Abstract
N6-methyladenosine (m6A) RNA methylation has recently been found involving in regulatory mechanism of the tumor progression. Our aim was to explore the biological function and clinical significance of the m6A methyltransferase METTL3 in intrahepatic cholangiocarcinoma (ICC). In this study, we revealed that METTL3 was upregulated and predicted poor prognosis of patients with ICC. Multivariate regression analysis demonstrated that METTL3 expression was an independent predictor for overall survival in patients with ICC. Moreover, METTL3 knockdown inhibited ICC progression, while METTL3 overexpression showed the opposite effect. METTL3 inhibitor STM2457 also showed anti-tumor effect in ICC. Mechanistically, METTL3 transcription was driven by H3K4me3 activation. Upregulation of METTL3 mediated m6A modification of IFIT2 mRNA and accelerated IFIT2 mRNA decay in a YTHDF2-dependent manner, which promoted the development of ICC and lead to poorer prognosis. In summary, our findings revealed that H3K4me3 activation-driven METTL3 transcription promotes ICC progression by YTHDF2-mediated IFIT2 mRNA degradation, suggesting that METTL3 may serve as a potential target for human ICC therapy.
Introduction
Intrahepatic cholangiocarcinoma (ICC) is the second most common primary malignant liver cancer, which accounts for ~10% of all such cancers [1, 2]. The prognosis of patients with ICC is poor. Nearly 70% of patients with ICC are already unresectable at the time of diagnosis. Even in patients undergoing curative surgical treatment, the 5-year overall survival (OS) rate is ~30%, and the 5-year recurrence rate is up to 70% [3]. Therefore, effective systemic therapy during the course of the disease is required for the ICC. The combination of gemcitabine and cisplatin is the current first-line therapy for patients with unresectable ICC, but its efficacy remains very limited [4, 5]. Due to the lack of effective treatment and poor prognosis of ICC, understanding the molecular mechanisms underlying ICC development is urgently needed.
Accumulating evidence has revealed that ICC pathogenesis is complicated, which involves epigenetic, genetic, and proteomic alterations [6, 7]. Epigenetic regulation is one of the most common pathways causing gene aberrant expression and facilitating ICC progression [8]. N6-methyadenosine (m6A) modification is the most prevalent mRNA modification in eukaryote [9]. m6A modification is dynamic and reversible, which is regulated by m6A “writer” proteins (Methyltransferases like 3 [METTL3], Methyltransferases like 14 [METTL14] and Wilms tumor 1 associated protein [WTAP]) and “eraser” proteins (Fat-mass and obesity-associated protein [FTO] and Alkylation repair homolog protein 5 [ALKBH5]). In addition, specific “reader” proteins (YTH domain-containing proteins, YTHDF1-3, and YTHDC1-2) can recognize m6A sites and affect RNA process including mRNA stability, decay, splicing, and translation [10, 11]. Aberrant m6A modifications have been found to be involved in the carcinogenesis of a variety of human tumors [12]. However, the mechanism of carcinogenesis influenced by m6A modification dysregulation in ICC remains unclear.
Here, we dissected the reason for the dysregulation of METTL3 in ICC and revealed the regulatory mechanism of the m6A modification mediated by METTL3 in ICC. We also demonstrated that METTL3 regulates IFIT2 expression in an m6A-YTHDF2-dependent manner, and METTL3 may be a novel prognostic predictor and therapeutic target for ICC.
Results
METTL3 expression was elevated in ICC and associated with poorer prognosis
The m6A levels are mainly regulated by m6A writers and erasers. Therefore, we detected mRNA expression of m6A modulators in ICC tissues. The results showed that methyltransferase METTL3 was significantly up-regulated in all ICC data sets, including GEPIA2 dataset (Fig. 1A), GSE107943 (Fig. 1B), and our dataset (Fig. 1C). We then measured the expression level of METTL3 in tumor tissues of 96 ICC patients by immunohistochemistry (IHC) staining (Fig. 1D) and compared the correlation between METTL3 expression and clinical characteristics (Supplementary Table 1). METTL3 expression was positively correlated with tumor size (P < 0.05) (Fig. 1E) and tumor, node, metastasis (TNM) stage (P < 0.05) (Fig. 1F). Kaplan–Meier analysis showed that ICC patients with high METTL3 expression had poorer DFS (P = 0.0025, Fig. 1G) and OS (P = 0.0015, Fig. 1H). Univariate regression analysis illustrated that tumor size, vascular invasion, nerve invasion, TNM stage, lymphatic metastasis, distant metastasis, and METTL3 expression were associated with OS in 96 ICC patients, and Multivariate regression analysis demonstrated that METTL3 expression was an independent predictor for overall survival in patients with ICC (HR = 2.105, 95% CI [1.246–3.557] (Supplementary Table 2 and Fig. 1I)). These results suggest that METTL3 is upregulated in ICC and might be an independent prognostic marker for ICC patients.