An Alternatively Spliced Variant Of METTL3 Mediates Tumor Suppression In Hepatocellular Carcinoma

Many post-transcriptional mRNA processing steps play crucial roles in tumorigenesis and the progression of cancers,such as N6-methyladenosine（m6A）modification and alternative splicing.Upregulation of methyltransferase-like 3（METTL3）,the catalytic core of the m6A methyltransferase complex,increases m6A levels and results in significant effects on the progression of hepatocellular carcinoma（HCC）.However,alternative splicing of METTL3 has not been fully investigated,and the functions of its splice variants remain unclear.Here,we analyzed both our and online transcriptomic data,obtaining 13 splice variants of METTL3 in addition to canonical full-length METTL3-A in HCC cell lines and tissues.Validated by RT-qPCR and western blotting,we found that METTL3-D,one of the splice variants expressing a truncated METTL3 protein,exhibits higher levels than METTL3-A in normal human livers but lower levels than METTL3-A in HCC tumor tissues and cell lines.Further functional assays demonstrated that METTL3-D expression decreased cellular m6A modification,inhibited the proliferation,migration and invasion of HCC cells and was negatively associated with the malignancy of patient tumors,exhibiting functions opposite to those of full-length METTL3-A.Our clinical data showed that HCC early-stage tumor samples exhibited higher levels of METTL3-D;by contrast,late-stage samples exhibited lower levels of METTL3-D.Further analyses of TCGA data also showed that HCC patients with higher levels of METTL3-D had longer survival times.This study demonstrates that the METTL3-D splice variant is a tumor suppressor that could potentially be used as a target for HCC therapy.Objective HCC is one of the most frequently occurring primary malignant liver tumors,and understanding the molecular mechanism of HCC development is crucial.Many studies have demonstrated the potential implications of m6A modification in diagnosing and treating liver cancer.m6A modification is catalyzed by a multicomponent enzyme complex,including METTL3.Although METTL3 has been studied extensively in recent years,the existence and function of its splice variants have not been reported.In this study,METTL3 splice variants will be identified and analyzed in normal liver and HCC tissues.Methods We first analyzed our next-generation RNA-seq data of the HCC cell line HepG2,fourteen transcripts from the METTL3 gene in HepG2 cells were constructed,all of which can be found in the online Ensembl database.To confirm the presence of these splice variants,we verified the existence of METTL3-A,-C and-D by RT-PCR using specific primers and Sanger sequencing and METTL3-A and-D by detecting full transcripts using public HepG2 third-generation sequencing data.The expression levels of METTL3 splice variants in hepatocellular carcinoma tissues and normal tissues were compared by Western blot and qPCR assays.Hepatocellular carcinoma cells overexpressing METTL3 splice variants were constructed,and then the effect of METTL3 splice variants on the development of hepatocellular carcinoma cells was examined using relevant cell analysis techniques;the effect of METTL3 splice variants on the proliferation ability of hepatocellular carcinoma cell lines was also analyzed by cell counting,CCK8 and MTT.Results Here,we analyzed both our and online transcriptomic data,obtaining 13 splice variants of METTL3 in addition to canonical full-length METTL3-A in HCC cell lines and tissues.Validated by RT-qPCR and western blotting,we found that METTL3-D,one of the splice variants expressing a truncated METTL3 protein,exhibits higher levels than METTL3-A in normal human livers but lower levels than METTL3-A in HCC tumor tissues and cell lines.Further functional assays demonstrated that METTL3-D expression decreased cellular m6A modification,inhibited the proliferation,migration and invasion of HCC cells and was negatively associated with the malignancy of patient tumors,exhibiting functions opposite to those of full-length METTL3-A.Our clinical data showed that HCC early-stage tumor samples exhibited higher levels of METTL3-D;by contrast,late-stage samples exhibited lower levels of METTL3-D.Conclusions:1、In addition to the presence of full-length METTL3-A,13 additional spliced isoforms of METTL3 exist in HCC tumor tissues and cell lines.2、The level of METTL3-D is higher than that of METTL3-A in human normal liver tissues but is lower than that of METTL3-A in HCC cell lines and patient tumors.3、In contrast to the promotion of m6A modification and mRNA decay/stability of downstream target genes by METTL3-A,METTL3-D inhibits cellular m6A modification and protects the mRNA decay/stability of downstream genes.4、METTL3-D is negatively associated with the malignancy of HCC tumors,including its positive correlation with longer patient survival times and negative correlations with the growth,migration and invasion of HCC cells.