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The N6-methyladenosine reader protein YTHDC2 promotes gastric cancer progression via enhancing YAP mRNA translation.

ABSTRACT: N6-methyladenosine (m⁶A) modification is the most prevalent internal modification in eukaryotic mRNA. YTH domain containing 2 (YTHDC2), a m⁶A binding protein, has recently been identified as a key player in human cancer. However, its contribution to gastric cancer (GC) remains unknown. Herein, we found that YTHDC2 was significantly upregulated in human GC tissues and associated with poor prognosis. CRISPR-Cas9 mediated YTHDC2 knockout notably inhibited GC cell viability, proliferation and invasion. Transcriptome analysis coupled with mechanism experiments revealed that yes-associated protein (YAP), the well-known oncogene, is the target of YTHDC2 in GC cells. Specifically, YTHDC2 recognized m⁶A-modified YAP mRNA at 5`-UTR, resulting in enhancing the translation efficiency of YAP, without affecting its mRNA level. In turn, YAP/TEAD directly targeted -843∼-831 region on the promoter of YTHDC2 and activated the transcription of YTHDC2, thus forming a positive regulatory loop. Further, using the xenograft tumor model, we found that knockout of YTHDC2 markedly reduced tumor size and lung metastasis nodules in vivo. And high YTHDC2 was strongly positively correlated with high YAP in clinical GC tissues. Collectively, our data demonstrate that YTHDC2 is a novel oncogene in GC, which provides the theoretical basis for the strategy of targeting YTHDC2 for GC patients.

SUBMITTER: Yuan W

PROVIDER: S-EPMC8681016 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundA number of studies have demonstrated that N6-methyladenosine (m6A) plays a vital role in the pathological process of various tumours. Recently, it was found that m6A writers or erasers affect the tumourigenesis of melanoma. However, the relationship between m6A readers such as YTH domain family (YTHDF) proteins and melanoma was still elusive.MethodsRT-qPCR, Western blot and immunohistochemistry were conducted to measure the expression level of YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) and lysyl oxidase-like 3 (LOXL3) in melanoma tissues and cells. The effects of YTHDF3 and LOXL3 on melanoma were verified in vitro and in vivo. Multi-omics analysis including RNA-seq, MeRIP-seq, RIP-seq and mass spectrometry analyses was performed to identify the target. The interaction between YTHDF3 and LOXL3 was verified by RT-PCR, Western blot, MeRIP-qPCR, RIP-qPCR and CRISPR-Cas13b-based epitranscriptome engineering.ResultsIn this study, we found that m6A reader YTHDF3 could affect the metastasis of melanoma both in vitro and in vivo. The downstream targets of YTHDF3, such as LOXL3, phosphodiesterase 3A (PDE3A) and chromodomain helicase DNA-binding protein 7 (CHD7) were identified by means of RNA-seq, MeRIP-seq, RIP-seq and mass spectrometry analyses. Besides, RT-qPCR, Western blot, RIP-qPCR and MeRIP-qPCR were performed for subsequent validation. Among various targets of YTHDF3, LOXL3 was found to be the optimal target of YTHDF3. With the application of CRISPR-Cas13b-based epitranscriptome engineering, we further confirmed that the transcript of LOXL3 was captured and regulated by YTHDF3 via m6A binding sites. YTHDF3 augmented the protein expression of LOXL3 without affecting its mRNA level via the enrichment of eukaryotic translation initiation factor 3 subunit A (eIF3A) on the transcript of LOXL3. LOXL3 downregulation inhibited the metastatic ability of melanoma cells, and overexpression of LOXL3 ameliorated the inhibition of melanoma metastasis caused by YTHDF3 downregulation.ConclusionsThe YTHDF3-LOXL3 axis could serve as a promising target to be interfered with to inhibit the metastasis of melanoma.

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The N6-methyladenosine reader protein YTHDC2 promotes gastric cancer progression via enhancing YAP mRNA translation.

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