NSUN2-mediated mRNA m5C Modification Regulates the Progression of Hepatocellular Carcinoma

Abstract

RNA modifications affect many biological processes and physiological diseases. The 5-methylcytosine (m⁵C) modification regulates the progression of multiple tumors. However, its characteristics and functions in hepatocellular carcinoma (HCC) remain largely unknown. Here, we found that HCC tissues had a higher m⁵C methylation level than the adjacent normal tissues. Transcriptome analysis revealed that the hypermethylated genes mainly participated in the phosphokinase signaling pathways, such as the Ras and PI3K-Akt pathways. The m⁵C methyltransferase NSUN2 was highly expressed in HCC tissues. Interestingly, the expression of many genes was positively correlated with the expression of NSUN2, including GRB2, RNF115, AATF, ADAM15, RTN3, and HDGF. Real-time PCR assays further revealed that the expression of the mRNAs of GRB2, RNF115, and AATF decreased significantly with the down-regulation of NSUN2 expression in HCC cells. Furthermore, NSUN2 could regulate the cellular sensitivity of HCC cells to sorafenib via modulating the Ras signaling pathway. Moreover, knocking down NSUN2 caused cell cycle arrest. Taken together, our study demonstrates the vital role of NSUN2 in the progression of HCC.

Keywords: 5-methylcytosine; Hepatocellular carcinoma; NSUN2; Ras pathway; Sorafenib.

Figure 1

mRNAs are frequentlym⁵C-hypermethylated in HCC tissuesA. Distribution pattern of the m⁵C sites on mRNAs in the HCC tissues (tumor) and the adjacent tissues (normal). B. Different proportions of m⁵C modifications in regions of mRNA between the HCC tissues and the adjacent tissues. C. The overall m⁵C modification level is higher in the HCC tissues than in the adjacent tissues, as determined by the BisSeq data analysis. Statistical significance was calculated by Wilcoxon test (****, P = 5.116E−09). D. Difference in the m⁵C modification levels between the HCC tissues and the adjacent tissues. E. Heatmap showing the differential m⁵C methylation levels between the HCC tissues and the adjacent tissues. HCC, hepatocellular carcinoma; m⁵C, 5-methylcytidine; CDS, coding sequence; UTR, untranslated region.

Figure 2

Multiplem⁵C-hypermethylated genes related to NSUN2 participate in the oncogenic pathwaysA. The distribution of mRNAs with a significant change in the m⁵C methylation level and the gene expression level in HCC tissues and the adjacent tissues. B. The KEGG analysis showed that m⁵C-hypermethylated genes with high expression levels in the HCC tissues were enriched in oncogenic signaling pathways. C. A relation analysis showed that the expression levels of GRB2, RNF115, and AATF were positively correlated with their m⁵C modification levels. D. The expression levels of GRB2, RNF115, and AATF were positively correlated with the NSUN2 expression level. E. The overall survival analysis indicates the correlation of the mRNA expression of GRB2, RNF115, and AATF with poor prognosis in HCC patients. P values were calculated by Student’s t-test. KEGG, Kyoto Encyclopedia of Genes and Genomes; TPM, transcripts per kilobase of exon model per million mapped reads.

Figure 3

NSUN2 is highly expressed in HCC and regulates mRNA m⁵C modificationA. The expression of NSUN2 mRNA was higher in HCC tissues than in the adjacent tissues determined by transcriptome analysis. B. Western blot analysis showed the higher expression of NSUN2 in HCC tissues than in the adjacent tissues. GAPDH was used as a reference control. “T” indicates a tumor smaple, and “N” indicates the adjacent tissue. C. Immunohistochemical analysis showed the higher expression of NSUN2 in HCC tissues than in the adjacent tissues. D. In HCC cells, UHPLC-MS/MS analysis showed that the down-regulation of NSUN2 significantly decreased the density of m⁵C/C in mRNAs. E. The real-time PCR analysis showed that the mRNA expression of GRB2, RNF115, and AATF was significantly decreased when NSUN2 was silenced in QGY-7703 cells. Data were represented by mean ± SD. Statistical significance was determined by Student’s t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). UHPLC-MS/MS, ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry.

Figure 4

NSUN2 affects the sensitivity of HCC cells to sorafenib by regulating the activity of the Ras pathwayA. Box plots showing the mRNA m⁵C levels of the Ras pathway-related genes. B. Heatmap showing the differential mRNA m⁵C levels of the Ras pathway-related genes in HCC tissues and the adjacent tissues. C. The overall survival analysis indicated that the high expression of NSUN2 and GRB2 was correlated with the worst prognosis in HCC patients (****, P < 0.0001). D. Western blot showing the Ras activity detected in wild-type QGY-7703 cells (WT), NSUN2-knockout cells (NSUN2-KO6/KO10), and NSUN2-rescued cells (NSUN2-Res). E. Western blot of Erk and p-Erk in NSUN2-knockout cells (NSUN2-KO6/KO10) and NSUN2-rescued cells (NSUN2-Res, NSUN2-C271A, and NSUN2-DM). NSUN2-Res, NSUN2-C271A, and NSUN2-DM indicate wild-type rescued, binding site mutant rescued, and binding site and catalytic site double mutant rescued, respectively. GAPDH was used as a reference control. F. Sorafenib treatment and flow cytometry analysis of the apoptosis of QGY-7703 cells when NSUN2 was knockout or rescued. G. The statistical analysis of the apoptosis ratio shown in (F). Data were represented by mean ± SD. Statistical significance was determined by Student’s t-test (*, P < 0.05; **, P < 0.01; ns, not significant). Raf RBD, Ras-binding domain of Raf; IP, immunoprecipitation; p-Erk, phosphorylated-Erk; PI, propidium iodide.

Supplementary Figure S1

Distribution characteristics of m5C in HCC and expression pattern of target genes. A. The type and proportion of RNA in the sequencing library. B. The sequences are proximal to the mRNA m⁵C sites in HCC tissues and the adjacent tissues. C. The m⁵C hypermethylated and highly expressed genes in HCC tissues were involved in different cellular processes, determined by the GO functional analysis. D. and E. The expression of target genes, including GRB2, AATF, RNF115, ADAM15, RTN3, and HDGF, was higher in HCC tissues than that in the adjacent tissues, determined by transcriptome analysis, ****, P < 0.0001. F. The overall survival analysis showed that the higher expression of ADAM15, RTN3, and HDGF was correlated with a poor prognosis in HCC patients; ADAM15 (*, P = 0.0097), RTN3 (**, P = 0.00018), HDGF (*, P = 0.0092). TPM, transcripts per kilobase of exon model per million mapped reads; HCC, hepatocellular carcinoma; GO, gene ontology.

Supplementary Figure S2

NSUN2 regulates downstream target gene expression, especially GRB2. A. The mRNA expression of m⁵C writers and readers in HCC tissues was higher than that in the adjacent tissues, determined by the transcriptomic analysis, ***, P < 0.001. B. In HCC cell lines, the UHPLC-MS/MS analysis showed that downregulation of NSUN2 did not change the density of m5C/C in total RNA. C. Real-time PCR analysis showed that the mRNA expression levels of GRB2, RNF115, and AATF were significantly lower in Huh7 NSUN2 knockdown cells, *, P < 0.05; **, P < 0.01. D. The m⁵C modification in the 3′ UTR and the expression of GRB2 mRNA were analyzed by IGV visualization in HCC tissues and the adjacent tissues. UHPLC-MS/MS, ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry; UTR, untranslated region; IGV, itegrative genomics viewer.

Supplementary Figure S3

Identification of NSUN2 knockout efficiency at the genome level and transcriptome level. A. NSUN2 knockout characteristics were identified by Sanger sequencing in the QGY-7703 cell genome. B. The mRNA expression levels of NSUN2 in NSUN2 knockout cells (KO6/KO10) and NSUN2 reconstituted cells were evaluated by real-time PCR. WT, wilg type ; KO, knock out ; Res, rescue.

Supplementary Figure S4

Apoptosis and cycle analysis of NSUN2 on HCC cells. A. and B. After sorafenib treatment, flow cytometry analyses of the apoptosis of HCC cells rate when NSUN2 knockdown. The statistical analyses of the apoptosis ratio are shown in (C), **, P < 0.01. D. Flow cytometry analyses of the cell cycle of Huh 7 cells. These cells were arrested in the G1 phase when NSUN2 was knocked down. The statistical analyses of the arrest ratio are shown in (E), *, P < 0.05. Statistical significance was calculated by Student’s t-test, mean ± SD. shNC, nonsense control-shRNA.