The RNA m5C Methyltransferase NSUN2 Promotes Progression of Hepatocellular Carcinoma by Enhancing PKM2-Mediated Glycolysis

Background: Hepatocellular carcinoma (HCC) is a leading cancer worldwide. The 5-methylcytosine (m5C) RNA methyltransferase NSUN2 has been involved in the cell proliferation and metastasis formation and is upregulated in a variety of cancers. However, NSUN2-mediated m5C modification has not been well studied in HCC. This study aimed to investigate the biological function and regulatory mechanism of NSUN2-mediated m5C modification in HCC.Methods: The association between NSUN2 expression and clinical characteristics was analyzed in 80 HCC patients. The function of NSUN2 in HCC was tested by in vitro and in vivo experiments. mRNA m5C-RIP-seq (m5C-RIP-sequencing) was performed in five paired human HCC and ANL tissues. PKM2 mRNA-the main target of NSUN2-was screened by overlapping the mRNA m5C-RIP-seq data in human HCC tissue and transcriptome sequencing data in NSUN2-silencing HCC cell lines and validated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and m5C-RIP-qPCR. The exact m5C site of PKM2 mRNA was validated by bisulfite-PCR. The role of NSUN2 in glycolysis was confirmed by detecting glucose and lactate level and extracellular acidification rate in the culture medium of HCC cells. Rescue assays were performed to prove whether NSUN2 could promote the progression of HCC through PKM2.Findings: NSUN2 is upregulated and associated with poor prognosis in HCC patients after hepatectomy. Up-regulated NSUN2 can elevate m5C mRNA levels in HCC and promote HCC growth and metastasis. m5C-RIP-Seq revealed that mRNA m5C is frequently hypermethylated and hypermethylated m5C correlates with mRNA overexpression and NSUN2-mediated m5C hypermethylation promotes metabolism in HCC. The results showed that PKM2, a terminal enzyme in the glycolytic pathway, as a downstream target of NSUN2-mediated m5C modification. Mechanistically, our data revealed that NSUN2 promotes HCC glycolysis and progression by upregulating PKM2.Interpretation: NSUN2-mediated m5C modification promotes glycolysis and progression of hepatocellular carcinoma by stabilizing PKM2 mRNA, and provide a potential prognostic factor and therapeutic target for HCC patients.Funding: This work was supported by the National Natural Science Foundation of China (Grant No.82002458 and 82102482).Declaration of Interest: The authors declare no competing interests.Ethical Approval: Human specimen collection was approved by the Ethics Committee of Eastern Hepatobiliary Surgery Hospital. Written informed consent was obtained from each patient according to the policies of the committee. The animal experiments in this study conformed to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (http://www.nc3rs.org.uk/arrive-guidelines) and were approved by the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (Shanghai, China).