What this is
- Pancreatic cancer has a high mortality rate and is resistant to chemotherapy.
- This research investigates the role of the demethylase ALKBH5 in pancreatic cancer.
- ALKBH5's downregulation is linked to poor outcomes and increased cancer cell proliferation.
- The study reveals that ALKBH5 inhibits tumor growth by regulating WIF-1 and .
Essence
- ALKBH5 overexpression sensitizes pancreatic ductal adenocarcinoma (PDAC) cells to chemotherapy and inhibits tumorigenesis. This occurs through decreased levels of WIF-1, which suppresses .
Key takeaways
- ALKBH5 levels are downregulated in gemcitabine-treated pancreatic cancer models. This downregulation correlates with increased cell proliferation and poor clinical outcomes.
- Silencing ALKBH5 enhances PDAC cell proliferation, migration, and invasion in vitro and in vivo, while its overexpression has the opposite effects.
- ALKBH5 regulates WIF-1 expression, which is crucial for inhibiting , thereby affecting tumor growth and metastasis in PDAC.
Caveats
- The study primarily focuses on ALKBH5's role without fully elucidating the underlying mechanisms of its effects on chemotherapy resistance.
- Further research is needed to clarify the interactions between ALKBH5, WIF-1, and in the context of pancreatic cancer.
Definitions
- mA (N-Methyladenosine): A common RNA modification that affects gene expression by regulating mRNA stability and translation.
- Wnt signaling: A complex network of proteins crucial for cell communication, influencing cell proliferation and differentiation, often implicated in cancer.
AI simplified
Background
Pancreatic cancer is one of the few malignancies with the mortality approaching the incidence, ranking the six and seventh leading cause of cancer death in China and worldwide, respectively [1–3]. It caused an estimation of 458,918 new cases and an associated 432,242 deaths globally in 2018 [4]. Despite improvements in surgical techniques and medical therapy, pancreatic cancer still has extremely poor diagnosis and prognosis, with a median survival of 5 to 8 months (less than 10% survival rate) [5, 6]. As the etiology for this highly lethal disease has yet to be well characterized, it is urgent to explore genetic or epigenetic factors that contribute to the initiation and development of this cancer so as to identify novel therapeutic targets or biomarkers.
N6 -Methyladenosine (m6A), the most abundant posttranscriptional methylation of mRNA in eukaryotes, occurs in approximately 25% of transcripts at the genome-wide level [7]. m6A-dependent mRNA modification regulates RNA splicing, translocation, stability, and translation into protein, which is vital in mammals and affects various biological processes including self-renewal and differentiation, tissue development, DNA damage response, primary microRNA processing, and RNA–protein interactions [7, 8]. This modification is reversible and relies on the RNA methyltransferases (writers), the demethylases (erasers), and m6A-binding proteins (readers), which are frequently upregulated in a variety of human cancers and might play a crucial role in the process of carcinogenesis [7–10].
As a demethylase, alkylation repair homolog protein 5 (ALKBH5) is implicated in mediating methylation reversal. Overexpression of ALKBH5 has been reported in multiple cancers, such as breast cancer, glioblastoma, ovarian cancer, and gastric cancer [9, 11–13]. As regards to pancreatic cancer, ALKBH5 expression was reported to be positively associated with overall survival in the TCGA and ICGC cohorts [14]. However, ALKBH5 was also found to be downregulated in pancreatic cancer tissues and inhibit pancreatic cancer motility by demethylating long non-coding RNA KCNK15-AS1 [15]. Accordingly, the exact role of ALKBH5 in tumorigenesis of pancreatic cancer deserves further investigation.
Aberrant mutational activation of the Wnt signaling pathway is pivotal during development and progression of pancreatic cancers, especially its most common type, pancreatic ductal adenocarcinoma (PDAC). Direct or indirect epigenetic alterations of the Wnt signaling pathway lead to increased cell proliferation and resistance of the tumor cells to chemotherapy, implying its potential value as a therapeutic target in pancreatic cancers [16–18].
In this study, we assessed m6A’s clinicopathological relevance to PDAC and explored the underlying mechanisms. The m6A eraser ALKBH5 was found to be downregulated in gemcitabine-treated patient-derived xenograft (PDX) model and its overexpression sensitized PDAC cells to chemotherapy. Additionally, ALKBH5 deficiency boosts PDAC cell proliferation, migration, and invasion both in vitro and in vivo, which is dependent on m6A modification of Wnt inhibitory factor 1 (WIF-1) and activation of Wnt signaling.
Materials and methods
Chemicals and antibodies
Lipofectamine 2000 transfection and TRIZOL LS reagents were bought from Invitrogen (Grand Island, NY, USA). The DAB substrate kit has been purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Abcam (Cambridge, MA, USA) provided antibodies towards ALKBH5, FTO, WTAP, VIRMA, RBM15, C-myc, Cyclin D1, MMP-2, MMP-9, E-cadherin, Fibronectin, Vimentin. METTL3, METTL14, WIF-1, and β-actin antibodies were got from Cell Signaling Technology (Danvers, MA, USA). Anti-α-catenin antibody was made by BD (Franklin Lakes, NJ, USA). Unless in any other case noted, all other used chemicals were from Sigma (St. Louis, MO, USA).
Cell culture
Human PDAC cell lines AsPC-1, PANC-1, BXPC-3, HPDE6-C7, Capan-1, CFPAC-1, Capan-2, and MIA Paca-2 were purchased from the ATCC (American Type Culture Collection). Cells were cultured in DMEM (Gibco, Grand Island, New York, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin and 1% streptomycin, and incubated in an incubator with 5% CO2 at 37 °C.
mA-seq 6
m6A-IP and library preparation were performed according to the reported protocol (Dominissini et al., 2012). Briefly, poly-A-purified RNA was fragmented and incubated with m6A primary antibody for 2 h at 4 °C. The mixture was then immunoprecipitated by incubation with Protein A beads (Thermo Fisher) for 2 h at 4 °C. Captured RNA was washed for 3 times, eluted with m6A nucleotide solution and purified by RNAClean and Concentrator kit (Zymo). Sequencing was carried out on Illumina HiSeq 2000 according to the manufacturer’s instructions.
MeRIP-qPCR
Intact poly-A-purified RNA was denatured to 70 C for 10 min, transferred immediately on ice and then incubated with m 6 A antibody in 1 ml buffer containing RNasin Plus RNase inhibitor 400 U (Promega), 50 mM Tris-HCl, 750 mM NaCl and 0.5% (vol/vol) Igepal CA-630 (Sigma Aldrich) for 2 h at 4 °C. Dynabeads Protein G (Invitrogen) were washed, added to the mixture and incubated for 2 h at 4 °C with rotation. m6A RNA was eluted twice with 6.7 mM N 6 -methyladenosine 5′-monophosphate sodium salt at 4 °C for 1 h and precipitated with 5 mg glycogen, one-tenth volumes of 3 M sodium acetate in 2.5 volumes of 100% ethanol at − 80 °C overnight. m6A enrichment was determined by qPCR analysis. Fragmented mRNA was directly incubated with m6A antibody containing buffer and treated similarly. The primer sequences used for qRT-PCR and MeRIP-PCR are in the Additional file 13: Table S2.
Immunohistochemistry
The tissues were fixed in 4% (wt/vol) paraformaldehyde in PBS overnight at 4 °C and embedded in paraffin wax, Paraffin-embedded tissues cut into 5 μm sections, after deparaffinization and rehydration, 0.01 M citrate (pH 6.0) was used for heat-induced antigen recovery. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 15 min at RT in the dark. Unspecific binding was blocked with 5% serum for 1 h at room temperature. Slides were incubated overnight at 4 °C in primary antibodies diluted with blocking solution. Slides were incubated at room temperature for 1 h with secondary antibodies added with avidin-biotin peroxidase complex, including biotinylated anti-rabbit antibodies in goats and anti-mouse antibodies in goats. Stained with DAB reagent and counterstained with hematoxylin. Finally, observation and statistical analysis were performed. Fluorescent images were captured and analyzed using Nikon Eclipse 90i microscope.
Statistical analysis
The experiments were conducted in triplicates, and the data are presented as mean ± SD. Comparisons between groups were performed by Student’s two-tailed t test. The overall survival rate curves of PDAC patients based on Kaplan-Meier method were plotted using the log-rank test. P values <0.05 were considered statistically significant.
The detailed methodology can be found in the Supporting Materials and Methods.
Results
ALKBH5 expression is downregulated in gemcitabine-treated PDX pancreatic cancer
ALKBH5 expression is downregulated in gemcitabine-treated PDX pancreatic cancer. ALKBH5 expression is downregulated in gemcitabine (Gem)-treated-patient-derived xenograft (PDX).Schematic representation of the gemcitabine (Gem)-treated-patient-derived xenograft (PDX) approach.Immunoblotting to measure the expression of N-methyladenosine (mA) demethylases (ALKBH5 and FTO) and methyltransferase complex composed of METTL3, METTL14, WTAP, VIRMA and RBM15, in cells isolated from PDX mice of 2 passages treated with saline (control) or gemcitabine.Quantification of ().Representative images of immunohistochemistry staining for ALKBH5 in tumor tissue from PDX treated with control or gemcitabine. H&E, hematoxylin and eosin.and () Gene expression of ALKBH5 in human PDAC compared to normal tissues from 2 GEO data sets.ALKBH5 expression in PDAC tissues compared with paired adjacent tissues in 57 patients () Kaplan-Meier analysis indicating overall survival of PDAC patients with high (red) (= 94) or low (black) (= 83) ALKBH5 expression. Scale bar = 200 μm in. The data are shown as the means ± S.D. *< 0.05; **<0.01 a b c b d e f g h d 6 6 n n P P
Overexpression of ALKBH5 results in sensitization of cancer cells to chemotherapy
Overexpression of ALKBH5 results in sensitization of cancer cells to chemotherapy.Colony formation of MIA Paca-2 cells transfected with control (upper panel) or ALKBH5 (bottom panel) vector with gemcitabine treatment of different doses (0, 10, 20 nM).Dose response curves of gemcitabine in BxPC-3 cells expressing the indicated vectors.Correlation of ALKBH5 expression and ICof gemcitabine.Quantification of Colony formation.Immunoblotting to measure ALKBH5 protein levels in AsPC-1 cells transfected with shCtr and/or shALKBH5 and/or ALKBH5 vectors. WT, wildtype; KD, ALKBH5 knockdown; OV, ALKBH5 overexpression.Quantification of Colony formation assay with cells described in ().Kaplan-Meier analysis indicating overall survival of xenograft mice injected with gemcitabine-treated AsPC-1 cells bearing ALKBH5 injected with gemcitabine-treated AsPC-1 cells bearing ALKBH5 shRNA (red) or control (green).Tumor growth rate of xenograft mice described above.Representative images of tumor volumes from xenograft mice described above. Representative images () and its quantification () of immunohistochemistry (IHC) staining for Ki67 in tumor sections from xenograft mice described above. Scale bar = 200 μm in. The data are shown as the means ± S.D. **<0.01 a b c d e f e g h i j k j 50 P
Knockdown of ALKBH5 significantly increased PDAC cell proliferation, colony formation, and migration
Knockdown of ALKBH5 significantly increased PDAC cell proliferation, colony formation, and migration.Immunoblotting (upper panel) and qRT-PCR (bottom panel) to measure ALKBH5 expression in AsPC-1 (left) and PANC-1 (right) cells transfected with shCtr and/or sh ALKBH5.MTT, () Colony formation, () Quantification of (), and () Migration (upper) and invasion (lower panel) assay of cells described in ().Representative ventral view images and its quantification () of bioluminescence from xenograft mice implanted with AsPC-1 cells described in ().Representative images (left) and its quantification (right) of immunohistochemistry (IHC) staining for Ki67 in tumor sections from xenograft mice described above.Representative ventral view images and its quantification () of bioluminescence for liver metastasis from xenograft mice described above.Table summarizing the result of liver metastasis. Scale bars, 50 μm () and 100 μm (). The data are shown as the means ± S.D. **<0.01 a b c d c e a f g a h i j k e h P
Overexpression of ALKBH5 inhibited PDAC cells proliferation, colony formation, cell migration and tumor growth in a nude mice model
Overexpression of ALKBH5 inhibited PDAC cells proliferation, colony formation, cell migration and tumor growth in a nude mice model.Immunoblotting (left panel) and qRT-PCR (right panel) to measure ALKBH5 expression in BxPC-3 (upper) and MIA Paca-2 (bottom) cells transfected with control and/or ALKBH5 vector.MTT, () Colony formation and its quantification, and () Migration (upper) and invasion (lower panel) assay of cells described in (A). (E) Representative ventral view images and its quantification () of bioluminescence from xenograft mice implanted with BxPC-3 cells described in ().Representative images (upper) and its quantification (bottom) of IHC staining for Ki67 in tumor sections from xenograft mice described above.Representative ventral view images and its quantification of bioluminescence for liver metastasis from xenograft mice described above. Scale bars, 50 μm () and 100 μm (). The data are shown as the means ± S.D. **<0.01 a b c d f a g h d g P
WIF-1 as a downstream target of ALKBH5-mediated m6A modification
WIF-1 as a downstream target of ALKBH5-mediated m6A modification.Megagene profiles depicting global changes in mA binding surrounding 5′ and 3′ end UTRs in BxPC-3 and MIA Paca-2 cells transfected with control and/or ALKBH5 vector. Right panel is a list of top 10 genes that most significantly changed in mA modification in either 5′ or 3′ end UTRs.Tracks of mA sequence of WIF-1 mRNA in cells described in (). () Venn diagram showing the overlap of genes differentially expressed in AsPC-1 and PANC-1 cells transfected with shALKBH5.Expression of WIF-1 mRNA in BxPC-3 cells transfected with control and/or ALKBH5 vector determined by qRT-PCR. Enrichment of a Wnt signaling gene expression signature in transformed () AsPC-1 () MIA Paca-2 cells described above a b a c d e f 6 6 6
WIF-1 as a downstream target of ALKBH5-mediated m6A modification.qRT-PCR to measure WIF-1 expression in AsPC-1 (left) and PANC-1 (middle) cells transfected with shCtr and/or sh ALKBH5, and MIA Paca-2 (right) cells transfected with control and/or ALKBH5 vector.Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of mA levels of WIF-1 pre-mRNA in transformed cells described above.Luciferase reporter assays showing the impact of ALKBH5 overexpression on WIF-1 promoters in BxPC-3 cells.qRT-PCR to measure WIF-1 expression in transformed AsPC-1 and PANC-1 cells described above with or without 3-deazaadenosine (DAA), the global methylation inhibitor. The data are shown as the means ± S.D. **<0.01; ## <0.01 a b c d 6 P
ALKBH5 suppresses C-MYC, Cyclin D1, MMP-2, and MMP-9 expression by inhibiting Wnt signaling
ALKBH5 suppresses C-MYC, Cyclin D1, MMP-2, and MMP-9 expression by inhibiting Wnt signaling.-qRT-PCR to measure C-MYC, cyclin D1, MMP-2, and MMP-9 mRNA levels in transformed AsPC-1, PANC-1, and BxPC-3 cells described above.Luciferase reporter assays in BxPC-3 and MIA Paca-2 cells showing the impact of ALKBH5 overexpression on the promoter activities of C-MYC in the absence or presence of LiCl, a Wnt signaling activator.,qRT-PCR to measure β-catenin mRNA levels in transformed BxPC-3, MIA Paca-2, AsPC-1, and PANC-1 cells described above.Luciferase reporter assays showing the impact of ALKBH5 overexpression or knockdown on the promoter activities of β-catenin in BxPC-3, MIA Paca-2, AsPC-1, and PANC-1 cells.Immunoblotting to measure WIF-1, β-catenin, C-myc, cyclin D1, MMP-2, and MMP-9 protein levels in transformed AsPC-1 and BxPC-3 cells described above.Representative images of IHC staining for ALKBH5, WIF-1, and β-catenin in tumor sections from human PDAC specimens.ALKBH5 protein levels were positively correlated with WIF-1 protein levels in human PDAC specimens.ALKBH5 protein levels were negatively correlated with β-catenin protein levels in human PDAC specimens. Scale bar = 200 μm (). The data are shown as the means ± S.D. **<0.01; ## <0.01 a c d e f g h i j k i P
WIF-1 is critical to ALKBH5 suppressed Wnt signaling
WIF-1 is critical to ALKBH5 suppressed Wnt signaling.Immunoblotting of lysates from BxPC-3 cells transfected with control, ALKBH5, shCtrl, and/or shWIF-1. Expression of ALKBH5 and WIF-1 were measured. β-actin was used as a loading control.MTT,Colony formation,Migration,Invasion, and () Immunoblotting of WIF-1 downstream targets in cells described in (). () Immunoblotting of lysates from AsPC-1 cells transfected with shCtrl, shALKBH5, control, and/or WIF-1 vector. Expression of ALKBH5 and WIF-1 were measured. β-actin was used as a loading control. () MTT, () Colony formation, () Migration () Invasion, and () Immunoblotting of WIF-1 downstream targets in cells described in ().Representative ventral view images and its quantification () of bioluminescence from xenograft mice implanted with AsPC-1 cells described in ().Quantification of IHC staining for Ki67 in tumor sections from xenograft mice described above. Scale bars = 50 μm (,,and). The data are shown as the means ± S.D. **<0.01; ## <0.01 a b c d e f a g h i j k l g m n g o d e j k P
Discussion
As the most abundant mRNA modification in mammals, m6A influences almost every step of RNA metabolism, contributing to a variety of biological processes involved in cancer, such as the self-renewal of cancer stem cell, cell proliferation, and resistance to radiotherapy or chemotherapy. m6A modification was reported to be involved in the DNA damage response following ultraviolet irradiation, which is regulated by the methyltransferase METTL3 (methyltransferase-like 3) and the demethylase FTO (fat mass and obesity-associated protein), suggesting m6A might be a promising target for combined therapy with radiotherapy or chemotherapy [20]. Moreover, other groups have implicated FTO and METTL3 in the chemo- and radio-resistance in human cervical squamous cell carcinoma (CSCC) and human pancreatic cancer cells, respectively [21, 22]. Similarly, the results presented here demonstrate decreased ALKBH5 and increased METTL3 protein levels in gemcitabine-treated PDXs, and that ALKBH5 deficiency renders notable resistance to gemcitabine treatment both in vitro and in vivo. In addition, our studies have implied a critical role for ALKBH5 in predicting prognosis of gemcitabine treatment. Unlike the readers and writers, only two m6A demethylases have been identified so far, namely, FTO and ALKBH5, both of which are Fe (ii) and α-ketoglutarate dependent [23]. Recently, downregulation of m6A erasers FTO and ALKBH5 has been found to facilitate PARPi resistance by increasing m6A modification in FZD10 mRNA to promote Wnt signaling in BRCA1/2-mutated ovarian cancer cells [24]. This finding further validated the importance of m6A in treatments associated with DNA damage responses, including radio-, chemo-therapy and therapies targeting mutations related to DNA damage repair. Since crystal structure of FTO and ALKBH5 was determined, subsequent drug development and validation might be expected [25, 26].
The Wnt/β-catenin pathway is a highly conserved pathway, and its aberrant activation contributes to progression of pancreatic cancers via increased cell proliferation and chemo-resistance of the tumor cells. PDAC patients often have several mutations of the Wnt ligands, and epigenetic silencing of extracellular Wnt inhibitors, such as WIF-1 and DICKKOPFs (DKKs) may prompt the stabilization and accumulation of β-catenin in cancers despite mutational activation of Wnt/β-catenin signaling [27–29]. Accumulating evidence has showed the epigenetic regulation of Wnt/β-catenin signaling in cancer, though, the impact of mRNA modification is still insufficiently studied. Until very recently, global analysis of m6A functions in 75 MeRIP-seq human samples using deep learning and network-based methods identified 709 functionally significant m6A-regulated genes, which were enriched in many critical biological processes including cancer-related pathways such as Wnt pathway [30]. YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) regulates tumorigenicity and cancer stem cell-like activity in human colorectal carcinoma by mediating Wnt/β-catenin pathway [31]. Reduction of RNA m6A methylation activates oncogenic Wnt/PI3K-Akt signaling to promote malignant phenotypes of GC cells [32]. METTL3 promotes osteosarcoma cell progression by upregulating the m6A level of LEF1 and activating Wnt/β-catenin signaling pathway [33]. Here, we clarified a novel mechanism of m6A modification that ALKBH5 inhibits Wnt signaling by downregulating m6A level of WIF-1, which attenuates PDAC cell proliferation, migration, invasion, tumorigenesis, and metastasis in vitro and in vivo. Our work demonstrates the downregulation of ALKBH5 in PDAC and attests its contribution to retention of malignant phenotypes in PDAC by gain of function and phenotypic rescue assays separately. We then screened and verified WIF-1 and Wnt signaling as the key target of ALKBH5-mediated m6A modification, and indicated that WIF-1 is critical to the tumor suppressive effect of ALKBH5 in PDAC. This may give rise to at least one piece of the jigsaw puzzle regarding to ALKBH5-mediated m6A modification in solid tumor, and open up new horizons for development of new treatment.
Although we provide robust evidence of ALKBH5’s effect on resistance to gemcitabine treatment, the underlying mechanism is not fully characterized. Previously, FTO was reported to enhance the chemo-radiotherapy resistance by reducing m6 A levels of β-catenin mRNA transcripts to regulate its expression [21]. YTHDF1 mediates Wnt/β-catenin pathway to regulate cancer stem cell-like activity [31], which is also a known factor affecting gemcitabine efficacy [34, 35]. Given the importance of Wnt signaling in regulating cancer stemness and chemo-resistance, ALKBH5’s function in resistance to gemcitabine might also rely on m6A modification of WIF-1 and subsequent repression of Wnt signaling, which deserves further investigation.
The graphical explanation of the mechanisms
Conclusions
In conclusion, we show that ALKBH5 overexpression sensitizes PDAC cells to gemcitabine treatment, and it represses PDAC tumorigenesis by reducing m6A levels of WIF-1 and hindering activation of Wnt signaling. These findings shed light on novel molecular mechanisms of PDAC tumorigenesis regulated by m6A modification and provide new insight into developing effective therapeutic strategies in the treatment of PDAC.
Supplementary information
Additional file 1: Figure S1. (A) Heatmap summarizing genes differentially expressed in gemcitabine treated compared to control PDX mice of the 3rd passage. (B) Volcano plot displaying differentially expressed genes. Up-regulated genes are highlighted in pink. Down-regulated genes are highlighted in black.Additional file 2: Figure S2. (A) TCGA database available from GISTIC for ALKBH5 expression multiple human cancers. (B-G) Kaplan-Meier analysis indicating overall survival of patients with high (red) or low (n = 83) ALKBH5 expression in multiple human cancers. (H) ALKBH5 expression was determined by immunohistochemistry assay.Additional file 3: Figure S3. Immunoblotting (A) and qRT-PCR (B) to measure ALKBH5 expression in 8 PDAC cell lines.Additional file 4: Figure S4. (A) Quantification of Fig.2a. (B-E) Colony formation of transformed cells described above with gemcitabine treatment.Additional file 5: Figure S5. Kaplan-Meier analysis indicating overall survival of PDAC patients with high (A) or low (B) ALKBH5 expression in the absence (red) or presence (blue) of gemcitabine treatment.Additional file 6: Figure S6. Immunoblotting (A) and qRT-PCR (B) to measure expression of EMT markers including E-cadherin, α-catenin, N-cadherin, Fibronectin, and Vimentin in transformed AsPC-1 and PANC-1 cells.Additional file 7: Figure S7. Immunoblotting (A) and qRT-PCR (B) to measure expression of EMT markers including E-cadherin, α-catenin, N-cadherin, Fibronectin, and Vimentin in transformed BxPC-3, and MIA Paca-2 cells.Additional file 8: Figure S8. Heatmap summarizing genes differentially expressed in AsPC-1-shCtr and AsPC-1-shALKBH5 cells (A), PANC-1-shCtr and PANC-1-shALKBH5 cells (B), MIA Paca-2-Vector and MIA Paca-2-ALKBH5 cells (C).Additional file 9: Figure S9. Luciferase reporter assays showing the impact of ALKBH5 overexpression on WIF-1 promoters in MIA Paca-2 cells.Additional file 10: Figure S10. (A) qRT-PCR to measure C-MYC, cyclin D1, MMP-2, and MMP-9 mRNA levels in transformed MIA Paca-2 cells described above.Additional file 11: Figure S11. Correlation between ALKBH5 and WIF-1(A), β-catenin (B), C-MYC (C), cyclin D1 (D), MMP-2 (E), and MMP-9 (E) at mRNA level.Additional file 12: Table S1 Correlations between ALKBH5 expression and clinicopathologic features in PDCA patients.Additional file 13: Table S2. Primer sequences used for qRT-PCR and MeRIP-PCR.