subcutaneous implantation model (left panel) and the respective quantitative analysis (right panel). * p < 0.05. h Statistical analysis of the average number of liver metastases per group in the orthotopic transplantation mouse model. Scale bar: 1 mm. * p < 0.05; ** p < 0.01. i Statistical analysis of the average number of liver metastases per group in the liver metastasis model. ** p < 0.01; ***p < 0.001

Fig. 4

Identification of METTL14 targets via RNA-Seq and m⁶A-Seq. a Differentially expressed genes with over 2-fold expression changes in PANC-1 cells treated with shMETTL14 compared with those treated with shCtrl. b Top consensus motif identified from m⁶A-Seq peaks in PANC-1-shCtrl and PANC-1-shMETTL14 cells. c, d Number of m⁶A peaks and m⁶A-modified transcripts identified from m⁶A-Seq peaks in PANC-1-shCtrl and PANC-1-shMETTL14 cells. e Schematic diagram of METTL14 downstream analysis

Fig. 5

PERP is the key target of METTL14 in pancreatic cancer. a qPCR analysis of METTL14 and PERP in PANC-1 cells expressing shCtrl or shMETTL14. ***p < 0.001. b Western blotting of METTL14 and PERP in PANC-1 cells expressing shCtrl, shMETTL14, vector or METTL14. c MeRIP-qPCR analysis of fragmented PERP RNA from PANC-1 (control and METTL14 depleted) cells. ** p < 0.01. d qPCR analysis of PERP mRNA levels in PANC-1 cells (control and METTL14 depleted) after actinomycin D treatment. e PANC-1 cells were pre-transfected with wild-type or mutated pmiR-RB-Report-PERP-3′UTR plasmids, and then treated as indicated. Renilla luciferase activity was normalized to firefly luciferase activity and expressed as the mean ± SD. * p < 0.05; ** p < 0.01; ***p < 0.001; n.s., no significance. f Correlation between METTL14 and PERP protein levels in pancreatic cancer specimens. Left - representative IF images of pancreatic cancer specimens. Scale bar, 20 μm. Right - percentage of PERP-positive cells among METTL14-positive versus METTL14-negative cells in selected microscope fields of each tumor (compared by the t-test). * p < 0.05. g qPCR analysis of PERP in PANC-1 cells (control and YTHDF2 depleted). (H) qPCR analysis of PERP in PANC-1 cells (control and YTHDF2 depleted) in the absence or presence of METTL14 overexpression. * p < 0.05; ** p < 0.01. i qPCR analysis of PERP mRNA levels in PANC-1 cells (control and YTHDF2 depleted) in the absence or presence of METTL14 overexpression, and after actinomycin D treatment

Fig. 6

PERP is involved in the METTL14-induced Pancreatic Cancer Cells’ Growth and Invasion. a Viability of PANC-1 cells with or without PERP knockdown in the absence or presence of METTL14 knockdown analyzed by the CCK8 assay. ***p < 0.001. b Representative images from the colony-forming assay (lower panel) and colony number analysis (upper panel) as indicated. All experiments were performed in triplicate and data are presented as the mean ± SD. **, p < 0.01; ***, p < 0.001. c PANC-1 cells with or without PERP knockdown in the absence or presence of METTL14 knockdown were analyzed in a transwell assay with Matrigel. All experiments were performed in triplicate and data are presented as the mean ± SD. Scale bar: 200 μm. ** p < 0.01; ***p < 0.001. d Western blotting of PERP and Flag in PANC-1 cells with or without PERP WT transfection in the absence or presence of METTL14 overexpression (left panel); western blotting of PERP and Flag in PANC-1 cells with or without PERP 3′-UTR transfection in the absence or presence of METTL14 overexpression (right panel). e Colony-forming assay in PANC-1 cells with or without PERP WT transfection in the absence or presence of METTL14 overexpression (left panel); colony-forming assay in PANC-1 cells with or without PERP 3′-UTR transfection in the absence or presence of METTL14 overexpression (right panel). ** p < 0.01; ***p < 0.001; n.s., no significance. f Transwell assay in PANC-1 cells with or without PERP WT transfection in the absence or presence of METTL14 overexpression (upper panel); transwell assay in PANC-1 cells with or without PERP 3′-UTR transfection in the absence or presence of METTL14 overexpression (lower panel). Scale bar: 200 μm. ** p < 0.01; ***p < 0.001; n.s., no significance