The RNA M5C methyltransferase NSUN2 promotes progression of hepatocellular carcinoma by enhancing PKM2-mediated glycolysis

Abstract

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. The 5-methylcytosine (m5C) RNA methyltransferase NSUN2 is involved in cell proliferation and metastasis and is upregulated in a variety of cancers. However, the biological function and regulatory mechanism of NSUN2-mediated m5C modification have not been well studied in HCC. Our results showed that NSUN2 is upregulated and associated with poor prognosis in HCC patients after hepatectomy. NSUN2 overexpression significantly promoted HCC growth and metastasis, whereas NSUN2 knockdown had the opposite effect. m5C RNA immunoprecipitation sequencing (m5C-RIP-Seq) revealed that m5C hypermethylation correlates with mRNA overexpression and that NSUN2-mediated m5C hypermethylation promotes metabolism in HCC patients. Mechanistically, our data revealed that PKM2, a terminal enzyme in the glycolytic pathway, is a downstream target of NSUN2-mediated m5C modification. Specifically, NSUN2 could stabilize PKM2 mRNA by increasing the m5C level of the m5C site C773 in the 3'-UTR of PKM2 mRNA. In addition, rescue assays revealed that NSUN2 promotes HCC glycolysis and progression by upregulating PKM2. In conclusion, this study revealed that NSUN2-mediated m5C modification promotes glycolysis and the progression of hepatocellular carcinoma by stabilizing PKM2 mRNA, and provides a potential prognostic factor and therapeutic target for HCC patients.

Fig. 1. Upregulated NSUN2 expression predicts poor prognosis in patients with HCC.

The expression of NSUN2 (A) and NSUN6 (B) mRNAs was analysed via RT-qPCR. The Wilcoxon signed-rank test was used. C NSUN2 protein expression was analysed via western blot analysis. Student’s t-test was used. D IHC staining of NSUN2. (Right) Histochemistry score of NSUN2 in 80-paired HCC tissues and ANL tissues. (Left) Representative samples. E Kaplan–Meier analysis of the overall survival and recurrence-free survival of 80 HCC patients. ANL adjacent noncancerous liver, HCC hepatocellular carcinoma, RT-qPCR quantitative reverse transcription PCR.

Fig. 2. NSUN2 promotes HCC growth and metastasis in vitro and in vivo.

A Cell Counting Kit-8 assays revealed that NSUN2 overexpression promoted the growth of HCC cells. B Transwell migration assays revealed that NSUN2 overexpression promoted the migration of HCC cells. C Cell Counting Kit-8 assay results showing that NSUN2 knockdown inhibited the growth of HCC cells. D Transwell migration assays revealed that NSUN2 knockdown inhibited the migration of HCC cells. E, F Subcutaneous xenografts from the indicated HCC cells were excised from nude mice, and tumour weights and tumour growth curves were generated. Eight mice were included in each group. G, H (Right) Luciferase signal intensities of mice over time after tail vein injection of the indicated HCC cells. (Left) Representative images. There were six mice in each group. For (A–D), the data are presented as the means ± SDs; n = 3. Student’s t-test was used. For (E–H), the data are presented as the means ± SEM. The Mann–Whitney U test was used.

Fig. 3. Overview of mRNA m5C in human HCC and ANL tissues.

A Dot blot analysis of 5-methylcytosine (m5C) in mRNAs from five paired HCC and ANL tissues (50 ng each). Student’s t-test was used. B The numbers of m5C peaks in HCC and ANL tissues. C The numbers of mRNAs with m5C peaks in HCC and ANL tissues. D Cluster analysis of the differential m5C methylation in five paired HCC and ANL tissues. E Correlation analysis between the m5C level and the transcript level. Spearman correlation was used. F Distribution of mRNAs whose m5C methylation and gene expression levels significantly changed. G KEGG pathway enrichment for the mRNAs with both upregulated expression and m5C levels. ANL adjacent noncancerous liver, HCC hepatocellular carcinoma.

Fig. 4. PKM2 mRNA is the main target of NSUN2.

A Cluster analysis of the differentially expressed mRNAs after NSUN2 was knocked down in Hep3B cells. B Screening of the potential target mRNAs of NSUN2 in human HCC. C RT-qPCR results showing the mRNA levels of the 11 candidate genes in 40-paired HCC and ANL tissues. D RT-qPCR analysis of the mRNA levels of the 7 candidate genes in HCC cells. E The protein expression of PKM2 in HCC cells was analysed by western blot analysis. F The enrichment of PKM2 mRNA in 20 paired HCC and ANL tissues was identified via m5C-RIP-qPCR using anti-IgG or anti-m5C antibodies. For (C, F), the Wilcoxon signed-rank test was used. For (D), Student’s t-test was used.

Fig. 5. NSUN2 could stabilise PKM2 mRNA by increasing its m5C level.

A The relative RNA level of PKM2 was analysed by RT-qPCR after treatment with actinomycin D at the indicated time points in HCC cells. B Enrichment of PKM2 mRNA in the indicated HCC cells was identified via m5C-RIP-qPCR using anti-IgG or anti-m5C antibodies. C Sanger sequencing was performed after bisulfite- PCR. Representative sequencing chromatograms showing the m5C site (chr15:72491773 (hg19) (named C773)) of PKM2 mRNA. D, E The m5C level of C773 in the indicated samples is shown. F Relative luciferase activity of the luciferase reporter gene with the wild-type C773 site (PKM2-Wt) or the mutant C733 site (PKM2-Mut) in the indicated HCC cells. For (A–C, E, F), Student’s t-test was used. For (D), the Wilcoxon signed-rank test was used.

Fig. 6. NSUN2 promotes HCC glycolysis and progression by upregulating PKM2.

A, F The relative glucose uptake of HCC cells was measured with a glucose colorimetric assay kit (K606-100, BioVision, USA). For (B, G), the relative lactate level of HCC cells was measured with a lactate colorimetric assay kit II (K627-100, BioVision, USA). For (C, H), the extracellular acidification rate (ECAR) of HCC cells was analysed via a Seahorse XF96 instrument (Seahorse Bioscience, USA). 2-DG, 2-deoxyglucose. For (D, I), the glycolytic proton efflux rate (glycoPER) of HCC cells was analysed via a Seahorse XF96 instrument (Seahorse Bioscience, USA). Rot/AA, rotenone + antimycin A; 2-DG, 2-deoxyglucose. The results were normalised per 10,000 cells. E Western blot analysis of the expression of the PKM2 monomer, dimer, tetramer and NSUN2 in SNU387 cells. J The growth ability of SNU387 cells was measured via a Cell Counting Kit-8 assay on the third day after co-transfection. K The ability of SNU387 cells to metastasize was measured via a Transwell assay. L Representative images for (K).

Fig. 7. Schematic diagram depicting the proposed mechanisms of NSUN2-mediated m5C modulation of PKM2 in HCC.

NSUN2 is upregulated in HCC, and upregulated NSUN2 can stabilise PKM2 mRNA by increasing the m5C level of the m5C site C773 at the 3′-UTR of PKM2 mRNA, thus contributing to increased glycolysis and subsequent HCC progression and migration. Created via BioRender software.