Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide, resulting in over 500,000 deaths per year (Torre et al., 2015). Despite the recent advances in surgical resection and liver transplantation, the 5-year survival rate of liver cancer patients is still less than 17% (Miller et al., 2016). In most cases, HCC is detected at advanced stage with limited therapeutic options. Only 10–20% of the tumors are considered surgically resectable at the time of diagnosis (El-Serag, 2011). Thus, a better understanding of the molecular mechanisms underlying the initiation and progression of HCC, as well as identification of prognostic biomarkers are still needed.

N6-methyladenosine (m6A) is the most abundant chemical modification in eukaryotic mRNA (Meyer et al., 2012). The m6A modification is a reversible process regulated by the balanced activities of m6A “writer” and “eraser” proteins (writers, erasers). m6A is installed by an RNA methyltransferase complex, the m6A writer, which is consisted of methyltransferase like 3 (METTL3), METTL14, Wilm’s tumor 1-associated protein (WTAP), KIAA1429, RNA binding motif protein 15 (RBM15), and zinc finger CCCH domain-containing protein 13 (ZC3H13) (Liu et al., 2014). Besides, METTL16 is a newly identified RNA methyltransferase independent from the methyltransferase complex. The reversible process is conducted by the m6A erasers, including fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5) (Jia et al., 2011; Zheng et al., 2013). The m6A-modified mRNAs are specifically recognized by the m6A readers YTH domain containing proteins YTHDC1-2, YTH-family proteins YTHDF1-3, insulin-like growth factor 2 mRNA-binding proteins IGF2BP1-3, as well as heterogeneous nuclear ribonucleoprotein HNRNPA2B1 and HNRNPC, leading to alterations of mRNA splicing, export, translation and degradation (Wang X. et al., 2014; Xiao et al., 2016; Meyer and Jaffrey, 2017). M6A modification is implicated in various biological processes including circadian rhythm (Hastings, 2013), lipid metabolism (Zhong et al., 2018), embryonic stem cell differentiation (Wang Y. et al., 2014), as well as carcinogenesis (He et al., 2018). Increasing evidence have indicated that dysregulation of m6A pathway is frequently found in malignancies such as breast cancer (Niu et al., 2019), glioblastomas (Zhang et al., 2017), acute myeloid leukemia (Paris et al., 2019), lung cancer (Liu et al., 2020), and HCC (Lan et al., 2019). It is reported that the m6A modification level of total RNA is decreased in HCC, and that METTL14 is the main factor involved in the aberrant m6A modification (Ma et al., 2017). The decreased METTL14 expression promoted tumor metastasis by reducing the m6A modification level and miR-126 expression. Another study demonstrated that the high expression of METTL3 in HCC led to increased tumor growth and m6A modification level, mediating degradation of the tumor suppressor SOCS2 through an m6A reader protein YTHDF2-dependent pathway (Chen et al., 2018). Knockdown of METTL14 significantly suppressed HCC cell proliferation and migration (Chen et al., 2018). These findings underscore the complexity of m6A modification and its regulatory enzymes in HCC. The role of m6A-related factors in carcinogenesis and progression of HCC remains to be elucidated. Hence, in the present study, we analyzed the clinical and sequencing data of HCC cohort from TCGA datasets, and investigated the alteration spectrum and prognostic values of 19 main m6A regulatory genes in patients with HCC.

Accumulating studies have reported the important role of m6A modification in tumorigenesis and progression (Wang et al., 2018). However, the alterations and biological role of m6A modification remain elusive in HCC. In this study, we analyzed the alterations of m6A regulatory genes in HCC, and found that the frequency of the alterations was much higher than that reported in acute myeloid leukemia (AML) (Kwok et al., 2017) and clear cell renal cell carcinoma (ccRCC) (Zhou et al., 2019), implying that dysregulation of m6A might play a more important role in HCC tumorigenesis. Among the 19 m6A regulatory genes, KIAA1429 and METTL16 had the most frequent CNV events, indicating the critical role of m6A writer genes in m6A modification of HCC. Moreover, we found that the alterations of KIAA1429 and YTHDF1 tend to gain of copy number, while METTL14 and YTHDF2 tend to loss of copy number. These findings were consistent with previous studies, which reported the upregulation of KIAA1429 (Lan et al., 2019) and YTHDF1 (Zhao et al., 2018), and downregulation of METTL14 (Ma et al., 2017) and YTHDF2 (Hou et al., 2019) in HCC.

Previous studies have suggested that the risk of developing HCC in males is higher compared with that in females (Yeh and Chen, 2010; Liu et al., 2019). Irrespective of the etiology, the morbidity of liver cancer in males is 2–4-fold higher compared with that in females (El-Serag, 2011). In this study, we found that the mutation or CNV events of m6A related genes were more common in male than that in female patients with HCC, suggesting a possible role for m6A modification in sex disparity. Moreover, we found that, for most of the m6A regulatory genes, their CNVs were significantly associated with histological grade or TNM stage, prompting that dysregulation of m6A methylation were closely correlated with HCC development. In addition, we observed the alteration of m6A regulatory genes was significantly associated with the alteration of TP53, an important tumor suppressor genes in HCC. It has been reported that silencing METTL3 in a HCC cell line HepG2 affects the expression and alternative splicing pattern of more than 20 genes involved in the TP53 signaling pathway (Dominissini et al., 2012). Thus, genetic alterations of m6A regulators may cooperate with TP53 signaling pathway in the pathogenesis of HCC.

We also evaluated the effect of m6A regulators’ alterations on the survival of HCC patients. We found that patients with any mutation of m6A regulatory genes had a worse OS and DFS. Further separate analysis showed that only METTL16 and ALKBH5 among the 19 regulators were associated with survival of HCC patients. Moreover, the result from the Multivariate Cox regression analysis indicated that copy number loss of METTL16 was an independent risk factor for DFS. It has been reported that several m6A regulators including METTL3 (Chen et al., 2018), METTL14 (Ma et al., 2017), WTAP (Chen et al., 2019), KIAA1429 (Lan et al., 2019), and YTHDF2 (Hou et al., 2019), play critical roles in the carcinogenesis and progression of HCC. Here, we report for the first time that the alterations of METTL16 and ALKBH5 are closely associated with worse clinical characteristics including survival, and loss of METTL16 is an independent risk factor of DFS, suggesting their potential roles in HCC carcinogenesis and progression.

Recently, the mRNA methylation complex containing METTL3, METTL14, and WTAP has been the subject of intense study in human cancers (He et al., 2019). METTL16 is an independent RNA m6A methyltransferase that can methylate both coding and non-coding RNA, but its biological role is rarely studied. Although many potential RNA targets of METTL16 have been identified by high throughput method, only a few, including U6 snRNA (Warda et al., 2017), Long non-coding RNA MALAT1 (Brown et al., 2016), and mRNA MAT2A (Pendleton et al., 2017) have been verified. The MAT2A gene encodes a methionine adenosyltransferase that catalyzes the production of S-adenosylmethionine, a key substrate required for RNA methylation. It is reported that METTL16 could induce splicing of MAT2A mRNA, which increases the stability and translation of MAT2A mRNA (Pendleton et al., 2017). Thus, loss of METTL16 may result in a transcriptome-wide loss of m6A methylation by modulating S-adenosylmethionine production (Koh et al., 2019). In this study, we observed a copy number loss with down-regulation of METTL16, which might explain the previous findings that the m6A level of total RNA was decreased in HCC (Ma et al., 2017; Hou et al., 2019).

Metabolic reprogramming has been recognized as a hallmark of cancer (Pavlova and Thompson, 2016). The cancer cells reprogram their metabolism to support their rapid proliferation. A previous TCGA analysis highlighted the critical role of metabolic reprogramming in the progression of HCC (Cancer Genome Atlas Research Network, 2017). In the present study, to explore the potential biological role of METTL16 in HCC, we performed GSEA analysis in the TCGA cohort and found that low METTL16 level was associated with the activation of multiple metabolic pathways, implying a potential role of METTL16 in metabolic reprogramming.

In conclusion, our present study determined the alterations of 19 m6A regulatory genes in HCC and their association with clinicopathological features including survival. We found significant associations between the genetic alterations and clinicopathological features as well as TP53 alteration. Notably, we found that loss of METTL16 predicted a worse survival and was an independent risk factor of DFS for HCC patients. Our study highlight METTL16 as a potential prognostic biomarker as well as potential target for HCC treatment. These findings provide a new insight into the underlying mechanism of m6A modification in tumorigenesis and development of HCC.

The datasets generated for this study can be found in The Cancer Genome Atlas (https://portal.gdc.cancer.gov/).

Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

PW and CZ conceived and designed the study and wrote the initial draft of the manuscript. PW, XW, and LZ collected and analyzed the data. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.