ARTICLE

RNA m6A methylation regulates the epithelialmesenchymal transition of cancer cells andtranslation of SnailXinyao Lin1,6, Guoshi Chai2,6, Yingmin Wu1,6, Jiexin Li1, Feng Chen1, Jianzhao Liu 3,4, Guanzheng Luo 2,3,

Jordi Tauler 3, Jun Du1, Shuibin Lin 5, Chuan He 3 & Hongsheng Wang 1

N6-Methyladenosine (m6A) modification has been implicated in the progression of several

cancers. We reveal that during epithelial-mesenchymal transition (EMT), one important step

for cancer cell metastasis, m6A modification of mRNAs increases in cancer cells. Deletion of

methyltransferase-like 3 (METTL3) down-regulates m6A, impairs the migration, invasion and

EMT of cancer cells both in vitro and in vivo. m6A-sequencing and functional studies confirm

that Snail, a key transcription factor of EMT, is involved in m6A-regulated EMT. m6A in Snail

CDS, but not 3’UTR, triggers polysome-mediated translation of Snail mRNA in cancer cells.

Loss and gain functional studies confirm that YTHDF1 mediates m6A-increased translation of

Snail mRNA. Moreover, the upregulation of METTL3 and YTHDF1 act as adverse prognosis

factors for overall survival (OS) rate of liver cancer patients. Our study highlights the critical

roles of m6A on regulation of EMT in cancer cells and translation of Snail during this process.

https://doi.org/10.1038/s41467-019-09865-9 OPEN

1 Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong510006, China. 2MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510275,China. 3 Department of Chemistry, Department of Biochemistry and Molecular Biology, and Institute for Biophysical Dynamics, The University of Chicago,929 East 57th Street, Chicago, IL 60637, USA. 4MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Scienceand Engineering, Zhejiang University, Hangzhou, Zhejiang 310027, China. 5 Department of Rehabilitation Medicine, Center for Translational Medicine, TheFirst Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. 6These authors contributed equally: Xinyao Lin, Guoshi Chai, Yingmin Wu.Correspondence and requests for materials should be addressed to H.W. (email: whongsh@mail.sysu.edu.cn)

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Among the more than 100 modifications in mRNA, N6-methyladenosine (m6A) has been identified since the1970s1. m6A modification is the most abundant in

eukaryotic mRNAs2, while thousands of RNA transcripts containm6A modifications with unique distribution patterns as well3. Animportant report by Jia et al.4 identified the first RNA deme-thylase, fat mass and obesity-associated protein (FTO), revealingthat RNA modification is reversible. Since then, the study ofdynamic of m6A modification has greatly expanded5–9.Methyltransferase-like 3 (METTL3), METTL14, and Wilm’stumor-1-associated protein (WTAP) have been proved to act asm6A methyltransferases (“writers”) in mammalian cells10,11.m6A-binding proteins with YTH domain, including cytoplasmicprotein YTHDF1, YTHDF2, YTHDF3, and nuclear proteinYTHDC1, have been identified to be the “readers” of m6A andmodulate mRNA stability and translation to mediate downstreameffects6,7,12,13.

Studies on biological functions of m6A modification in mRNAhave arisen in recent years14. It has been reported that m6Amodification is involved in various biological processes, includingheat-shock response12, ultraviolet-induced DNA damageresponse15, maternal mRNA clearance5, neuronal functions and sexdetermination16, cortical neurogenesis17, haematopoietic stem andprogenitor cell specification18, and T-cell homeostasis19. Although,m6A modification has been associated with tumorigenesis andprogression of cancer cells, the molecular mechanisms involvedhave not been described yet. Recently, FTO has been shown to bean oncogenic factor in acute myeloid leukemia, which functions asN6-methyladenosine RNA demethylase20. Inhibition of FTO sup-presses tumor progression and prolongs lifespan of glioblastomastem cells (GSC)-grafted mice substantially21. Also, FTO inhibitionexerts a broad anti-leukemic activity in vitro and in vivo22. Anotheridentified m6A demethylase, ALKBH5, maintains tumorigenicity ofglioblastoma stem-like cells by sustaining FOXM1 expression andcell proliferation program23. ALKBH5-mediated m6A-demethylation of NANOG mRNA is involved in hypoxia-inducedbreast cancer stem cell phenotype24. Together, these results suggestthat m6A modification found in mRNA may regulate the pro-gression of cancer development.

The metastasis of cancer cells is responsible for more than 90%of cancer-associated mortality25, and epithelial–mesenchymaltransition (EMT) is considered to be an important step for cancercell metastasis. During EMT process, epithelial cells lose polarityand gain invasive properties to become mesenchymal stem cells26.Loss of E-cadherin (E-Cad, encoded by the CDH1 gene) has beenconsidered as the most fundamental event in EMT program27.Repression of E-Cad expression, by EMT-inducing transcriptionfactors (EMT-TFs), such as Snail, Slug, Twist, and Zeb, results inpromotion of metastasis in cancer cells28. Snail (encoded bySNAI1), acts as a key transcriptional repressor of E-cad expres-sion in EMT, confers tumor cells with cancer stem cell-like traitsand promotes drug resistance, tumor recurrence, and metas-tasis29. Recent evidences indicated that epigenetic modificationsregulate progression of EMT and cancer development30. Snailrecruits chromatin modifiers, such as lysine-specific demethylase1 (LSD1)31 and Suv39H1 histone methyltransferases32, and trig-gers both transient and long-lasting chromatin changes of CDH1promoter33. However, the precise role of RNA epigenetic factorson progression of EMT has not been reported yet.

This study revealed a significant increase of m6A levels incancer cells undergoing EMT. However, METTL3-deleted cellswere less inducible for EMT. The m6A-seq and m6A-RIP-qPCRresults showed that EMT induced elevation of m6A in CDS ofSNAI1, the key inducer of EMT, and promoted YTHDF1-mediated Snail translation. Our study revealed that m6A regu-lated the progression of EMT and translation of Snail.

ResultsEMT in cancer cells is regulated by m6A levels of mRNAs.Although EMT can be induced by various extracellular ligands,TGF-β has been considered as the major inducer of this trans-differentiation process of cancer cells34. We treated HeLa andHepG2 cells with 10 ng/ml TGF-β for 3 days. Both TGF-β treatedHeLa and HepG2 cells became scattered and adopted thefibroblast-like morphology described for mesenchymal cells(Supplementary Fig. 1A). TGF-β treatments significantlyincreased wound healing (Supplementary Fig. 1B) and in vitroinvasion ability (Supplementary Fig. 1C) of both HeLa andHepG2 cells. Moreover, upregulation of FN1 (fibronectin) andMMP2 mRNA and downregulation of CDH1 (E-Cad) mRNAwere observed by qRT-PCR (Supplementary Fig. 1D). TheseTGF-β-induced changes in expression of EMT markers werefurther confirmed by western blot analysis (SupplementaryFig. 1E). All these data indicated that cancer cells, treated withTGF-β, were undergoing EMT processes.

We then investigated the variations of m6A levels in mRNAs ofcancer cells undergoing EMT. By using LC-MS/MS, we identifiedthat the m6A levels of mRNAs isolated from HeLa and HepG2cells, treated with TGF-β, were statistically (p < 0.05, t test) moreabundant than that of their corresponding control cells (Fig. 1a).The m6A/A levels of mRNA from HeLa and HepG2 cellsundergoing EMT increased 20.0% and 14.9%, respectively. Thiswas further confirmed by the results obtained in dot-blot analysis(Supplementary Fig. 2A). Similarly, LC/MS/MS showed that m6Alevels of mRNAs isolated from Huh7 and A549 cells, treated withTGF-β, were statistically (p < 0.05, t test) more abundant thanthat of their corresponding control cells (Supplementary Fig. 2B).Collectively, these data showed that cancer cells undergoing EMTincreased m6A levels of mRNAs.

To characterize the roles of m6A in EMT process, we usedMettl3Mut/− HeLa cells (Supplementary Fig. 2C) generated in ourprevious study35 by using the CRISPR/Cas9 editing systemaccording to the published protocol35,36. The results showed thatMettl3Mut/− cells had significantly lower levels of m6A than wild-type cells (Supplementary Fig. 2D), which also confirmed theroles of METTL3 as m6A “writer” of mRNA. We evaluated theEMT-related characteristics of Mettl3Mut/− cells. The resultsshowed that both wound healing (Fig. 1b) and in vitro invasionabilities (Fig. 1c) ofMettl3Mut/− HeLa cells were suppressed whencompared with wild-type cells. Similarly, sh-Mettl3 or si-Mettl3-mediated knockdown of METTL3 suppressed the in vitroinvasion of Huh7 and HepG2 cells, respectively (SupplementaryFig. 2E). The mRNA and protein levels of both MMP2 and FNwere downregulated, while E-Cad mRNA and protein levels wereupregulated in Mettl3Mut/− HeLa cells (Fig. 1d, e). In addition,western blot analysis confirmed that METTL3 knockdowndecreased MMP2 and FN, while increased E-Cad, in both Huh7and HepG2 cells (Supplementary Fig. 2F, H). These datasuggested that deletion of METTL3 can suppress the EMT ofcancer cells.

To verify the role of m6A levels in EMT, we overexpressedALKBH5, a key m6A demethylase of mammalian mRNA37, inHeLa cells. Our data showed that overexpression of ALKBH5decreased mRNA of FN and MMP2 in HeLa cells (SupplementaryFig. 2G). Western blot analysis showed that overexpression ofALKBH5 increased E-Cad while it decreased MMP2 and FNin HeLa cells (Fig. 1f). Furthermore, TGF-β-induced down-regulation of E-Cad and upregulation of FN were rescued inMettl3Mut/− HeLa (Fig. 1g) and Mettl3 knockdown Huh7(Supplementary Fig. 2H) cells. The clinical roles of METTL3were further investigated. TCGA data showed that expression ofMETTL3 in liver cancers was significantly higher than theirmatched adjacent normal tissues (Fig. 1h). Moreover, the

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expression of METTL3 was negatively correlated with the CDH1mRNA in 364 liver cancer patients (Fig. 1i). It has been reportedthat TGF-β can induce the mRNA methylation in humanembryonic stem cells (hESCs) via the interaction between Smad2/3 and the METTL3–METTL14–WTAP complex38. LC/MS/MSshowed that the Smad2/3 inhibitor SB431542 (SB) can blockTGF-β-induced upregulation of m6A in HeLa cells (Fig. 1j).Together, our data indicated that the total m6A levels in mRNAmay regulate EMT progression of cancer cells.

Variations of m6A-regulated genes in cells undergoing EMT.To investigate the variations of m6A modification in specificgenes, we mapped m6A methylomes of HeLa cells undergoingEMT (EMT cells) by m6A sequencing (m6A-seq), with inde-pendent biological replicates. Principal component analysis(PCA) showed that two repeats of each sample clustered together,suggesting the good reproducibility among the two replicates ofeach group (Supplementary Fig. 3A). GGAC motif was highlyenriched within m6A sites in both control and EMT cells

(Fig. 2a). m6A peaks were especially abundant in the vicinity ofstart and stop codons (Fig. 2b).

m6A-seq analysis identified 11,687 and 11,785 m6A peaks from6836 to 7230 m6A-modified transcripts in control and EMT cells,respectively. In EMT cells, 4347 new m6A peaks and 4249disappearing peaks were observed, while the remaining 7438peaks were found in both control and EMT cells (SupplementaryFig. 3B). For m6A-regulated genes, m6A-seq identified 569 newmodifying genes and 472 genes loss of m6A modification inEMT cells, while the other 2357 genes were found in both controland EMT cells (Supplementary Fig. 3C). The results indicatedthat EMT cells unique peaks and genes were expected to containgenuine targets. We further analyzed the total m6A distributionpatterns of mRNAs according to the m6A-seq results. Similarpatterns of the total m6A distribution in control and EMT cellswere observed (Fig. 2c), showing that m6A peaks were mainlyenriched in exon regions. Furthermore, we investigated thedistribution of unique EMT peaks. Compared with the 4249missing peaks, which displayed identical distribution to total

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Fig. 1 EMT in cancer cells is regulated by m6A levels of mRNAs. a HeLa and HepG2 cells were treated with or without 10 ng/ml TGF-β for 3 days, the m6A/A ratio of the total mRNA were determined by LC–MS/MS. b Wound healing of wild-type (control) or Mettl3Mut/− cells was recorded (left) andquantitatively analyzed (right). c Wild-type orMettl3Mut/− cells were allowed to invade for 24 h and tested by CytoSelect™ 24-well Cell Invasion assay kits(8 µm, colorimetric format); d, e mRNA (d) and protein (e) expressions of MMP2, FN, and E-Cad in wild-type and Mettl3Mut/− HeLa cells were measuredby qRT-PCR and western blot analysis, respectively. f HeLa cells were transfected with pcDNA/ALKBH5 or a vector control for 48 h, protein expressionwas determined by western blot analysis (left) and quantitatively analyzed (right). g Wild-type or Mettl3Mut/− cells were treated with or without 10 ng/mlTGF-β for 3 days, protein expression was determined by western blot analysis (left) and quantitatively analyzed (right). h The expression of METTL3 inliver cancer and its matched adjacent normal tissues of 50 patients from TCGA database. i Correlation between METTL3 and CDH1 in liver cancer patients(n= 364) from TCGA database. j HeLa cells were pretreated with or without Smad2/3 inhibitor SB431542 (10 μM) and then further treated with 10 ng/mlTGF-β for 3 days, the m6A/A ratio of the total mRNA were determined by LC–MS/MS. Data are presented as means ± SD from three independentexperiments. *p < 0.05, **p < 0.01, NS, no significant, by Student’s t test. Red bar= 200 μm

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peaks, 4347 unique peaks in EMT cells showed a distinct pattern,in which a relative increase of m6A deposit appeared in both 5′and 3′ UTR (Supplementary Fig. 3D).

Comparing the control to EMT cells, a total of 128 genes,showing a 1.5-fold m6A change in the expression level, wereidentified. Gene ontology (GO) enrichment analysis of these 128genes indicated that a handful of genes were associated to theformation of the adherens junction and actin cytoskeleton incancer cells undergoing EMT (Fig. 2d). Collectively, the m6A-seqdata revealed that m6A modifications occur on a handful ofgenes related to the cell junction, adhesion, and migrationduring EMT.

Snail is involved in m6A-regulated EMT in cancer cells. Tocharacterize potential targets involved in m6A-regulated EMT ofcancer cells, we identified four genes overlapping between 84EMT-related genes with key functions (listed in SupplementaryTable 1) and the 61 m6A-regulated genes (greater than 2.0-foldm6A change) during EMT (Fig. 3a). Among the four identifiedgenes, Snail has been suggested as an important transcriptionfactor controlling EMT39. Our data confirmed that SNAI1mRNA was modified by m6A and showed significant enrichmentof m6A in its CDS and 3′UTR regions during EMT progression(Fig. 3b, c), which is consistent with the published reports usingzebrafish embryonic cells5 and human HEK293T cells40,41. Byapplying m6A RNA-immunoprecipitation (RIP) qPCR, we

verified a 12-fold m6A antibody enriched SNAI1 mRNA(Fig. 3c). During EMT, m6A levels of SNAI1 mRNA were sig-nificantly increased, and relative m6A enrichment fold inEMT cells was about 2.3-fold greater than in control cells(Fig. 3c). However, neither m6A pull down enrichment nor m6Aupregulation during EMT was observed for the negative candi-date gene ZEB1, which is another transcription factor of EMT aswell (Supplementary Fig. 4A, B).

Next, we investigated protein expression of Snail in Mettl3Mut/−

and ALKBH5 overexpressing HeLa cells. We observed a decreaseof Snail protein levels in HeLa (Fig. 3d) and HepG2 (Supple-mentary Fig. 4C) cells without changes in ZEB1 expression, inboth contexts of deletion of METTL3 and overexpression ofALKBH5. To confirm the roles of m6A on Snail expression, wetreated wild-type and Mettl3Mut/− HeLa cells with TGF-β. Theresults showed that TGF-β-induced expression of Snail wasreduced in Mettl3Mut/− cells (Supplementary Figure 4D), whichindicated that m6A participated in TGF-β-induced expression ofSnail in cancer cells. Although the promotion roles of Snail inEMT and cancer progression have been well illustrated27,42,43, wefurther investigated the roles of Snail in m6A-triggered EMT ofcancer cells. Overexpression of Snail attenuated the suppressionof wound healing via METTL3 deletion in HeLa cells (Fig. 3e).Consistently, overexpression of Snail antagonized the down-regulation of FN and upregulation of E-Cad occurred inMettl3Mut/− cells (Fig. 3f; Supplementary Fig. 4E). These data

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Fig. 2 Variations of m6A-regulated genes in cells undergoing EMT. a Predominant consensus motif GGAC was detected in both the control and EMT cellsin m6A-seq. b Density distribution of m6A peaks across mRNA transcripts. Regions of the 5′ untranslated region (5′UTR), coding region (CDS), and 3′untranslated region (3′UTR) were split into 100 segments, then percentages of m6A peaks that fall within each segment were determined. c Proportion ofm6A peak distribution in the 5′UTR, start codon, CDS, stop codon or 3′UTR region across the entire set of mRNA transcripts. d A cluster profiler identifiedthe enriched gene ontology processes of 128 genes, which showed 1.5-fold m6A expression upregulation in EMT cells compared with control cells

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suggested that Snail is involved in m6A-regulated EMT incancer cells.

m6A triggers translation of Snail mRNA in cancer cells. Themechanisms regulating Snail expression through m6A methyla-tion were further investigated. First, we tested the expression ofprecursor (pre-) and mature (mat-) mRNA of Snail. Surprisingly,expression of both precursor (pre-) and mature (mat-) mRNA ofSnail in Mettl3Mut/− cells were significantly enhanced (Fig. 4a).However, there was no difference on promoter activity in wild-type and Mettl3Mut/− cells (Supplementary Fig. 5A), suggestingthat transcription of Snail was not affected by m6A. By separatingthe nuclear and cytoplasmic RNAs (Supplementary Fig. 5B), wefound that there was no difference on subcellular localization ofSnail mRNA between wild-type and Mettl3Mut/− cells (Fig. 4b).We treated wild-type and Mettl3Mut/− HeLa cells with Act-D toblock transcription. The results showed that half-life of bothprecursor (Fig. 4c) and mature (Fig. 4d) mRNA in wild-type cellswere significantly shorter than in Mettl3Mut/− cells. It indicatedthat m6A modification may trigger the splicing of precursormRNA and the degradation of mature mRNA of Snail in cancercells. This was confirmed by the results indicating that Snail was atarget of YTHDF2 (Supplementary Fig. 5C), which is responsible

for m6A-mediated mRNA destabilization of transcripts6. Over-expression of YTHDF2 can decrease the expression and stabilityof mature Snail mRNA in HeLa cells (Supplementary Fig. 5D).

We observed increasing of mature SNAI1 mRNA levels whiledecreasing of protein levels of Snail in Mettl3Mut/− cells, then wehypothesized that downregulation of Snail protein in Mettl3Mut/−

cells may be due to a difference on protein stability or translationefficiency. To verify this, both wild-type and Mettl3Mut/− HeLacells were treated with protein translation inhibitor cycloheximide(CHX). The results from western blot analysis showed that half-lives of Snail proteins were similar between wild-type andMettl3Mut/− HeLa cells (Fig. 4e), suggesting that m6A-inducedSnail expression was not related to protein stability.

Furthermore, Mettl3Mut/− HeLa cells were pretreated withMG132 to inhibit proteasome activity or CHX to blocktranslation and then treated with TGF-β. The data showed thatin the presence of CHX, but not MG-132, attenuated TGF-β-induced Snail expression in Mettl3Mut/− HeLa cells (Fig. 4f). Weconstructed the pmirGLO-Snail luciferase reporter by ligatingSnail CDS to the mutiple cloning site (MCS) site. The dual-luciferase assay showed that translation efficiency of Snail inMettl3Mut/− HeLa cells was significantly lower than that in HeLaWT cells (Fig. 4g). It indicated that m6A-induced Snail expressionwas related to regulation of translation.

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Fig. 3 Snail is involved in m6A-regulated EMT in cancer cells. a Overlapping of 2.0-fold m6A expression changes in EMT cells and EMT-related functionalgenes. b m6A peaks were enriched in CDS and 3′UTRs of SNAI1 genes from m6A RIP-seq data. Squares marked increases of m6A peaks in cancer cellsundergoing EMT; c m6A RIP-qPCR analysis of SNAI1 mRNA in the control and EMT undergoing HeLa cells. d Protein expression of Snail in Mettl3Mut/− orAKLBH5 transfected (24 h) HeLa cells and the control. e The wound healing of wild-type or Mettl3Mut/− HeLa cells transfected with or without pcDNA/Snail for 48 h were recorded (left) and quantitatively analyzed (right). fWild-type orMettl3Mut/− HeLa cells were transfected with or without pcDNA/Snailfor 48 h, expression of Snail, FN and E-Cad were measured by western blot analysis. Data are presented as means ± SD from three independentexperiments. *p < 0.05, NS, no significant, by Student’s t test. Red bar= 200μm

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Next, we separated the RNA fractions: non-translating fraction(< 40 S), translation initiation fraction (including 40S ribosomes,60S ribosomes, 80S monosomes, and < 80S) as well as translationactive polysomes (> 80S) from wild-type and Mettl3Mut/− HeLacells by ribosome profiling (Fig. 4h). Polysome profilingsupported the decreases of 80S monosome assembly andpolysomes in Mettl3Mut/− cells (Fig. 4h). qRT-PCR showed thatSnail mRNA in translation-active polysomes (> 80S) of Mettl3-Mut/− cells were significantly lower than in wild-type cells(Fig. 4i). In EMT cells, ribosome profiling was similar to that inwild-type cells (Supplementary Fig. 5E). Remarkably, an increaseof SNAI1 mRNA in polysomal fractions (> 80S) isolated fromcancer cells undergoing EMT was observed (Fig. 4j), indicatingthat translation of SNAI1 mRNA was improved during EMTprocess. To further confirm our observations, we targeted cap-independent/dependent translation initiation to investigatewhether it was involved in m6A methylation-regulated SNAI1translation. Both Mettl3Mut/− and wild-type cells were treated

with rapamycin, which inhibits cap-dependent but not cap-independent translation44. However, no obvious differences ofSnail1 levels were observed between wild-type and Mettl3Mut/−

cells (Supplementary Fig. 5F). Maintenance of Snail1 expressionin the presence of rapamycin was not due to an increase of Snail1protein stability, as co-treatment of wild-type cells with proteinsynthesis inhibitor CHX resulted in a rapid disappearance ofSnail1 (Supplementary Fig. 5F), suggesting that Snail expressionwas regulated through cap-independent translation45.

m6A-methylated CDS regulates translation of Snail. m6A-RIP-seq data showed that m6A peaks in CDS regions ranged fromchr20:48,600,490 to 48,600,630, while in 3′UTR regions rangedfrom chr20: 48,604,770 to 48,605,060 (Fig. 3b). One GGAC motifin CDS and two in 3′UTR of Snail mRNA were identified, asshown in Fig. 5a, which was consistent with positions andamounts of peaks identified by m6A RIP-seq. To characterizem6A methylation in Snail mRNA, we used m6A antibody to

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Fig. 4 m6A triggers translation of Snail mRNA in cancer cells. a Precursor and mature mRNA of SNAI1 in wild-type (WT) and Mettl3Mut/− cells. b Therelative levels of the nuclear versus cytoplasmic SNAI1 mRNA in wild-type (WT) or Mettl3Mut/− cells. c, d Wild-type (WT) or Mettl3Mut/− cells werepretreated with Act-D for 90min, then precursor (c) or mature (d) SNAI1 mRNA were analyzed at indicated times. e Wild-type (WT) or Mettl3Mut/− cellswere treated with CHX for the indicated times, and protein expression of Snail was analyzed by western blot analysis (left) and quantitatively analyzed(right); fMettl3Mut/− cells were pretreated with CHX or MG-132 for 6 h and then further treated with or without 10 ng/ml TGF-β for 48 h, the expression ofSnail was detected by western blot analysis (left) and quantitatively analyzed (right). g Wild-type (WT) or Mettl3Mut/− cells were transfected withpmirGLO-Snail reporter for 24 h, and the translation efficiency of Snail is defined as the quotient of reporter protein production (F-luc/R-luc) divided bymRNA abundance7. h Polysome profiling of wild-type (WT) orMettl3Mut/− HeLa cells were analyzed. i Analysis of Snail mRNA in non-ribosome portion (<40S), 40S, 60S, 80S, and polysome for the Mettl3Mut/− cells compared to control cells. j Analysis of Snail mRNA in non-ribosome portion (< 40S), 40S,60S, 80S, and polysome for EMT undergoing HeLa cells compared with control cells. Data are presented as means ± SD from three independentexperiments. *p < 0.05, NS, no significant, by Student’s t test

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immunoprecipitated fragmented RNA isolated from HeLa cells(Supplementary Fig. 6A). qRT-PCR showed that distributions ofm6A methylation in Snail mRNA, from high to low, were: CDS, 3′UTR and 5′UTR, in wild-type HeLa cells (SupplementaryFig. 6B). Consistently, the m6A enrichment of Snail CDS wassignificantly downregulated in Mettl3Mut/− cells (SupplementaryFig. 6B), suggesting that m6A methylation in CDS regions mightbe more dynamic than those in 3′UTR.

To study the potential roles of m6A methylation on the SNAI13′UTR region, we generated luciferase reporters containing a

firefly luciferase, followed by the wild-type SNAI1 3′UTR, mutant1 or mutant 2 3′UTR (GGAC to GGCC, Fig. 5b). The SNAI1 3′UTR-reporter luciferase assay showed that Snail 3′UTR fromWT-3′UTR, mut1–3′UTR, and mut2–3′UTR had no translationaldifference between wild-type and Mettl3Mut/− cells (Fig. 5c).Consistently, co-transfection of ALKBH5 did not alter expressionlevels of WT-3′UTR, mut1–3′UTR, or mut2–3′UTR in HeLa cells(Supplementary Fig. 6C). This suggested that m6A methylationon 3′UTR might not be involved in the m6A modification-regulated Snail expression.

chr20:48,600,490

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Fig. 5 m6A methylated CDS regulates translation of Snail. a Schematic representation of positions of m6A motifs within Snail mRNA. b Schematicrepresentation of mutated (GGAC to GGCC) 3’UTR of pmirGLO vector to investigate the m6A roles on Snail expression. c pmirGLO-3′UTR or pmirGLO-3′UTR-Mut1/2 reporter was transfected into wild-type or Mettl3Mut/− HeLa cells for 24 h. d pcDNA-Snail-CDS-WT, pcDNA-Snail-mut1/2, or pcDNA/ZEB1were transfected into HeLa cells for 24 h. Protein expression was measured by western blot analysis (left) and quantitatively analyzed (right). e Schematicrepresentation of mutation in CDS to investigate the m6A roles on Snail expression. f After transfected with pcDNA-Snail-CDS-WT or pcDNA-Snail-mut1/2, HeLa cells were further treated with or without 10 ng/ml TGF-β for 48 h. The expression of Snail was measured by western blot analysis (left) andquantitatively analyzed (right). g YTHDF1 RIP-qPCR analysis of SNAI1 mRNA in wild-type or Mettl3Mut/− HeLa cells. h Binding of YTHDF1 with the CDS or3’UTR in wild-type or Mettl3Mut/− cells were analyzed by YTHDF1 RIP-qPCR using fragmented RNA. i After pre-transfected with siNC or si-YTHDF1 for 12h, HeLa cells were further treated with or without 10 ng/ml TGF-β for 48 h. Protein expression was measured by western blot analysis (left) andquantitatively analyzed (right). j eEF-1 and eEF-2 RIP-qPCR analysis of SNAI1 mRNA in wild-type orMettl3Mut/− HeLa cells. k Binding between YTHDF1 witheEF-1 or eEF-2 in wild-type or Mettl3Mut/− HeLa cells was checked by immunoprecipitation (left) and quantitatively analyzed (right). Data are presented asmeans ± SD from three independent experiments. *p < 0.05, NS, no significant, by Student’s t test

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We then investigated whether m6A methylation in CDS canregulate the expression of Snail. First, we generated an expressionconstruct containing Snail CDS alone. We co-transfected SnailCDS with pcDNA/ZEB1, expression of which was not regulatedby m6A methylation, to normalize the transfection efficiency. Theresults showed that expression of Snail in Mettl3Mut/− cellsremarkably decreased, while expression of ZEB1 was not affected,indicating that m6A methylation in CDS was critically involved inm6A-mediated Snail expression (Fig. 5d). Second, we mutated thepotential m6A motif GGAC to GGCC (CDS-mut1). Accordingly,the nearby GGAA was mutated to GGCA for comparison (CDS-mut2, both mutants were located at chr20:48,600,490–48,600,630, Fig. 5e). Our data showed that Snail levelwas partially recruited in Mettl3Mut/− cells overexpressingpcDNA-Snail-CDS-mut1, but not CDS-mut2 (Fig. 5d). HeLacells transfected with pcDNA-Snail-CDS-WT or pcDNA-Snail-CDS-mut1/mut2 were further treated with TGF-β. Western blotresults showed that TGF-β-induced Snail expression of pcDNA-Snail-CDS-mut1 was lower than pcDNA-Snail-CDS-WT orpcDNA-Snail-CDS-mut2 (Fig. 5f). Together, our data indicatedthat m6A in SNAI1 CDS was the main location for expressionregulation.

YTHDF1, acting as a m6A “reader”, recognizes m6Amethylated mRNA and promotes the translation of its targets7.In order to verify whether YTHDF1 participates in m6Amethylation of SNAI1 mRNA, RIP-qPCR was used to investigatethe interaction between SNAI1 mRNA and YTHDF1. Our datashowed that YTHDF1 enriched in SNAI1 mRNA remarkably,while this relative enrichment (IP vs. input) was significantlysuppressed in Mettl3Mut/− cells (Fig. 5g). The results of RIP-PCRfurther revealed that YTHDF1 preferred to interact with the CDSregion of Snail mRNA rather than 3′UTR region (Fig. 5h). InMettl3Mut/− cells, binding between YTHDF1 with CDS, but not 3′UTR, of SNAI1 mRNA, was significantly decreased in compar-ison with HeLa cells. To confirm the roles of YTHDF1 in m6A-regulated Snail expression, we knocked down expression ofYTHDF1 in HeLa cells (Supplementary Fig. 6D). Our datashowed that si-YTHDF1 attenuated TGF-β-induced expression ofSnail in HeLa cells (Fig. 5i). Similarly, overexpression of YTHDF1increased protein expression of Snail in both HeLa and HepG2cells, while it had no effect on the expression of ZEB1(Supplementary Fig. 6E).

Since the translation elongation in eukaryotes is carried out withtwo elongation factors: eEF-1 and eEF-246, we measured thevariation of eEF-1- and eEF-2-binding Snail mRNA in Mettl3Mut/−

HeLa cells. Our data showed that the binding between Snail mRNAand eEF-2, but not eEF-1, in Mettl3Mut/− HeLa cells wassignificantly less than that in HeLa cells (Fig. 5j). Furthermore,co-IP analysis indicated that the binding between YTHDF1 andeEF-2, while not eEF-1, was significantly decreased in Mettl3Mut/−

HeLa cells compared with that in WT HeLa cells (Fig. 5k). This datasuggested that YTHDF1 and eEF-2 are likely responsible for them6A-induced translation elongation of Snail mRNA in cancer cells.

m6A regulates in vivo cancer progression. We used three animalcohorts to study the potential effects of m6A methylation oncancer cells in vivo metastasis. First, both sh-control and sh-Mettl3 Huh7 cells were injected subcutaneously into nude femalemice. Mice were killed when the tumor volumes were about 100mm3 for each group. IHC results showed that METTL3 depletionled to a lower level of Snail, FN, and Vim in xenograft tumortissues (Fig. 6a), which suggested that the knockdown of METTL3can decrease the EMT potential and Snail expression of Huh7cells when the tumor volumes were comparable. To furtherdetermine the impacts of m6A methylation on in vivo metastasis,

both sh-control and sh-Mettl3 Huh7 cells were injected into theBALB/c nude mice, respectively, by tail vein injection to analyzelung colonization. Eight weeks after injection, the experiment wasterminated and lungs were analyzed to report the presence ofmetastatic tumors. As shown in Fig. 6b, the number of lungtumors derived from METTL3 knockdown Huh7 cells was sig-nificantly decreased compared with control cells, suggesting thatMETTL3 deficiency suppressed tumor metastasis in vivo. Toinvestigate whether Snail was involved in m6A regulated in vivometastasis, HeLa WT, Mettl3Mut/− HeLa, HeLaSnail, and Mettl3-Mut/− HeLaSnail stable cells were injected into nude mice viaintravenous tail vein. In vivo data showed that the number of lungtumors derived from Mettl3Mut/− HeLa cells was significantlydecreased compared with HeLa WT cells; however, over-expression of Snail can attenuate the suppression effect ofMettl3Mut/− HeLa cells on in vivo lung colonization (Fig. 6c). Itsuggested that Snail was involved in the in vivo metastasis effectof METTL3.

Our data suggested that knockdown of METTL3 can decreasethe expression of Snail and the in vitro invasion and EMT ofHepG2 and Huh7 cells (Supplementary Figs. 2E, 2F, 4C), we thenquestioned the possibility of a link between m6A methylation andclinical development of liver cancer. Increased expression ofMETTL3 (Fig. 1h) and YTHDF1 (Fig. 6d) was observed in livercancer tissues as compared with matched adjacent normal tissues.Significant rising expression levels of YTHDF1 from T1 toT4 stage of liver cancer tissues were observed (Fig. 6e), implyingan increasing tendency of YTHDF1 expression during malignanttransformation. In addition, expression of YTHDF1 waspositively correlated with the SNAI1 mRNA in liver (Fig. 6f)cancer patients. Rising expression levels of METTL3 from thenormal liver to cirrhosis to liver cancer tissues were observed(Fig. 6g). Considering that Mettl3 induced Snail via translation,we checked their protein expression in 100 cases of liver cancertissues by use of tissue microarray. IHC analysis confirmed thatthe expression of Mettl3 was significantly (p < 0.05, t test)positively associated with that of Snail in the measured 100 livercancers (Fig. 6h). Using the online bioinformatics toolKaplan–Meier plotter47, we found that liver cancer patients withincreased expression of METTL3 (Fig. 6i), YTHDF1 (Fig. 6j), andSnail (Fig. 6k) showed reduced overall survival (OS).

DiscussionIncreasing evidences suggested that m6A mRNA modificationparticipates in a number of biological functions and in progres-sion of cancer cells15,20,21. In this study, we demonstrated thatm6A can regulate the progression of EMT and expression ofSnail. In brief, m6A levels in mRNA of cancer cells undergoingEMT increased significantly compared with control cells. Dele-tion of METTL3 or overexpression of ALKBH5 suppressed thein vitro migration, invasion, and EMT of cancer cells. m6A-seqand GO analysis showed that genes related to cell migration andadherens junctions are dynamically regulated by m6A duringEMT. Particularly, Snail, a key transcription factor of EMT, wasm6A methylated in its CDS and 3′UTR regions. Gain and loss offunction studies revealed that m6A in CDS of Snail can trigger itstranslation elongation via interaction with YTHDF1 and eEF-2.Deletion of METTL3 suppressed the in vivo metastasis of cancercells, which could be attenuated by the overexpression of Snail.Consistently, clinical data showed that tumor tissues containedhigher METTL3 and YTHDF1 levels than adjacent normal tis-sues, which induced worse prognosis for liver cancer patients.

The roles of mRNA modification in controlling the cancerprogression have just begun to be studied. FTO, the first char-acterized m6A demethylase, has been reported to enhance

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1.0

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Fig. 6 m6A methylation regulates in vivo cancer progression. a IHC (Snail, FN, and Vim)-stained paraffin-embedded sections obtained from sh-control andsh-Mettl3 Huh7 xenografts when the tumor volumes were about 100mm3 for each group. b The sh-control and sh-Mettl3 Huh7 cells were injected intothe nude mice by tail vein injection. Representative images of metastatic lung tumors and the H&E staining results were shown (left), and the number oflung tumors was quantitatively analyzed (right). c HeLa WT,Mettl3Mut/− HeLa, HeLaSnail, andMettl3Mut/− HeLaSnail stable cells were injected into the nudemice by tail vein injection. Representative images of metastatic lung tumors and the H&E staining results were shown (left), and the number of lung tumorswas quantitatively analyzed (right). d Expression of YTHDF1 in paired human liver (n= 26) cancer tissues and adjacent normal mucosa tissues from TCGAdatabase. e YTHDF1 expression in liver cancers of T1 (n= 153), T2 (n= 77), T3 (n= 65), and T4 (n= 13) stages from TCGA database. f Correlationbetween YTHDF1 and SNAI1 in liver cancer patients (n= 80) from TCGA database. g METTL3 expression in HCC tumor tissues (n= 35), liver cirrhosistissues (n= 13), and normal liver tissues (n= 10) from Oncomine database (Wurmbach liver cancers). h Correlation between Mettl3 and Snail protein inliver cancer patients (100) by use of tissue microarray (CS03–01–008, Alenabio, Xian, Shanxi, China). i, j, k Kaplan–Meier survival curves of OS based onMETTL3 (i), YTHDF1 (j), or SNAI1 (k) mRNA expression in HCC patients. The log-rank test was used to compare differences between two groups. Dataare presented as means ± SD from three independent experiments. *p < 0.05, **p < 0.01, NS, no significant, by one-way ANOVA with Bonferroni test. Redbar= 200 μm

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leukemic oncogene-mediated cell transformation and leukemo-genesis via regulation of expression of its targets, such as ASB2and RARA, by reducing their m6A levels20. Another m6Ademethylase ALKBH5 can also trigger tumorigenicity of gliomastem-like cells (GSC) by sustaining FOXM1 expression and cellproliferation23. METTL3, which is the methyltransferase ofmRNA m6A, exhibits several functions in cancer cells, such as:maintaining myeloid leukemia48, promoting liver cancer pro-gression49, playing essential role in GSC maintenance andradioresistance50, positively regulating proliferation of hemato-poietic stem/progenitor cells (HSPCs)51, and triggering thetranslation in cancer cells52. Contradictorily, METTL3 acts as atumor suppressor in renal cell carcinoma53. METTL14, whichforms a stable heterodimer core complex with METTL3 tofunction in cellular m6A deposition, can suppress metastaticpotential by modulating the primary microRNA 126 processingin an m6A-dependent manner54. In this study, we provided thecausative link between mRNA m6A methylation and EMT. Wefound that levels of m6A in mRNA increased in cancer cellsundergoing EMT, while targeted deletion of METTL3 impairedthe migration and EMT of cancer cells. GO analysis revealed thatmodulation of mRNA m6A levels impacted multiple aspects ofcancer progression, including migration, self-renewal, andtumorigenesis. Our results described the roles of m6A andMETTL3 in cancer progression, and also created the possibility todevelop therapeutic strategies against cancer metastasis by tar-geting m6A modification and its related targets.

m6A modification modulates all stages in the life cycle of RNA,such as RNA processing, nuclear export, and translation mod-ulation13,55. First, splicing regulators and a m6A “reader”HNRNPC could affect the abundance as well as alternative spli-cing of target mRNAs in a m6A switch-regulated manner56.Second, m6A was found to promote RNA export from thenucleus to cytoplasm, which was evidenced by the fact thatMETTL3 knockdown delayed mRNA export57, while inhibitionof ALKBH5 enhanced mRNA export to the cytoplasm37. Third,m6A modification can trigger mRNA degradation by promotingdeadenylation of RNAs through the first characterized m6A“reader” protein YTHDF26. Half-life of Snail mRNA in Mettl3-Mut/− cells was significantly longer than in control cells, whichindicated a negative impact of m6A on mRNA stability. FTOmediated repression of ASB2 and RARA stability20 and ALKBH5modulated FOXM1 expression via m6A modifications in 3′UTRof targets23. However, our luciferase/mutation assays showed thatm6A in 3′UTR had no effect on Snail protein expression, sug-gesting that m6A regulation on mRNA stability is more compli-cated than expected and could be related to target or locationspecificity.

We revealed that m6A triggered translation of Snail mRNAthrough YTHDF1 and eEF-2 binding with methylated mRNA.YTHDF1 interacts with EIF3 to promote the rate-limiting step oftranslation for m6A-modified mRNAs by increasing their asso-ciation with polysomes7. Our data showed that YTHDF1 prefersto bind with the CDS of Snail mRNA and then triggers itsassociation with polysome in cancer cells undergoing EMT. Themutation of GGAC motif can partially rescue m6A-regulatedtranslation. Similarly, in METTL3 deletion acute myeloid leuke-mia (AML) cells, c-MYC, BCL2, and PTEN protein expressionwere reduced despite a 2–5 log2-fold increase in mRNA expres-sion51. A specific shift of SP1 and SP2 transcripts toward lower-molecular-weight polysomes has been observed in METTL3depletion cancer cells48. Alternatively, METTL3 can localize topolysomes and functionally associate with translation initiationfactors to promote translation in an YTHDF1-independentmanner52. In Mettl3Mut/− cells, the binding between SnailmRNA and eEF-2 or between YTHDF1 and eEF-2 was decreased.

eEF-2 is essential for the GTP-dependent translocation of theribosome along mRNA58. Our results also showed that m6A-regulated Snail translation is cap-independent, which was evi-denced by the fact that rapamycin had no effect on Snailexpression in wild-type and Mettl3Mut/− cells. However, todeterminate if promoter-bound METTL3 augments translation48

will need further studies.In summary, we provide compelling in vitro and in vivo evi-

dences demonstrating that m6A can regulate the progression ofEMT, cancer metastasis, and Snail translation. Our presentresearch provided the mechanism of regulation of cancer pro-gression by modulation of EMT through RNA m6A modification.It should be noted that the effects of Mettl3-modified SnailmRNA might be heterogeneous in different tumor models, fur-ther, Snail is not always associated with the induction ofmesenchymal features59,60 and EMT is not necessary for thegeneration of metastases in some cancers59,61. The roles of m6Ain cancer metastasis and Snail expression in other types of cancerneed further investigation.

MethodsCell culture, treatments, and transfection. Human cancer HeLa, HepG2, Huh7,and A549 cells were purchased from the American Type Culture Collection(ATCC, Manassas, VA, USA) and maintained in our lab with Dulbecco’s modifiedEagle medium (DMEM, GIBCO, Carlsbad, CA, USA) with 10% fetal bovine serum(FBS) and 1% penicillin/streptomycin (Invitrogen). Cells were routinely tested formycoplasma contamination using MycoAlert Mycoplasma Detection Kit. Toestablish the model of cancer cells undergoing EMT, cells were treated with 10 ng/ml TGF-β for 3 days. To generate Mettl3Mut/− HeLa cells, the CRISPR-cas9 editingsystem was used according to the published protocol36. The sh-Control and sh-Mettl3 Huh7 cells were kindly provided by Prof Jun Cui at Sun Yat-sen University.To generate Snail stable overexpression cells, the wild-type and Mettl3Mut/− HeLacells were transfected with pcDNA/Snail or vector control and via liposome-mediated transfection. The transfected cells were selected with G418 (700 μg/ml)for 2 weeks. The survived cells were picked out and seeded into 96-well plate forformation of cell clones and further expansion. The expanded monoclonal cellpopulations were named as HeLa WT, Mettl3Mut/− HeLa, HeLaSnail, and Mettl3-Mut/− HeLaSnail cells, respectively. The expression of Snail and Mettl3 was checkedby western blot analysis. For overexpression of METTL3, Snail, ALKBH5,YTHDF1, or YTHDF2, cells were transfected with plasmids by lipofectamine 2000according to the manufacturers’ instructions. For YTHDF1 or METTL3 knock-down, three synthesized duplex RNAi oligos targeting human YTHDF1 orMETTL3 mRNA sequences from Sigma were used. The most efficient one wasselected for following studies. A scrambled duplex RNA oligo (5′-UUCUCC-GAACGUGUCACGU) was used as a RNA negative control (si-NC). Lipofecta-mine RNAiMAX (Invitrogen) was used for siRNA transfection. Workingconcentration for siRNA was 50 nM.

Wound-healing and in vitro invasion assay. For wound-healing assay, cells wereseeded and cultured until a 90% confluent monolayer was formed. Cells were thenscratched by a sterile pipette tip and treated as indicated in the text in the FBS-freemedium. Cell migration distances into the scratched area were measured in 10randomly chosen fields under a microscope. Transwell assay was conducted usingCytoSelect™ 24-well Cell Invasion assay kits. Briefly, polycarbonate filters (8-μmpore size, Corning) coated with 50% Matrigel (BD bioscience, Bedford, MA) wereused to separate upper and lower chambers. In total, 1 × 105 cells in 200 μl of theculture medium (supplemented with 0.1% FBS) were added into the upperchamber, while 600 μl medium supplemented with 10% FBS were added into thelower chamber and served as a chemotactic agent. After incubation, cells invadinginto the lower chamber were fixed, stained, and counted under the uprightmicroscope (five fields per chamber).

RNA extraction and real-time PCR for gene expression. RNA extraction withTrizol (Invitrogen) and real-time PCR were performed according to the protocolused in our previous study18. Primers of targeted genes were as follow: GAPDH,forward 5′-GTC TCC TCT GAC TTC AAC AGC G-3′ and reverse 5′-ACC ACCCTG TTG CTG TAG CCA A-3′ MMP2, forward 5′-AGC GAG TGG ATG CCGCCT TTA A-3′ and reverse 5′-CAT TCC AGG CAT CTG CGA TGA G-3′ FN1,forward 5′-ACA ACA CCG AGG TGA CTG AGA C-3′ and reverse 5′-GGA CACAAC GAT GCT TCC TGA G-3′ CDH1, forward 5′-GCC TCC TGA AAA GAGAGT GGA AG-3′ and reverse 5′-TGG CAG TGT CTC TCC AAA TCC G-3′ Pre-Snail, forward 5′-GGG ACA GTT GAA CCC CTC AG-3′ and reverse 5′-GGAGTC AAT GAG GAC CGC AA-3′ (mat-) Snail, forward 5′-TGC CCT CAA GATGCA CAT CCG A-3′ and reverse 5′-GGG ACA GGA GAA GGG CTT CTC-3′ZEB1, forward 5′-GGC ATA CAC CTA CTC AAC TAC GG-3′ and reverse

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5′-TGG GCG GTG TAG AAT CAG AGT C-3′ METTL3, forward 5′-CTA TCTCCT GGC ACT CGC AAG A-3′ and reverse 5′-GCT TGA ACC GTG CAA CCACAT C-3’YTHDF1, forward 5′- CAA GCA CAC AAC CTC CAT CTT CG-3′ andreverse 5′-GTA AGA AAC TGG TTC GCC CTC AT-3′. For quantitative RT-PCR,GAPDH was used as endogenous control for the cytoplasmic RNA, while 18S RNAwas selected as endogenous control for the nuclear RNA.

Western blot analysis. Western blot analysis was performed as previouslydescribed32. The antibodies used in this study were MMP2 (Bioworld, BS1236,1:500), FN (Novus, NBP1-91258, 1:500), E-Cad (Bioworld, 3195S, 1:500), ALKBH5(Abcam, ab69325, 1:500), Snail (Bioworld, BS1853, 1:500), ZEB1 (Millipore,ABD53, 1:1000), eEF-1 (Proteintech, 11402–1-AP, 1:500), eEF-2 (Proteintech,20107–1-AP, 1:500), YTHDF1 (Abcam, ab99080, 1:500), and GAPDH (BOSTER,BM3876, 1:1000). The uncropped gel images are shown in Supplementary Figs 7and 8.

LC–MS/MS assay. mRNA was purified from the total RNA using oligo dTmagnetic beads. About 200 ng of purified mRNAs were incubated with nuclease P1(0.5 U, Sigma) in 25 -μl reaction system containing 10 mM NH4OAc (pH= 5.3) at42 °C for 1 h, followed by addition of NH4HCO3 (1 M, 3 μL) and alkaline phos-phatase (1 μL, 1 U/μL; Sigma) and incubation at 37 °C for 2 h. After neutralized by1 μl HCl (3 M), samples were diluted to 50 μl and filtered by 0.22 μm filter (Mil-lipore). All samples (10 μl for each injection) were separated by a C18 column(Agilent) using reverse-phase ultra-performance liquid chromatography and ana-lyzed by an Agilent 6410 QQQ triple-quadrupole LC mass spectrometer using apositive electrospray ionization mode. All nucleosides were quantified by use ofretention time and ion mass transitions of 268.0 to 136.0 (A) and 282.1 to 150.0(m6A). Quantification was calculated using standard curves from standards run-ning in the same batch. Ratio of m6A to A was calculated based on calibrationcurves.

Dot-blot assay. mRNA was enriched using GenElute messenger RNA (mRNA)Miniprep Kit (Sigma‐Aldrich) and RiboMinus Transcriptome Isolation Kit(human/mouse; ThermoFisher Scientific). After 5 min of denaturation at 70 °C,equal amounts of serial-diluted mRNA were added into an Amersham HybondN+membrane (GE Healthcare) and cross-linked into auto-cross-linker threetimes under auto-cross-linking mode. Membrane was blocked with 5% milk in 1×PBST for 1 h at room temperature and then was inducted with anti‐m6A antibody(SySy) overnight (4 °C). Membrane was washed according to the standard protocol.Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Cell SignalingTechnology) was diluted 1:5000 and incubated with the membranes for 1 h at roomtemperature. Membranes were developed with a 3,3′‐diaminobenzidine peroxidasesubstrate kit (Yeasen Biotechnology, Shanghai, China) to detect the signal.

m6A sequencing (m6A-seq) and data analysis. The total polyadenylated RNAwas isolated from HeLa cells treated with or without 10 ng/ml TGF-β for 3 daysusing TRIZOL reagent followed by isolation through FastTrack MAGMaxi mRNAisolation kit (Invitrogen). RNA fragmentation, m6A-seq, and library preparationwere performed according to the manufacturer’s instructions and previouslypublished protocol7. NEBNext Ultra Directional RNA Library Prep Kit (NewEngland BioLabs, Ipswich, MA, USA) was used for library preparation. Eachexperiment was conducted with two biological replicates. m6A-seq data wereanalyzed according to protocols described before7. Significant peaks with FDR <0.05 were annotated to RefSeq database (hg19). Sequence motifs were identified byusing Homer. Gene expression was calculated by Cufflinks using sequencing readsfrom input samples. Cuffdiff was used to find DE genes.

m6A-RT-PCR. m6A-RT-PCR was conducted according to previously describedprotocol with a slight modification20. Briefly, the total RNA was extracted usingTrizol and fragmented by RNA fragmentation reagents (Thermo, AM8740) or not.After saving 50 ng of the total RNA as input, the remaining RNAs (2 μg) were usedfor m6A-immunoprecipitation with m6A antibody (Synaptic Systems) in 500 μL ofIP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris, pH 7.4, 100 U RNase inhibitor)to obtain m6A pull down portion (m6A IP portion). m6A RNAs were immuno-precipitated with Dynabeads® Protein A (ThermoFisher Scientific) and eluted twicewith elution buffer (5 mM Tris-HCL pH 7.5, 1 mM EDTA pH 8.0, 0.05% SDS, 20mg/ml Proteinase K). m6A IP RNAs were recovered by ethanol precipitation, andRNA concentration was measured with Qubit® RNA HS Assay Kit (ThermoFisherScientific). Then 2 ng of the total RNA and m6A IP RNA were used as templates inqRT-PCR, as described above. RT-PCR for Snail was conducted using the followingprimers: 5′UTR, forward 5′-CGG CCT AGC GAG TGG TTC T-3′ and reverse 5′-TCG AGG CAC TGG GGT CG-3′ CDS, forward 5′-GCC AAT TGC CTG GGCCTC C-3′ and reverse 5′-GGC TGG GAG CCT TTC CCA CTG-3′ 3′UTR, forward 5′-TGG ATA CAG CTG CTT TGA GC-3′ and reverse 5′-TGT GAG GGG AGT GGG GTC-3′. Housekeeping gene HPRT1 was chosen asinternal control since HPRT1 mRNA did not have m6A peaks from m6A profilingdata6.

RIP–RT-PCR. For RNA immunoprecipitation, cells were irradiated twice with 400mJ/cm2 at 254 nm by Stratalinker on ice and lysed in high salt lysis buffer (300 mMNaCl, 0.2% NP-40, 20 mM Tris-HCl PH 7.6, 0.5 mM DTT, protease inhibitorcocktail (1 tablet/50 ml), and RNase inhibitor (1:200)) at 4 °C for 30 min. Aftertreatment with, or without, 1 U RNase T1 for 15 min at 24 °C, 10% supernatantwas collected as input. Remaining supernatant was incubated with anti-YTHDF1antibody, or IgG-conjugated protein A/G Magnetic Beads in 500 mL 1× IP buffersupplemented with RNase inhibitors at 4 °C overnight. Bound RNAs wereimmunoprecipitated with beads and treated with 100 µl of elution buffer (5 mMTris-HCL pH 7.5, 1 mM EDTA pH 8.0, 0.05% SDS, 20 mg/ml Proteinase K) for 2 hat 50 °C, and RNA was recovered with phenol:chloroform extraction followed byethanol precipitation. IP enrichment ratio of a transcript was calculated as ratio ofits amount in IP to that in the input, yielded from same amounts of cells.

RNA stability. To measure RNA stability in HeLa wild-type and Mettl3Mut/− cells,actinomycin D (Act-D, Catalog #A9415, Sigma, USA) at 5 μg/ml was added to cells.After incubation at the indicated times, cells were collected, and RNA was isolatedfor real-time PCR. Half-life (t1/2) of precursor and mature Snail mRNA werecalculated using ln2/slope and GAPDH was used for normalization.

Protein stability. To measure protein stability, HeLa wild-type and Mettl3Mut/−

cells were treated with cycloheximide (CHX, final concentration 100 μg/ml) duringindicated times. The expression of Snail was measured through western blotanalysis.

Snail promoter activity assay. Promoter activity of Snail in HeLa wild-type andMettl3Mut/− cells was measured according to our previously described protocol62.Briefly, cells were transfected with pGL3-Basic-Snail-luc containing sequence−797/+ 59 of Snail promoter. Transfection efficiency was normalized by co-transfection with pRL-TK. Transcriptional activity was determined by a lumin-ometer, using a dual-luciferase assay kit. The results were displayed as the ratiosbetween the activity of reporter plasmid and pRL-TK.

Luciferase reporter assay. Luciferase assay was performed using reporter lysisbuffer (Catalog #E3971, Promega, USA) and luciferase assay reagent according tothe manufacturer’s instructions. Briefly, HeLa wild-type and Mettl3Mut/− cells weretransfected with pmiRGLO, pmiRGLO-WT-3′UTR, pmiRGLO-Mut1-3′UTR, orpmiRGLO-Mut2-3′UTR in a 12-well plate. After transfection for 6 h, each cell linewas re-seeded into a 96-well plate. After 24 h incubation, cells were analyzed withthe Dual-Glo Luciferase Assay system (Promega). Renilla Luciferase (R-luc) wasused to normalize firefly luciferase (F-luc) activity to evaluate reporter translationefficiency.

Experimental animals and xenograft models. Female BALB/c nude mice (4-week-old) were purchased from Sun Yat-sen University (Guangzhou, China)Animal Center and raised under pathogen-free conditions. All animal experimentscomplied with Zhongshan School of Medicine Policy on Care and Use ofLaboratory Animals. For subcutaneous transplanted model, sh-control and sh-METTL3 Huh7 cells (5 × 106 per mouse, n= 5 for each group) were diluted in 200μL of PBS+ 200 μL Matrigel (BD Biosciences) and subcutaneously injected intoimmunodeficient female mice to investigate tumor growth. When tumor volume,in each group, reached ~100 mm3, mice were killed, and tumors were removed andweighed for use in histology and further studies. The tumor volume was calculatedusing the formula V= 1/2 × larger diameter × (smaller diameter)2.

For in vivo lung metastasis model, mice were injected with sh-control and sh-METTL3 Huh7 cells, or HeLa WT, Mettl3Mut/− HeLa, HeLaSnail, and Mettl3Mut/−

HeLaSnail stable cells (1 × 106 per mouse, n= 10 for each group). Eight weeks afterinjection, mice were killed and metastatic lung tumors were analyzed.

Immunohistochemistry (IHC). Immunohistochemistry (IHC) was performed tomeasure expression of target protein according to our previous study63.

Kaplan–Meier survival analysis and clinical expression analysis. Kaplan–Meierplotter (http://kmplot.com/analysis/) is a comprehensive online platform that canassess the effect of 54,675 genes on survival based on 10,293 cancer samples64. Weused Kaplan–Meier plotter to assess the prognostic value of METTL3, YTHDF1,and SNAI1 expression in patients with liver cancers. mRNA expression ofMETTL3 and YTHDF1 in cancer tissues and matched adjacent normal tissues ofliver cancers were obtained from TCGA (The Cancer Genome Atlas) database.METTL3 expression in HCC tumor tissues (n= 35), liver cirrhosis tissues (n= 13)and normal liver tissues (n= 10) from Oncomine database (Wurmbach livercancers). mRNA expression of YTHDF1 in liver cancers of T1 (n= 153), T2 (n=77), T3 (n= 65), and T4 (n= 13) stages from TCGA database.65

Tissue microarray analysis. The protein expression of Mettl3 and Snail in 100cases of liver cancer patients by use of tissue microarray (CS03–01–008, Alenabio,Xian, Shanxi, China) according to our previous study66. The clinical slides of

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Mettl3 and Snail were analyzed separately by two pathologists without knowing thepatients’ clinical information. The intensity was scored on a scale of 0–3 as negative(0), weak (1), medium (2), or strong (3). The extent of the staining, defined as thepercentage of positive stained areas of tumor cells per the whole-tumor area, wasscored on a scale of 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%).An overall protein expression score (overall score range, 0– 12) was calculated bymultiplying the intensity and positivity scores. For statistical purposes, the stainingscore was further categorized as low (0–5), medium (6–8), and high (9–12).

Statistical analyses. Data were reported as mean ± SD from at least three inde-pendent experiments unless otherwise specified. Values for capillary tube forma-tion and luciferase activity assays are from three independent experiments thatwere performed in duplicate. Data were analyzed by two-tailed unpaired Student’s ttest between two groups and by one-way ANOVA followed by Bonferroni test formultiple comparison. Statistical analysis was carried out using SPSS 16.0 forWindows. All statistical tests were two sided. A p-value of < 0.05 was considered tobe statistically significant. Data are presented as means ± SD from three indepen-dent experiments. *p < 0.05, **p < 0.01. NS, no significant.

Reporting summary. Further information on research design is available inthe Nature Research Reporting Summary linked to this article.

Data availabilityThe high-throughput m6A sequencing data have been deposited in the GEO databaseunder the accession code GSE112795. The data underlying Figs 1h, 1I, 6E~G, and 6I~Kreferenced during the study are available in a public repository from the TCGA website.All the other data supporting the findings of this study are available within the article andits Supplementary Information files and from the corresponding author upon reasonablerequest. A reporting summary for this article is available as a Supplementary Informationfile.

Received: 11 May 2018 Accepted: 27 March 2019

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AcknowledgementsThis research was supported by the National Natural Science Foundation of China(Grant nos. 81673454, 81672608, 31801197, and 81572270), the Guangdong Natural

Science Funds for Distinguished Young Scholar (No. 2014A030306025), the GuangdongProvince Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation(2017B030314026), and the Fundamental Research Funds for the Central Universities(Sun Yat-sen University) (No. 16ykpy09). We thank the help of Miss Hailing Shi, MrPhillip Hsu, Dr Ziyang Hao, Dr Boxuan Zhao, Mr Tong Wu, and Mr Lisheng Zhang atUniversity of Chicago for experimental skills.

Author contributionsConception and design: H.W., C.H., Y.W., J.D. and G.L.; acquisition of the data: X.L., H.W., Y.W., G.C., J. L, F.C.; analysis and interpretation of the data: H.W., G.L. and C.H.;writing, review, and/or revision of the paper: H.W., C.H., J. Li, J.T. and S.L.

Additional informationSupplementary Information accompanies this paper at https://doi.org/10.1038/s41467-019-09865-9.

Competing interests: The authors declare no conflict of interest.

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