Novel insights into the interplay between m6A modification and noncoding RNAs in cancer

1Up to now, more than 100 kinds of RNA modifications have been confirmed. Among them, m6A RNA methylation is one of the most thoroughly studied modifications. m6A RNA modification occurs by methylation of the sixth N atom of adenine (A) in mRNAs or ncRNAs. m6A modification sites tend to be found in the stop codons and 3′-Untranslated region (3′-UTR) of mRNA with a typical consensus sequence RRACH (R = G or A and H = A, C, or U). Accumulating data show that m6A RNA methylation acts by modulating circadian rhythm, gene expression, cell differentiation, stress response, inflammatory response, and carcinogenesis. According to the global cancer statistics, there were estimated 18.1 million new cases and 9.6 million deaths in 2018. Recent studies have shown that m6A modification acts a vital role in the diagnosis, treatment and prognosis of cancer patients as well as in carcinogenesis. It also regulates fly sex, virus genome, meiosis of yeast, tissue differentiation, germination, and collateral generation of Arabidopsis.

2Noncoding RNAs (ncRNAs) including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) act pivotal roles in cancer. m6A modification can affect ncRNA splicing and maturation involved in carcinogenesis (Table 1). In this review, we summarize the latest progress about the interplay between m6A modification and ncRNAs in cancer.

3Molecular compositions of m6A RNA methylation include m6A methyltransferase, m6A demethylase, and m6A recognition factors (Fig. 1). m6A methyltransferases, called “writers” contain methyltransferase-like 3 (METTL3), METTL14, Wilms tumor 1-associated protein (WTAP), KIAA1429, METTL16 and RNA-binding motif protein 15/15B (RBM15/15B). METTL3 regulates the circadian clock of hepatic lipid metabolism and hematopoiesis. METTL3/14 depletion promotes myeloid differentiation and suppresses the progression of acute myeloid leukemia (AML). METTL16 maintains the levels of methyl donor S-adenosylmethionine (SAM). WTAP connects METTL3/14 to form a complex, anchored to the nucleus to catalyze m6A methyltransferase.

4m6A methylation is dynamic and can be reversed by m6A demethylase, also named as m6A “erasers”, containing fat mass and obesity-associated protein (FTO) and alkB homologue 5 (ALKBH5). FTO shares the motifs with Fe (II)- and 2-oxoglutarate-dependent oxygenase and is related to increased fat mass. FTO harbors an efficient oxidative demethylation activity and reduces the m6A levels of mRNAs. ALKBH5 is responsible for RNA splicing and stability and causes the degradation of abnormal transcripts in spermatocytes and round spermatids.

5m6A recognition factors, known as “readers,” consist of YT521-B homology (YTH) domain family (YTHDF1/2/3), YTH domain-containing proteins (YTHDC1/2), heterogeneous nuclear ribonucleoprotein (HNRNP) protein families, eukaryotic translation initiation factor 3 (eIF3), and insulin-like growth factor-2 mRNA-binding proteins 1/2/3 (IGF2BP1/2/3). m6A recognition factors act in oligodendrocyte progenitor cells and oligodendrocyte fate. YTHDF1 controls pre-crossing axon guidance in the spinal cord by regulating m6A-modified Robo3.1. HNRNPA2B1 can initiate the immune response to DNA viruses by regulating interferon-α/β and stimulator of interferon genes (STING)-dependent antiviral signaling.

6As is known to us, the dysregulation of miRNAs is involved in various bio-behaviors, such as mouse prenatal development, immune response, inflammatory response and carcinogenesis. METTL3 or HNRNPA2B1 facilitates pri-miRNA processing by recruiting RNA-binding protein DiGeorge syndrome critical region 8 (DGCR8). METTL3 suppresses osteogenic processes by promoting the maturation of miR-7212-5p and downregulating its target fibroblast growth factor receptor 3 (FGFR3).

7m6A methylation can modify the maturation of miRNAs involved in cell proliferation and tumorigenesis (Fig. 2). miR-25-3p acts as a pivotal role in pancreatic ductal adenocarcinoma (PDAC). Cigarette smoke condensate (CSC) mediates METTL3 to promote miR-25-3p maturation in PDAC tumorigenesis. METTL3 also enhances the binding of pri-miR-221/222 with DGCR8 involved in the proliferation of bladder cancer. m6A modification affects arsenite-induced carcinogenesis via modifying multiple miRNAs (miR-106b, miR-18a/b, miR-3607, miR-423, miR-30a, miR-320b/d/e).

8METTL14 promotes the maturation of pri-miR-126 and suppresses the invasion and metastasis of hepatocellular carcinoma (HCC). METTL3 facilitates the maturation of pri-miR-1246 to enhance the metastasis of colorectal cancer (CRC). METTL3 also accelerates the maturation of miR-143-3p, leading to the formation of METTL3/miR-143-3p/vasohibin-1 axis to favor the metastasis of lung cancers.

9LncRNAs, a subgroup of non-coding RNAs over 200 nucleotides in length can be modified by m6A methylation in cancer (Fig. 3). m6A methylation facilitates lncRNA X-inactive specific transcript (XIST)-mediated transcriptional repression. YTHDC1 preferentially recognizes the m6A residues of XIST and RBM15/15B and participates in XIST-mediated gene silencing. However, RBM15/m6A-MTase complex is reported to act a minor role in XIST-mediated gene silencing. YTHDF2 recognizes m6A methylation site of lnc-Dpf3 to promote its degradation and enhances the binding of lnc-Dpf3 with hypoxia-inducible factor 1-alpha (HIF-1α), leading to the suppression of the glycolysis and migration of dendritic cells. METTL3 can modify metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) to form the METTL3/MALAT1/miR-145/focal adhesion kinase (FAK) axis, contributing to the aggravation of renal fibrogenesis in obstructive nephropathy.

10ALKBH5 has been found upregulated in glioblastoma and prompts the proliferation of glioblastoma stem-like cells (GSCs). A lncRNA antisense to forkhead box M1 (FOXM1-AS) promotes the interaction of ALKBH5 with forkhead box M1 (FOXM1) nascent transcripts to increase FOXM1 expression and GSCs tumorigenesis. LncRNA Differentiation antagonizing non-protein coding RNA (DANCR) contributes to the tumorigenesis of multiple cancers. IGF2BP2 serves as an m6A reader to modify DANCR and favors the oncogenicity of pancreatic cancer. MALAT1, the first lncRNA to be found associated with lung cancer, possesses a triple helix structure at its 3’end. METTL16 interacts directly with MALAT1 triple helix and promotes cancer cell proliferation.

11Long non-coding RNA 00958 (LINC00958) is upregulated by METTL3 and facilitates HCC cell migration and invasion by sponging miR-3619-5p. METTL3/14 enhance the migration of head and neck squamous cell carcinoma (HNSCC) by upregulating lncRNA activating regulator of DKK1 (LNCAROD). METTL3-family with sequence similarity 225 member A (FAM225A)-integrin β3 (ITGB3)-FAK/PI3K/Akt axis facilitates the metastasis of nasopharyngeal cancer. METTL3 mediates lncRNA RP11–138 J23.1 (RP11) or MALAT1-miR-1914-3p-Yes associated protein (YAP) axis to enhance the migration and invasion of CRC and non-small cell lung cancer (NSCLC). METTL14 increases the m6A levels of XIST and suppresses the invasion of CRC. ALKBH5 favors the invasion and metastasis of gastric cancer (GC) by demethylating lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1). YTHDF1 restrains esophageal squamous cell carcinoma (ESCC) by interacting with long intergenic non-protein coding RNA 278 (LINC00278), but ALKBH5 harbors an opposite function.

12CircRNAs, a novel subset of ncRNAs generated by back-splicing, play a crucial role in protein translation. METTL3 and YTHDC1 are associated with the metabolism of circular RNA zinc finger protein 609 (circ-ZNF609) and promote its production. Minigenes of ribosomes-circRNAs (Ribo-circRNAs) can facilitate protein translation in drosophila heads and circ-ZNF609 boosts protein translation and myoblasts cell proliferation. m6A methylation has been reported to affect protein translation of cricRNAs. m6A motifs are enriched in circRNAs, and a single m6A site is regarded as a trigger to initiate the translation of circRNAs. m6A regulators METTL3/14, FTO, YTHDF3, and initiation factor eIF4G2 are involved in m6A-driven protein translation. Mammalian cells can recognize the m6A modification on circRNAs to inhibit innate immunity by abrogating immune gene activation and adjuvant activity.

13In addition, the dysregulation of circRNAs is associated with the progression of multiple cancers, such as breast cancer, gastric cancer (GC), gallbladder cancer and cervical cancer. YTHDC1 interacts with circRNA NOP2/Sun RNA methyltransferase 2 (circNSUN2) to facilitate its cytoplasmic export, which leads to colorectal liver metastasis by forming a circNUSN2/IGF2BP2/high mobility group AT-hook 2 (HMGA2) RNA-protein ternary complex in the cytoplasm. m6A modification can be involved in the progression of GC by regulating circRNA poliovirus receptor-related 3 (circPVRL3).

14NcRNAs have the capabilities to affect m6A levels involved in multiple biological processes (Table 2). miRNAs can modulate the binding between METTL3 and its target mRNAs to participate in the reprogramming efficiency of mouse embryonic fibroblasts (MEFs). miR-149-3p inhibits adipogenesis lineage differentiation and potentiates osteogenic lineage differentiation by targeting FTO. miR-1266 inhibits CRC progression by targeting FTO. miR-145 suppresses the proliferation of HCC by targeting YTHDF2. Similarly, miR-33a and miR-448 suppress the proliferation of NSCLC by targeting METTL3 and eIF3a. METTL3 is also downregulated by miR-600, which induces the apoptosis of lung cancer. miR-141 suppresses the proliferation of pancreatic cancer by forming the miR-141/IGF2BP2/P13K/Akt axis. Hepatitis B X-interacting protein (HBXIP) inhibits let-7 g expression to upregulate IGF2BP2, thus leading to the formation of a positive feedback loop of HBXIP/let-7 g/IGF2BP2/HBXIP to accelerate cell proliferation in breast cancer. miR-497 partially reverses transforming growth factor beta 1 (TGFβ1)-induced epithelial-mesenchymal transition (EMT) and pulmonary fibroblast proliferation through inhibiting eIF3a in alveolar epithelial cells.

15lncRNAs also regulate m6A methylation in cancer. LncRNA derived from hepatocytes (lnc-HC) interacts with HNRNPA2B1 to inhibit cholesterol metabolism in hepatocytes. Long intergenic non-protein coding RNA 470 (LINC00470) interacts with METTL3 to suppress the stability of phosphatase and tensin homolog (PTEN) to facilitate GC progression. LncRNA miR503 host gene (miR503HG) also interacts with HNRNPA2B1 to promote its degradation through an ubiquitin-proteasome pathway in HCC. Similarly, long intergenic non-protein coding RNA 1234 (LINC01234) interacts with HNRNPA2B1 to facilitate cell proliferation and inhibit cell apoptosis in NSCLC. Lin-28 homolog B antisense RNA 1 (LIN28B-AS1) interacts with IGF2BP1 to promote the proliferation and metastasis of lung adenocarcinoma (LUAD). Long intergenic Noncoding RNA for IGF2BP2 Stability (LINRIS) promotes CRC proliferation by stabilizing IGF2BP2. The antisense RNA of growth arrest special 5 (GAS5-AS1) depends on ALKBH5 to suppresses the growth and metastasis of cervical cancer. Growth arrest special 5 (GAS5) can suppress YAP-mediated YTHDF3 to restrain the proliferative behavior of CRC. Antisense strand of the GATA binding protein 3 gene (GATA3-AS) enhances the interaction between KIAA1429 and GATA binding protein 3 (GATA3) pre-mRNA, leading to the formation of the GATA3-AS/KIAA1429/GATA3 axis in HCC.

16m6A methylation serves as new biomarkers for diagnosis and prognosis in cancer. m6A regulators METTL3, YTHDC2 and HNRNPC are used to predict the prognosis in patients with HNSCC. Upregulated METTL3/FTO or downregulated YTHDF2 and METTL14 can indicate a poor survival in GC, CRC, and HCC. Low expression of METTL14 is associated with tumor differentiation, clinical stage, and microvascular invasion. Low expression of ALKBH5 or FTO predicts an unfavorable marker in lung cancer and HCC. IGF2BP2 is considered as a prognostic marker in pancreatic cancer, esophagogastric junction adenocarcinoma and CRC.

17m6A methylation also participates in drug resistance and cancer treatment. METTL3 stabilizes YAP and Rho GTPase activating protein 5 (ARHGAP5) to induce cisplatin resistance in NSCLC and in GC. HNRNPA2B1 is overexpressed in tamoxifen-resistant breast cancer and reduces 4-hydroxytamoxifen sensitivity. In addition to METTL3 and METTL14, FTO and YTHDF2 are overexpressed in AML. A recent study shows that FTO inhibitor (FB23) and its derivative (FB23–2) promote myeloid differentiation and apoptosis in AML by targeting FTO. m6A methylation is also involved in estimating tumor microenvironment and TME infiltration characterization so as to provide insights into an effective immunotherapy for cancer. YTHDF2 is correlated with inflammation infiltration, vascular reconstruction and distant metastasis and predicts a poor prognosis in HCC.

18In summary, the role of m6A modification in clinical application has been widely validated. As for the core members of m6A methylation, METTL3/14 exert their functions in many biological processes. METTL3/14 can be regarded as the most important and promising m6A regulator and arouse our attention about their modifications on ncRNAs and the clinical application in cancer diagnosis.

19Accumulating studies have been focused on how m6A methylation modifies the stability, splicing and translation of ncRNAs or ncRNAs regulate m6A regulators in cancer. The interaction between m6A methylation and ncRNAs can impact the different life activities including cancer cell proliferation, invasion and metastasis. As for the clinical application of m6A methylation, they can be regarded as the potential targets for cancer diagnosis, prognosis and treatment. The latest findings show that lncRNA long intergenic non-protein coding RNA 266–1 (LINC00266–1) interacts with IGF2BP1 by encoding a 71-amino acid peptide, named RNA-binding regulatory peptide, thereby promoting tumorigenesis. However, the specific binding sites between m6A methylation and ncRNAs need be further investigated.