The m6A Methyltransferase METTL3 Promotes Pancreatic Cancer Metastasis and Invasion

Research Article

Austin J Gastroenterol. 2023; 10(2): 1125.

The m6A Methyltransferase METTL3 Promotes Pancreatic Cancer Metastasis and Invasion

Hao Sun1; Shaolong Hao2; Yu Ji2; Xinyu Zhao3; Zhaoyu Sun4; Lang Ji1; Wei Han1,2; Yuchuan Ding5

1Central Laboratories, Beijing Luhe Hospital, Capital Medical University, Beijing, China

2Department of General Surgery, Beijing Luhe Hospital, Capital Medical University, Beijing, China

3Department of Critical Care, Beijing Lu He hospital, Capital Medical University, Beijing, China

4Medical image science, The fifth people’s hospital of Jinan, Jinan, Shandong, China

5Department of Neurosurgery, Wayne State University School of Medicine, USA

*Corresponding author: Hao Sun Central Laboratories, Beijing Luhe Hospital, Capital Medical University, Beijing, China. Email: m18810253032@163. com

Received: September 13, 2023 Accepted: October 13, 2023 Published: October 20, 2023


Background: Pancreatic Cancer (PC) poses a formidable challenge to human health due to its high degree of malignancy, difficult diagnosis and treatment, and poor prognosis. RNA N6-methyladenosine (m6A) is a key step of posttranscriptional modulation that is involved in governing gene expression. The m6A modification catalyzed by METTL3 has been widely recognized as a critical epigenetic regulation process for tumorigenic properties in various cancer cell lines, including pancreatic cancer, but its specific mechanism has remained elusive.

Methods: Transcriptional profiles and clinical data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were analyzed. Through Gene Set Enrichment Snalysis (GSEA) analysis, Western blot, and qPCR, we confirmed the high expression of METTL3 and the increased m6A methylation level in tumor tissues. And METTL3 high expression decreased survival. We constructed stable METTL3 knockout cell lines and found that METTL3 knockout inhibited tumor migration and invasion.

Results: According to the analysis of the TCGA clinical database, METTL3 exhibits higher expression in tumor tissues compared to normal tissues. METTL3 expression was higher in pancreatic cancer tumor tissues compared to paracancerous tissue. Gene Set Enrichment Analysis (GSEA) revealed that METTL3 promotes pancreatic cancer progression, potentially through its role in methylation. Western blot and qPCR experiments confirmed these findings. Constructing stable METTL3 knockout cell lines revealed that METTL3 knockout inhibited tumor migration and invasion. Furthermore, our results confirmed that METTL3 impacts the malignant progression of pancreatic cancer via the VEGF/VEGFR pathway.

Keywords: PDCA; METTL3; m6A; Metastasis; Invasion


Pancreatic cancer is a malignancy of the digestive tract that exhibits insidious and atypical clinical symptoms, rendering it challenging to diagnose and treat effectively. Around 90% of cases are Pancreatic Ductal Adenocarcinoma (PDAC) which ductal adenocarcinomas arising in the glandular duct epithelium. The development of pancreatic cancer is closely linked to lifestyle factors such as smoking, drinking, and diet. Unfortunately, early diagnosis of pancreatic cancer is uncommon, with high mortality rates associated with surgical intervention and low rates of complete remission [1,2]. Notably, pancreatic cancer boasts the lowest 5-year survival rate of all tumors (less than 8%) [3,4]. As such, early diagnosis and prompt intervention for patients with pancreatic cancer are essential to optimize treatment outcomes.

N6-Methyladenosine (m6A) is the most common methylation modification that occurs in eukaryotic mRNA sequences following transcription. It is enriched in the consensus RRACH motif located near the 3' non-coding region, the 5' non-coding region, and the stop codon. (R represents A or G, while H denotes A, C, or U). The process of m6A methylation is highly dynamic and involves three key types of enzymes: Writers, Erasers, and Readers [5,6]. methyltransferases are responsible for catalyzing adenylate to modify m6A on mRNA while demethylases work to remove the m6A modified base. Reading proteins, on the other hand, are responsible for identifying the base of m6A modification and activating downstream regulatory pathways. Through synergistic action with downstream recognition proteins, regulation of mRNA splicing, nucleation, localization, translation, and stability can be achieved [5,7], The functional regulation of transcriptional and post-transcriptional processes in eukaryotic cells is therefore achieved, with significant effects on stress response, cell reproduction, stem cell differentiation, development, and tumor occurrence [8,9].

METTL3 as a crucial component of the m6A methylated transferase complex, is a methyltransferase involved in the process of RNA modification through the addition of m6A. It forms stable heterodimers with methyltransferase 14(METTL4) with Wilm's Tumor 1-Associated Protein (WTAP) as a cofactor, thus constituting the main structure of m6A methylated transferase [10,11]. METTL3, also referred to as MT-A70, has two critical domains that bind S-Adenosylmethionine (SAM) and catalyze the formation of m6A, respectively [12,13]. Additionally, both METTL3 and METTL14 are core proteins of m6A methyltransferase complex [14]. Research has shown that METTL3 can impact the progression of various types of cancers such as gastric cancer [15,16], lung cancer [17], pancreatic cancer [18] and other tumors by affecting RNA methylation modification. . However, the mechanism by which METTL3 promotes malignant progression of pancreatic cancer remains understudied, making our results important for early diagnosis of pancreatic cancer and providing targeted therapy for METTL3.


Human Specimens

The study was performed after approval by the Institutional Ethical Board of our affiliated hospital. All enrolled individuals provided informed consent. Paired distant normal tissues away from the tumor margins at least 5 cm were served as controls.

Cell Lines and Transfection

PANC-1, SW1990 were were purchased from Guangzhou Genio Biotech Co., Ltd. The SW1990 and PANC-1 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Millipore Sigma) supplemented with 10% heat-inactivated fetal bovine serum FBS (Thermo Fisher Scientific, Inc.) and 1% antibiotics (penicillin-streptomycin; Thermo Fisher Scientific, Inc.). All cells were performed in a humidified incubator at 37°C and in a moist incubator stabilized at 5% CO2. To knock down endogenous METTL3. Lipofectamine™ 3000 (Thermo Fisher Scientific, Inc.) was used to transfect the PC cell lines with a small interfering siMETTL3 [19].

RNA Extraction and Quantitative Real-Time PCR

A TRIzol® RNA extraction kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used for isolation of total RNA from cell and tissue according to the manufacturer's instruction. Subsequently, we used a First-Strand cDNA Synthesis Kit ((Takara Biotechnology Co., Ltd.) to obtain the complementary DNA (cDNA). For real-time PCR (qRT-PCR), amplifications were performed by the real-time PCR Master Mix (SYBR® Primx Ex Taq™ (TIi RNaseH Plus) (cat. no. RR420A; Takara Bio, Inc.). We used 2 - ΔΔCt method to analyze the mRNA expression. GAPDH expression was used as internal controls. The primer sequence of the genes was shown is shown in Table 1.