PC tissues along with adjacent normal samples were obtained from the Department of General Surgery, the First Affiliated Hospital of Soochow University. The overall experiment design and approach was approved by the Ethical Committee of the First Affiliated Hospital of Soochow University, and written informed consent was successively obtained from all participating patients before the study.

The human PC cell lines PANC1 and CFPAC1 (GenePharma, Shanghai, China) were cultured with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% antibiotics. The culture dishes were placed in an incubator at 37°C with 95% relative humidity and 5% CO₂ partial pressure. The DMEM was changed every 2 days.

Target genes small interference fragment and negative control fragment (si-NC) were purchased from Synbio Technologies (Suzhou, China). Cells were evenly plated and cultured to a density of about 70% and then transfected with 50 nM siRNA using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) to downregulate the target gene expression level according to the manufacturer’s instructions. After transfection, the cells were harvested for further experiments, and all the siRNA sequences are shown in S1 Table.

Cell viability was detected with a Cell Counting Kit-8 (CCK-8) kit (Dojindo, Kumamoto, Japan) according to the protocol. Approximately 5×10³ PANC1 and CFPAC1 cells were seeded in 96-well plates per well after treated with or without gemcitabine for 48 hours, we checked relevant reports and conducted preliminary experiments, and finally set the gemcitabine concentration to 10 µM [11]. After incubated for 24, 48, 72, and 96hours, 10 µL CCK-8 was added and incubated at 37℃ for 2 hours, and the absorbance was detected at a wavelength of 450 nm.

A 5-ethynyl-2-deoxyuridine (EdU) analysis kit (RiboBio, Guangzhou, China) was applied to detect the cell proliferation capability. Stable transfected PC cells were incubated with diluted 5 µM EdU reagent for 3 hours. Then, cells were permeabilized and fixed for 30 minutes, then we used Apollo and DAPI regents to stain cell DNA and nuclei. Finally, EdU- and DAPI-positive images were taken with a fluorescence microscope.

After treated with or without gemcitabine for 48 hours, appropriate 5×10⁴ cells were evenly cultured in the upper chamber of a Transwell plate (Corning, Corning, NY) with serum-free DMEM, and 700 µL of DMEM containing 10% fetal bovine serum was added to the lower chamber. Then monitor cell growth status daily to ensure no apparent cells death. After normal culture for 48 hours, the cells were fixed with 4% paraformaldehyde and stained with crystal violet for 20 minutes, and the cells that migrated through the upper chamber membrane were counted. Images were acquired by inverted microscopy.

We used EpiQuik m6A RNA Methylation Quantification Kit (colorimetric) (Epigentek, Farmingdale, NY) to quantify the overall m6A methylation level in PC cells. First, we extracted total RNA, and approximately 200 ng RNA was separated as an initial input. Then, the sample RNA, standard positive/negative control, and binding solution were added to the wells for 1 hour for assay and capture of the RNA according to the manufacturer’s instructions. After the samples were washed, the detection antibody and enhancer solution were added, and color developer solution and stop solution were added prior to measurement of the absorbance. The values were calculated using linear regression equations.

Total RNA was extracted from PC cells or tissues with TRIzol for quantitative analysis. RNA was first reverse transcribed to cDNA with Vazyme reverse transcriptase at 42°C for 45 minutes following the manufacturer’s protocol, and PCR was then performed using SYBR Green qPCR Master Mix (GenePharma). Human ACTB was used as the housekeeping gene for mRNA expression. After 40 cycles of denaturation, annealing, and extension, the relative RNA levels were determined with the 2^{^-ΔΔCT} method relative to the control. All the primers are listed in S1 Table.

A radio immunoprecipitation assay lysate mixture containing 1% phenylmethylsulfonyl fluoride was used to lyse and obtain total protein from PC cells. After quantification, electrophoresis was performed to separate the proteins, and then, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes in transfer buffer. Then, the membranes were blocked in 5% skim milk at 37°C for 1 hour and incubated with primary antibodies overnight at 4℃. The next day, the membranes were incubated with an HRP-labeled secondary antibody for 1 hour at room temperature in the dark. Finally, immunoreactions were observed with a chemiluminescence detection system in a dark room.

Immunoprecipitation assays of m6A and WTAP were performed with an RIP kit (BersinBio Biotech, Guangzhou, China). In brief, approximately 1×10⁷ PC cells were collected and lysed for 30 minutes. DNA impurities were removed with DNase according to the protocol, and the supernatant was collected after centrifugation. The supernatant was then divided into two equal parts: one was incubated with anti-m6A or anti-WTAP primary antibodies overnight at 4℃ in a vertical orientation, and the other was used as input. Then the samples were incubated with target magnetic beads for 1 hour. After washing, RNA was extracted, and MYC expression was detected.

To comprehensively observe the expression relationship between WTAP and the target gene MYC, immunofluorescence staining assay was conducted. First, after deparaffinization and antigen retrieval, the sections were fixed with 4% paraformaldehyde and blocked with 5% BSA (Solarbio, Beijing, China). Then, the sections were stained with WTAP primary antibody (Abcam, Cambridge, MA) and MYC primary antibody (Sigma, Darmstadt, Germany) at 4℃ overnight and incubated with the secondary antibody afterward. Finally, the cell nuclei were stained with DAPI, anti-quenching agent was added, and the sections were imaged with a confocal laser scanning microscope.

To evaluate the effect of gemcitabine on the tumor formation ability and gemcitabine/WTAP/MYC axis in vivo, we injected 2×10⁶ PANC1 cells at logarithmic growth phase into the axilla of 4-week-old nude mice for subcutaneous tumor formation. The mice were randomly divided into phosphate buffered saline (PBS) group and gemcitabine treatment group. PBS or gemcitabine was injected into the mice via the abdominal cavity at a concentration of 50 mg/kg once every 5 days. The tumor dimension was measured every 5 days. After 30 days, the tumors were excised and weighed to compare the tumor size and volume, WTAP and MYC mRNA and protein expression was detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence. The care of laboratory animals was in accordance with the guidelines and ethical requirements of the Laboratory Animal Centre of Soochow University.

We used GraphPad 8.0 (GraphPad Software Inc., San Diego, CA) for data analysis and image generation. For statistical analysis, a two-tailed Student’s t test between two groups was performed. The Kaplan-Meier method for survival analysis. Correlational analysis of gene expression was conducted with linear regression. The scientific data are reported as the mean±standard deviation from three duplicate experiments. And the results were considered statistically significant when the p-value < 0.05.

To explore the regulatory activity of gemcitabine on m6A RNA modification, cells were treated with gemcitabine to check the cell viability changes. CCK-8 and EdU assays revealed that gemcitabine inhibited cells viability (Fig. 1A and B). Transwell assays revealed the inhibition of cell migration ability by gemcitabine (Fig. 1C), revealing that gemcitabine was an effective treatment for PC. Subsequently, m6A quantification demonstrated that gemcitabine decreased the m6A modification level (Fig. 1E and F). Considering the inhibitory effect of gemcitabine on m6A level, we wanted to explore m6A regulators that were regulated by gemcitabine. qRT-PCR demonstrated that m6A writers and erasers were changed to varying degrees by gemcitabine, among which the writer WTAP was significantly decreased (fold change=0.37, p < 0.001) (Fig. 1D), suggesting that RNA methylation might be a target for gemcitabine. TCGA analysis revealed the similar upregulation of WTAP in PC (Fig. 2A). Taken together, we took WTAP as the target of gemcitabine for further research.

Analysis of expression pattern of WTAP revealed its high expression in PC (Fig. 2A), we then divided 173 PC specimens into WTAP high and low expression groups according to the median WTAP level, Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene ontology (GO) analysis revealed the important implications of WTAP on immunity and metabolism pathways (Fig. 2B and C). qRT-PCR and western blot analysis showed that WTAP expression level was reduced by gemcitabine and WTAP-si in PANC1 cells (Fig. 2D-G). CCK-8 and Transwell assays revealed that WTAP-si decreased cell viability and migration (Fig. 2H and I), which was also confirmed with EdU assays (Fig. 2J and K). Our results verified that important interaction between gemcitabine and WTAP might be significant for the clinical treatment of PC.

After identifying the regulation of gemcitabine on WTAP, we investigated which target was regulated by gemcitabine/WTAP. We first predicted possible downstream target genes of WTAP and detected their expression changes in WTAP knockdown cells. Experimental data showed that after WTAP knockdown in PANC1, MYC, and HER2 were downregulated with fold changes of 0.25 and 0.34, respectively, while CDKN2A was upregulated (Fig. 3A). WTAP-immunoprecipitation (IP) assay showed that CDKN2A and MYC, but not HER2, were significantly enriched, suggesting that CDKN2A and MYC might be WTAP targets. Then, m6A-IP revealed that CDKN2A, HER2, and MYC were all enriched in PANC1(Fig. 3B), this lead us to speculate that CDKN2A and MYC might be methylated by WTAP but HER2 might be methylated by other m6A regulators. TCGA analysis also preliminarily verified the positive correlation between WTAP and MYC (Fig. 3C). We knocked down WTAP with siRNA in PANC1 and observed decreased MYC protein expression (Fig. 3D). Immunofluorescence analysis of PC specimens also verified the co-expression relationship between WTAP and MYC (Fig. 3E). We further experimentally verified that the level of m6A modification was also inhibited after WTAP knockdown (Fig. 3F). To give an insight into the influence of Gem and WTAP on MYC, we treated WTAP-si PANC1 cells with or without Gem to monitor MYC expression verification, we verified Gem could restrain both WTAP and MYC expression in WTAP-si cells (Fig. 3G), CCK-8 and Transwell assay verified WTAP knockdown could enhance the inhibition of Gem on cell proliferation (Fig. 3H and K).

The above results suggested that MYC is a crucial factor in PC progression. In addition, given that MYC is an indispensable proto-oncogene in various biological processes, such as metabolism [12] and the cell cycle [13], we believe that m6A-MYC is a novel treatment target in PC.

Studies found that IGF2BP1 could recognize m6A modification on MYC to alter MYC stability [14,15], so we speculated IGF2BP1 was a significant reader for m6A-MYC in PC. We found IGF2BP1 knockdown in PANC1 cells could also enhance the inhibition of Gem on MYC stability (Fig. 3I) and cell viability (Fig. 3J and L). What’s more, IGF2BP1 knockdown had no effect on m6A modification but inhibited MYC expression (Fig. 3M and N), Thus, we speculated that gemcitabine could interfere with WTAP-MYC-IGF2BP1 axis to inhibit PC progression.

Next, we performed in vivo experiments to further verify gemcitabine on WTAP expression and the tumorigenic ability. Gemcitabine decreased the tumor volume, central necrosis rate, and tumor weight (Fig. 4A-C), WTAP and MYC also showed a downward trend after treated with gemcitabine (Fig. 4D and E). In conclusion, our experiments showed that gemcitabine inhibits WTAP expression and reduces the m6A modification on MYC and its stability, resulting in a decrease in MYC protein expression, thereby interfering with related pathways and inhibiting the progression of PC in vivo (Fig. 4F).