Abstract
RNA methylation is an important epigenetic modification. Recent studies on RNA methylation mainly focus on the m6A modification of mRNA, but very little is known about the m5C modification. NSUN2 is an RNA methyltransferase responsible for the m5C modification of multiple RNAs. In this study, we knocked down the NSUN2 gene in HepG2 cells by CRISPR/Cas9 technology and performed high-throughput RNA-BisSeq. An important tumor-related lncRNA H19 was identified to be targeted by NSUN2. Studies have shown that the expression of H19 lncRNA is abnormally elevated and has a carcinogenic effect in many types of tumors. Our results demonstrated that m5C modification of H19 lncRNA can increase its stability. Interestingly, m5C-modified H19 lncRNA can be specifically bound by G3BP1, a well-known oncoprotein which further leads to MYC accumulation. This may be a novel mechanism by which lncRNA H19 exerts its oncogenic effect. Besides, both the m5C methylation level and the expression level of H19 lncRNA in hepatocellular carcinoma tissues were significantly higher than those in adjacent non-cancer tissues, which were closely associated with poor differentiation of hepatocellular carcinoma (HCC). In conclusion, we found that H19 RNA is a specific target for the NSUN2 modifier. The m5C-modified H19 lncRNA may promote the occurrence and development of tumors by recruiting the G3BP1 oncoprotein. Our findings may provide a potential target and biomarker for the diagnosis and treatment of HCC.
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Introduction
In recent years, the association between abnormal RNA modifications and tumorigenesis has attracted widespread attention. Several studies have clarified that N6-methyladenosine (m6A) modification of RNA plays an important role in the development of cancers, such as lung cancer [1], hepatocellular carcinoma [2], and glioblastoma [3]. As a widespread modification, 5-methylcytosine (m5C) in DNA and RNA has been found for decades. The function of DNA m5C modification in carcinogenesis has been studied extensively. However, very little is known about the function of RNA m5C modification in tumorigenesis. NSUN2(NOP2/Sun domain family, member 2), also named Misu [4], is one of the major m5C-modifying methyltransferases in mammals and plays an important role in RNA metabolic processes, such as RNA processing [5], RNA stability [6,7,8], RNA export [9], and mRNA translation [10, 11].
A previous study demonstrated that NSUN2 is a downstream target gene of MYC, and is essential for MYC-induced proliferation and cell-cycle progression [4]. Similar to MYC, NSUN2 is highly expressed in a variety of tumors. Immunohistochemical studies confirmed that NSUN2 protein levels are significantly increased in various +tumors, including the esophagus, liver, pancreas, cervix, prostate, kidney, bladder, thyroid, and breast cancers compared to normal controls [12]. In breast epithelial cells, overexpression of NSUN2 was shown to promote cell proliferation, migration, and invasion while NSUN2 knockdown inhibited these processes in vitro and in vivo [13]. The close association of NSUN2 with tumor features suggests that it may be a key factor in the study of the relationship between RNA m5C modification and tumorigenesis.
NSUN2 was shown to account for the formation of 5-methylcytosine (m5C) in a variety of RNA species, including tRNA, rRNA, mRNA, lncRNA, and mitochondrial RNAs [6, 14,15,16]. Long non-coding RNAs (lncRNAs) are a class of RNA molecules that are longer than 200 nucleotides in length and lack protein-coding capacity. As the most abundant non-coding RNA species, a large number of m5C modifications are detected in lncRNAs [14]. In two well-studied lncRNAs, XIST and HOTAIR, methylated cytosines were found to be located within or near functionally important regions that are known to mediate interactions with chromatin-associated protein complexes [17].
Functional studies have revealed that lncRNAs are involved in multiple biological processes and disease-related pathways, such as proliferation [18], differentiation [19], stem cell pluripotency [20], tumorigenesis, and metastasis [21]. A recent study on esophageal squamous cell carcinoma (ESCC) identified lncRNA NMR (NSun2 methylated lncRNA), which is highly expressed in ESCC and plays a key regulatory role in the tumorigenesis and drug resistance of ESCC [22]. NMR is methylated by NSUN2 and might competitively inhibit the methylation of potential mRNAs. Thus, it was speculated that NSUN2 may be implicated in tumorigenesis through its methylated lncRNAs.
In this study, we constructed an NSUN2-deficient hepatocellular carcinoma cell line and investigated the effect of NSUN2 on cell proliferation, migration, and invasion in vitro and in vivo. Furthermore, we provide a landscape of NSUN2-mediated m5C modification in hepatocellular carcinoma cells, and identified H19 lncRNA as a target of NSUN2. Recent studies have confirmed that H19 lncRNA has an oncogenic function in a variety of adult cancers, such as hepatocellular carcinoma [23], colorectal cancer [24], pancreatic cancer [25], and glioma cell [26]. Our further results show that methylation of H19 RNA by NSUN2 can affect the stability of H19 and its interaction with the G3BP1 oncoprotein. In addition, hypermethylation and overexpression of lncRNA H19 were identified in liver cancer tissues compared to matched non-cancerous liver tissues, and a higher level of H19 RNA m5C methylation was correlated with HCC progression. Thus, our findings demonstrate a regulatory mechanism involving NSUN2 and its mediation of RNA m5C modification in the progression of human HCC, indicating that this interaction may serve as a prognostic biomarker in HCC patients.
Results
NSUN2 deficiency inhibits the proliferation of HCC cells
To explore the functional role of NSUN2 in hepatocellular carcinoma, NSUN2-deficient HepG2 cells were generated. The genotype of NSUN2-deficient cells was assessed by PCR, and the gene silencing efficiency of cells was determined using RT-qPCR and western blotting. As a result, we obtained an NSUN2-deficient HepG2 cell line with a biallelic insertion of a transcriptional terminator signal (Fig. 1a). The results of RT-qPCR and western blotting showed that over 90% of the NSUN2 gene expression was repressed (Fig. 1b, c).
a Genotyping of NSUN2-deficient HepG2 cells based on the PCR method. Because of the insertion of Neo and Puro into the double allele, two longer bands could be amplified in NSUN2-deficient cells compared with normal cells. b NSUN2 mRNA level in HepG2 and NSUN2-deficient HepG2 cells were assessed by real-time qPCR. Data are represented as the mean ± SEM (n = 3 independent experiments). c NSUN2 protein level in HepG2 and NSUN2-deficient HepG2 cells were assessed by western blotting. Three independent experiments were performed for quantification (mean ± SEM). d Cell proliferation was determined by CCK-8 assay. Data are represented as the mean ± SEM (n = 6 independent experiments). e Representative picture of the colony formation assay result. Three independent experiments were performed for quantification (mean ± SEM). f FACS analysis was subjected to assess the cell-cycle distribution. OD optical density; HepG2-NKD, NSUN2-deficient HepG2 cells. P values were calculated by Student’s t test. *p < 0.05, **p < 0.01.
To further characterize NSUN2-knockdown phenotypes, CCK-8 and colony formation assays were employed to detect the impact of NSUN2 depletion on the proliferation of HepG2 cells. The CCK-8 assay was performed at 24, 48, 72, and 96 h after cell seeding. Compared to normal HepG2 cells, a significantly decreased viability was observed in NSUN2-deficient HepG2 cells after 24 h (Fig. 1d). The results of the colony formation assay were similar. As shown in Fig. 1e, the clone number in NSUN2-deficient HepG2 cells was significantly lower than that in wild-type HepG2 cells. Quantification of clone number revealed that NSUN2 deficiency inhibited the colony formation ratio by an average of 14% compared to that in wild-type HepG2 cells. To address whether NSUN2 deficiency affects the cell division cycle of HepG2 cells, the cell-cycle distribution of the cells was analyzed by FACS. As shown in Fig. 1f, NSUN2-deficient HepG2 cells had smaller G1 and S populations and larger G2/M populations than wild-type cells.
NSUN2 deficiency inhibits the migration, invasion, and angiogenesis of HCC cells
To investigate whether NSUN2 deficiency affects the metastasis and invasion of HepG2 cells, a wound healing assay was used to analyze the effect of NSUN2 depletion on the migration ability of HepG2 cells. As shown in Fig. 2a, NSUN2-deficient HepG2 cells displayed significant delays in wound closure resulting from diminished cell migration. Quantification of wound size revealed that wound closure rate was inhibited by an average of 72.5% in NSUN2-deficient cells compared to wild-type HepG2 cells. A transwell invasion assay was used to detect the invasion ability of NSUN2-deficient HepG2 cells in vitro. NSUN2 depletion significantly reduced the number of invaded cells (Fig. 2b). The inhibition of HepG2 cell invasion in response to NSUN2 deficiency was higher than 50%. Moreover, a tube formation assay was used to detect the effect of NSUN2 knockout on the angiogenesis of HepG2 cells in vitro. The results indicated that NSUN2 depletion decreased cell cord formation on Matrigel, with significantly reduced junctions, cord length, tubules, and tubule area (Fig. 2c).
a Cell migration was determined by wound healing assay. b Trans-well invasion assay. Three independent experiments were performed for quantification (mean ± SEM). c Tubule formation assay was used to test the effect of NSUN2 deficiency on angiogenesis ability of cells in vitro. d Xenograft nude mouse model was inoculated with NSUN2-deficient HepG2 cells or the normal HepG2 cells (n = 6/group), at day 24 after inoculation. Three independent experiments were performed for quantification (mean ± SEM). HepG2-NKD, NSUN2-deficient HepG2 cells. The data are represented as mean ± SEM, obtained in three independent experiments. P values were calculated by Student’s t test. *p < 0.05, **p < 0.01.
To further determine whether NSUN2 deficiency inhibits tumorigenicity in hepatocellular carcinoma in vivo, we established a xenograft model by inoculating nude mice with NSUN2-deficient HepG2 cells. As shown in Fig. 2d, the tumors formed by NSUN2-deficient HepG2 cells were significantly smaller than those formed by wild-type HepG2 cells. These results are consistent with the results in vitro and further confirm that NSUN2 plays an important role in promoting the tumorigenicity of hepatocellular carcinoma cells.
NSUN2 deficiency significantly influences the expression profiles of HepG2 cells
Based on the cell phenotype alteration after NSUN2 depletion, further experiments were performed to determine the molecular basis for the phenotype alterations. Genome-wide RNA-sequencing (RNA-Seq) was employed to detect NSUN2 deficiency induced genome-wide expression alterations. In total, 2681 differentially expressed genes (DEGs) were found between NSUN2-deficient HepG2 and wild-type HepG2 cells (Fig. 3a, b, Additional file 1). Among them, 1754 genes were upregulated and 927 genes were downregulated. Functional enrichment analysis indicated that the DEGs were mainly involved in biological processes such as cell migration, angiogenesis, cell adhesion, and cell communication. KEGG analysis showed that these DEGs were enriched in 32 pathways, most of which were related to carcinogenesis, including the pathways in cancer, Rap1 signaling pathway, PI3K-Akt signaling pathway, proteoglycans in cancer, and TGF-beta signaling pathway (Fig. 3c).
a Volcano plot of differentially expressed genes (DEGs) after NSUN2 depletion. b Heat-map expression profile of 2681 significantly dysregulated RNAs between HepG2 and NSUN2-deficient HepG2 cells. c Statistical analysis of KEGG pathway enrichment for the DEGs. d Histogram showed the number of total m5C sites and annotated RNAs in HepG2 cells and NSUN2-deficient HepG2 cells. e Statistical analysis of KEGG pathway enrichment for the differentially methylated genes (DMGs).
NSUN2 depletion decreases RNA m5C abundance in HepG2 cells
Genome-wide RNA bisulfite sequencing (RNA-BisSeq) analysis was performed to map and compare transcriptome 5-methylcytosine sites between NSUN2-deficient HepG2 cells and wild-type HepG2 cells (Additional file 2, Fig S1). RNA-BisSeq analysis identified a total of 11,027 m5C candidate sites in wild-type cells, while the number of m5C candidate sites in NSUN2-deficient HepG2 cells was considerably reduced to only 3182 m5C candidate sites (Fig. 3d, Additional file 3). The methylation levels of the candidate methylation sites ranged from 20 to 30%. Sequence context analysis of m5C sites revealed no preferred motif (Additional file 2, Fig S2).
A total of 11,788 m5C candidate sites within 1208 genes were shown to be differentially methylated between HepG2 and NSUN2-deficient HepG2 cells. Differentially methylated genes (DMGs) screened by RNA-BisSeq were further subjected to GO and KEGG pathway enrichment analyses to identify the biological role of NSUN2-mediated RNA m5C methylation. The significantly enriched GO terms of DMGs between NSUN2-deficient HepG2 and wild-type cells were associated with translational initiation (ontology: biological process), membrane (ontology: cellular component) and poly(A) RNA binding (ontology: molecular function). KEGG pathway analysis show enrichment of these DMGs in 20 pathways, including spliceosome, proteoglycans in cancer, cell cycle, estrogen signaling pathway, etc. (Fig. 3e).
To verify the candidate m5C sites identified by RNA-BisSeq, several differentially methylated m5C sites (DMSs) were randomly selected for subsequent bisulfite-PCR amplification and Sanger sequencing. The results indicated that all of the Cs were converted to Ts except the sites identified by RNA-BisSeq (Additional file 2, Fig S3). The methylation levels of the candidate DMSs were different when comparing NSUN2-deficient HepG2 cells and wild-type HepG2 cells, which was consistent with the RNA-BisSeq results.
H19 lncRNA is a substrate of NSUN2 and its m5C modification affects the half-life of H19 RNA
Compared to the results of the transcriptome-wide RNA-BisSeq and RNA-Seq analyses, 52 RNAs showed a significant change in both m5C methylation and RNA expression following NSUN2 depletion, including H19 lncRNA (Additional file 2, Table S1). NSUN2 deficiency resulted in a complete loss of methylation at the H19 RNA C986 site, and this was accompanied a decrease in the H19 RNA level. RT-qPCR results confirmed that H19 expression was reduced by 80% after NSUN2 depletion (Fig. 4a). Bisulfite-PCR sequencing (BPS) using H19-specific primers revealed that methylation of the H19 C986 site was completely ablated in NSUN2-deficient HepG2 cells (Fig. 4b). Additional results from the BPS of H19 RNA confirmed that the methylation level of H19 RNA C986 was reduced from 45 to 1% after NSUN2 depletion (Fig. 4c). Moreover, the re-expression of NSUN2 restored the expression and methylation of H19 RNA to some degree in NSUN2-deficient HepG2 cells (Fig. 4d).
a Deficiency of NSUN2 decreased the level of H19 RNA in HepG2 cells, while restore NSUN2 could recover the level of H19 RNA in NSUN2-deficient HepG2 cells. b Sanger-based validation of m5C sites within H19 RNA. cDNA was amplified by PCR using specific primers for bisulfite-treated RNAs. c Quantitative determination of methylation level of H19 RNA C986 site in normal and NSUN2-deficient HepG2 cells through pyrosequencing. d Pyrosequencing result demonstrated restore of NSUN2 could recover the methylation level of H19 C986 site in NSUN2-deficient HepG2 cells. Mock, cells transfected with empty pcDNA3.1 vector; pNSUN2, cells transfected with pcDNA3.1-NSUN2 vector; HepG2-NKD, NSUN2-deficient HepG2 cells. The results are expressed as the mean ± SEM from three independent experiments, **p < 0.01, ***p < 0.001.
NSUN2-mediated RNA methylation has been reported to affect RNA stability. Thus, we wondered whether H19 lncRNA methylation mediated by NSUN2 could affect H19 lncRNA levels. To test this notion, NSUN2-deficient HepG2 cells were subjected to Actinomycin D (ActD) treatment followed by RT-qPCR to analyze the H19 RNA half-life. As shown in Fig. 5a, the half-life of H19 RNA was markedly shortened after NSUN2 depletion. Furthermore, NSUN2 re-expression restored the half-life of H19 RNA to a certain extent in NSUN2-deficient HepG2 cells (Fig. 5b). As a negative control, the depletion or re-expression of NSUN2 did not affect the half-life of ACT-B mRNA. Furthermore, the specific role of the H19 m5C site in regulating its stability was determined using a luciferase reporter assay with a gene construct containing the wild-type H19 fragment (H19-Wt) or fragment with a mutated m5C site (H19-Mut). As expected, mutation of m5C site significantly inhibited luciferase activity in normal HepG2 cells but not NSUN2-deficient HepG2 cells (Fig. 5c). And, the luciferase activity of H19-Wt was significantly higher in normal HepG2 cells than that in NSUN2-deficient HepG2 cells. Thus, these results suggest that NSUN2-mediated RNA methylation may stabilize H19 lncRNA.
a NSUN2 deficiency decreased the half-life of H19 RNA in HepG2 cells. Cells were exposed to actinomycin D (5 μg/ml), whereupon the cellular RNA was isolated at times indicated. Real-time qPCR against GAPDH was performed to assess the half-lives of H19 RNA and β-actin mRNA b Restore of NSUN2 could recover the half-life of H19 RNA at a certain level in NSUN2-deficient HepG2 cells. Three independent experiments were performed for quantification (mean ± SEM). c HepG2 cells or NSUN2-deficient HepG2 cells were transfected with plasmids pimrGLO-H19-wt or pimrGLO-H19-mut. After 48 h, Firefly luciferase activity was determined and normalized against Renilla luciferase activity. Data are represented as the mean ± SEM from three independent experiments. d H19 expression in 55 pairs of liver cancer tissues and matched no-cancerous liver tissues. The relative expression levels were normalized to the expression amount of the GAPDH gene. e H19 RNA methylation status in 55 pairs of liver cancer tissues and matched no-cancerous liver tissues. f Pearson’s correlation analysis demonstrating that H19 expression is positively correlated with the m5C level in 110 samples (55 liver cancer tissues and 55 matched no-cancerous liver tissues). g NSUN2 mRNA level in 55 pairs of liver cancer tissues and matched no-cancerous liver tissues. h NSUN2 mRNA level in 16 pairs of liver cancer tissues and matched no-cancerous liver tissues with H19 C986 site methylation level difference over 10%. The relative expression levels were normalized to the expression amount of the GAPDH gene. mock, cells transfected with empty pcDNA3.1 vector; pNSUN2, cells transfected with pcDNA3.1-NSUN2 vector; HepG2-NKD, NSUN2-deficient HepG2 cells. *p < 0.05, **p < 0.01.
H19 expression and RNA m5C modification are linked to poor differentiation of HCC
We next determined the expression and methylation levels of H19 RNA in HCC tissues and their matched normal tissues derived from 55 HCC patients. The results of the RT-qPCR showed that H19 expression was significantly increased in HCC tissues compared to matched non-cancerous liver tissues (Fig. 5d). Bisulfite-PCR pyrosequencing (BPP) results demonstrated that the methylation level at the H19 C986 site in HCC tissues was significantly higher than that in matched non-cancerous liver tissues (Fig. 5e). Moreover, the H19 RNA level was significantly correlated (p < 0.01, r = 0.511) with its RNA methylation level in these tissues (Fig. 5f). The mRNA level of NSUN2 was also determined by RT-qPCR. The result showed no significant change in NSUN2 mRNA level between HCC tissues and matched non-cancerous liver tissues (Fig. 5g). However, when compared the tissues with H19 C986 methylation level difference over 10%, the NSUN2 mRNA level was significantly higher in HCC tissues than in matched non-cancerous liver tissues (Fig. 5h).
Further analysis revealed that the m5C methylation level and expression level of H19 RNA in HCC patients are significantly (p < 0.001) associated with the differentiation stages of tumors (Tables 1 and 2), indicating that abnormal NSUN2-mediated m5C modification of H19 lncRNA links to poorly differentiated hepatocellular carcinoma. Meanwhile, no significant differences (p > 0.05) were identified between the RNA m5C modification of H19 and other clinicopathological characteristics, including patient age, sex, extrahepatic metastasis, liver cirrhosis, and tumor size (Table 1 and Table 2). Taken together, our findings suggest that RNA methylation level of H19 is increased in HCC and is positively correlated with its expression, which significantly links to malignant HCC.
Methylated H19 lncRNA interacts with oncoprotein G3BP1
The m5C modification within XIST and HOTAIR lncRNAs was shown to modulate their interaction with PRC2. Thus, we hesitated that m5C-modified H19 lncRNA may regulate its interaction with other proteins. An H19 lncRNA-binding protein, G3BP1 was identified by chromatin isolation by RNA purification (CHIRP) using H19-specific probes and subsequent mass spectrometry analysis (Fig. 6a, Additional file 2, Fig S4, Additional file 4). Moreover, G3BP1 pulled down by the H19 RNA probe was detectable in wild-type HepG2 cells but not in NSUN2-deficient HepG2 cells, meanwhile, no significant change was detected in G3BP1 protein level between wild-type HepG2 cells and NSUN2-deficient HepG2 cells (Fig. 6b), suggesting that the interaction between H19 RNA and G3BP1 protein might be influenced by NSUN2. This notion was confirmed by RNA-binding protein immunoprecipitation (RIP) assay using a G3BP1 antibody (Fig. 6c). The fact that G3BP1 bound specifically to H19 lncRNA in wild-type HepG2 cells but not in NSUN2-deficient HepG2 cells suggests that the interaction of H19 lncRNA with G3BP1 protein may depend on NSUN2-mediated RNA methylation. Further rEMSA experiment was done using purified recombinant human G3BP1 protein and H19 RNA probes with or without m5C modification. The result showed that G3BP1 recombinant protein preferably binds to m5C-modified oligonucleotides (Fig. 6d), indicating the binding of G3BP1 protein and H19 RNA was regulated by m5C modification.
a Proteins retrieved by H19 probes visualized by silver staining. b G3BP1 protein level in HepG2 and NSUN2-deficient HepG2 cells were assessed by western blotting. c RIP was performed using HepG2 or NSUN2-deficient HepG2 cell lysate and either anti-G3BP1 or Normal Rabbit IgG as the immunoprecipitating antibody. Purified RNA was analyzed by real-time qPCR or RT-PCR using primers specific for H19 or GAPDH. Data are represented as means ± SEM from three independent experiments. d In vitro protein-RNA-binding assays (rEMSA) were performed using H19 oligonucleotides with or without m5C as well as the indicated amounts of purified G3BP1 protein (200–400 ng/reaction). HepG2-NKD, NSUN2-deficient HepG2 cells.
Discussion
Dysregulation of epigenomic modifications, including DNA methylation, histone modifications, and RNA methylation, plays an important role in tumorigenesis. In recent years, accumulating evidence has demonstrated that RNA m6A modification is implicated in the initiation, progression and drug response of multiple human cancers [27,28,29]. NSUN2, an m5C methyltransferase of mammalian RNA, was reported to be highly expressed in multiple tumors. In the present study, we found that depletion of NSUN2 led to inhibition of the proliferation, migration, and invasion of hepatocellular carcinoma cells in vitro. Consistently, the in vivo orthotopic tumor xenograft results also confirmed that NSUN2 deficiency suppressed HCC growth in nude mice. Furthermore, NSUN2 was shown to methylate H19 lncRNA and affect its stability. The Ras-GTPase-activating protein-binding protein 1 (G3BP1) was confirmed to bind methylated H19 RNA based on the presence of NSUN2. Moreover, lncRNA H19 was shown to be overexpressed, and its overexpression accompanied by hypermethylation in HCC tissues compared to matched normal liver tissues; in addition, high levels of H19 RNA methylation and expression were correlated with HCC poor differentiation. It is well known that tumor poor differentiation is an important feature of malignant tumors, which directly affects tumor progression and prognosis. Taken together, these results suggest that abnormal NSUN2-mediated RNA m5C modification represents a mechanism for the progression of human maglinant HCC, and it may serve as a prognostic biomarker to distinguish HCC patients.
Interestingly, NSUN2 is a direct target gene of MYC and is upregulated upon MYC activation. RNAi of NSUN2 blocked MYC-induced keratinocyte proliferation and cell-cycle progression. The growth of human squamous cell carcinoma xenografts was also inhibited by NSUN2 knockdown [4]. Here, we showed that NSUN2 deficiency decreased the proliferation, migration, and invasion of HepG2 cells. Moreover, NSUN2 deficiency resulted in smaller G1 and S populations and a larger G2/M population than those observed in normal cells, suggesting that NSUN2 depletion might block cells in the G2 phase and therefore suppress the proliferation of HepG2 cells. Previous reports have shown that NSUN2 is dynamically expressed during the cell division cycle, with the lowest level in the G1 phase and the highest level in the S phase [4, 30]. The dynamic distribution and expression of NSUN2 during the cell-cycle imply that NSUN2 may play an important role in regulating cell division. Wang et al. found that NSUN2-mediated methylation of CDK1 mRNA regulates CDK1 translation and thus affects the cell cycle [10]. Furthermore, in our study, functional enrichment analysis of DMGs revealed that many DMGs, including HSP90AA1, CKAP5, TUBB, and CDK2, were enriched in the cell-cycle pathway (hsa04110) and related processes (such as GO:0000086-G2/M transition of mitotic cell cycle). These results suggest that NSUN2 may regulate the cell cycle through RNA methylation, thereby affecting the proliferation of cancer cells.
Transcriptome sequencing identified 2681 DEGs and 1208 DMGs in HepG2 cells. Among them, 52 genes including the lncRNA H19 had changes in both RNA expression and methylation level. In many recent studies, H19 was identified as an onco-lncRNA and to promote tumorigenesis, such as in glioblastoma [31], colorectal cancer [32], and gastric cancer [33]. In HepG2 cells, H19 RNA was methylated at C986 site and this methylation was completely lost after NSUN2 depletion. Moreover, restore of NSUN2 could recover this methylation to some degree, but not the original level. We speculated this may be mainly caused by the NSUN2 overexpression method we used, the transient transfection efficiency of HepG2 cells is relatively low, and a large number of cells with no restore of NSUN2 pulled down the recovery level of H19 methylation.
Depletion of NSUN2 lead to reduced methylation of H19 RNA, which was also accompanied by decreased expression of H19. Many previous reports have shown that NSUN2-mediated RNA m5C modification may exert its function mainly by affecting translation of the RNA rather than by altering the RNA level [10, 11]. A recent study showed that in human urothelial carcinoma of the bladder (UBC), NSUN2-mediated m5C modification stabilizes oncogene RNA through the “reader” YBX1, and hypermethylated m5C levels correlate with oncogene mRNA overexpression in UBC cells [8]. NSUN2 may also play a similar role in lncRNAs. Here, we showed that NSUN2 deficiency significantly decreased the half-life of H19 RNA, which might be regulated by NSUN2-mediated m5C modification, since mutation of the methylation site could significantly inhibit luciferase activity of reporter gene in normal HepG2 cells but not NSUN2-deficient HepG2 cells.
H19 lncRNA has been confirmed to be highly expressed in many organs from early embryonic development to the fetal stage, but not after birth [34]. However, reactivation of H19 expression has been detected in many adult malignant tumors, including breast cancer [35], lung cancer [36], and bladder cancer [37]. Similarly, our results demonstrated that H19 is frequently upregulated in HCC tissues compared to matched non-cancerous liver tissues, and a higher level of H19 expression is correlated with HCC poor differentiation. Moreover, hypermethylation of H19 RNA is observed in HCC tissues compared to matched normal liver tissues, which is also correlated with HCC malignancy. Unexpectedly, the expression and methylation level of H19 RNA did not show significant correlation with clinical indicators such as tumor size and metastasis. It seems that H19 may not be the best target for the function of NSUN2 in tumor which NSUN2 deficiency significantly inhibited cancer cell proliferation and migration. We speculate that this may be due to improper sample selection. For example, in 55 clinical cases, 41 cases did not show metastasis, while only 14 cases were metastatic. In addition, the limited sample size may also be one of the reasons for this phenomenon. Moreover, NSUN2 may participate in regulating tumor progression through multiple mechanisms, which needs more research to explore. Maybe, further study in more tumor samples and multiple types of tumors can solve this. Abnormally high level of lncRNA and DNA m5C hypermethylation have been used as diagnostic tools in many types of tumors. Our study provided a mechanism by which RNA m5C methylation may act as a new diagnostic marker for tumors.
Most lncRNAs function by binding to proteins. A previous study demonstrated that methylated cytosine of the lncRNAs XIST and HOTAIR affects their recruitment of the chromatin-modifying complex PRC2 [17]. We tested whether NSUN2 influenced the ability of H19 RNA to bind with other proteins. Through ChIRP assays, we found that G3BP1 specifically binds to H19 RNA in normal HepG2 cells but not in NSUN2-deficient HepG2 cells, and RIP assays also confirmed this specific interaction. Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1), a known oncoprotein, is overexpressed in a variety of human cancers, such as breast cancer [38], renal cell carcinoma [39], and hepatocellular carcinoma [40]. G3BP1 was shown to be repelled by m6A and to positively regulate mRNA stability in an m6A-regulated manner [41]. Besides, a previous study found that G3BP1 protein was also pulled by the affinity chromatography of RNA m5C [9]. Thus, we hypothesized that G3BP1 may also bind to H19 RNA by recognizing m5C, and this was confirmed by further rEMSA assay. G3BP1 specific binds m5C modified H19 oligos, but not oligos without m5C. These results therefore suggested that NSUN2-mediated methylation of H19 RNA regulated its interaction with G3BP1 protein.
G3BP1 regulates a variety of carcinogenesis-related pathways, including Ras [42], Wnt/β-catenin, PI3K/AKT [43], and NF-κB/Her2 signaling pathways [44]. NSUN2 may affect these signaling pathways by regulating the binding of H19 RNA to G3BP1 and then affect the progression and malignancy of cancer cells. Moreover, G3BP1 was shown to bind MYC mRNA and promote its decay [45]. Methylated H19 RNA may compete with MYC mRNA for binding to G3BP1, promoting tumor cell progression. Hypomethylated H19 RNA will lose binding ability of G3BP1, and lead to an increase in binding ability between G3BP1 and MYC mRNA, which increases the decay rate and decreases the mRNA level of MYC, thus suppressing cell proliferation. In addition, MYC has been shown to bind to the promoter region of H19 and promote the transcription of H19 RNA [31]. The reduction of MYC expression may further decrease the H19 RNA level. Thus, we propose a regulatory axis: MYC-NSUN2-H19-G3BP1-MYC (Additional file 2, Fig. S5). In tumors, high expression of NSUN2 leads to increased methylation levels of H19 RNA. Hypermethylated H19 RNA may competitively bind to G3BP1, which further delays MYC mRNA decay and leads to a further increase in MYC levels, resulting in consequent deterioration of tumors. Nevertheless, this axis remains to be fully characterized in future studies.
In conclusion, our results revealed that the NSUN2-mediated m5C modification of H19 lncRNA exert an important function in the progression and malignancy of hepatocellular carcinoma. H19 RNA m5C methylation might be a new target and biomarker for HCC treatment and diagnosis.
Materials and methods
Cell culture and transfection
The human hepatocellular carcinoma cell line HepG2 was purchased from ATCC (HB-8065). For the details of cell culture, see the Supplementary of Materials and methods.
Plasmid construction
All primers used for plasmid construction are shown in Additional file 2, Table S2. For the details, see the Supplementary of Materials and methods.
Generation of NSUN2-deficient HepG2 cells
The NSUN2-deficient HepG2 cell line was generated via a CRISPR-Cas9-mediated method as previously described [46]. For the details, see the Supplementary of Materials and methods.
Human liver cancer specimens
Fifty-five pairs of HCC tissues and matched non-cancerous liver tissues were surgically removed following the guidelines approved by the Institutional Review Board of Chinese PLA General Hospital. For the details, see the Supplementary of Materials and methods.
RNA preparation and real-time PCR
The specific primers used for amplification are shown in Additional file 2, Table S3. For the details of qRT-PCR, see the Supplementary of Materials and methods.
CCK-8 assay
Cell viability was determined using the CCK-8 assay. For the details, see the Supplementary of Materials and methods.
Colony formation assay
Colony formation assay was performed as previously described with several modifications [47]. For the details, see the Supplementary of Materials and methods.
Wound healing assay
A wound healing assay was performed as previously described with several modifications [48]. For the details, see the Supplementary of Materials and methods.
Transwell invasion assay
Transwell invasion assay was performed as previously described with several modifications [13]. For the details, see the Supplementary of Materials and methods.
Xenografted tumor model
All mice experiments were performed according to the institutional guidelines and were approved by the Institutional Animal Care and Use Committee. For the details, see the Supplementary of Materials and methods.
Tube formation assay
The impact of NSUN2 deletion on in vitro angiogenesis of HepG2 was determined by tube formation assay. For the details, see the Supplementary of Materials and methods.
Gene expression profiling
Gene-expression profiling were described in previous studies [47]. For the details, see the Supplementary of Materials and methods.
Library construction and sequencing of bisulfite-converted RNAs
RNA-BisSeq were described in previous studies [47]. Primers for the validation of the candidate m5C sites are listed in Additional file 2 and Table S4. For the details, see the Supplementary of Materials and methods.
Bisulfite-PCR pyrosequencing
Primers for amplification and sequencing and nucleotide dispensation orders are listed in Additional file 2 and Table S5. For the details, see the Supplementary of Materials and methods.
RNA half-life measurement
The stability of the H19 RNA was assessed by the addition of actinomycin D (ActD, 5 μg/ml) into the cell culture medium. For the details, see the Supplementary of Materials and methods.
Luciferase reporter assay
HepG2 cells and NSUN2-deficient HepG2 cells were seeded into 24-well plates 24 h before transfection with pmirGLO-derived vectors. Forty-eight hours after transfection, cell lysates were collected and the firefly and Renilla luciferase activities were measured with a Dual Luciferase Reporter Assay system (Vazyme) following the manufacturer’s instructions. All firefly luciferase measurements were normalized to Renilla luciferase measurements from the same sample.
Antibodies and western blot analysis
Anti-NSUN2 (Abcam, UK, ab272624, 1:1000), anti-G3BP1 (Santa Cruz, America, sc-365338, 1:500), and anti-GAPDH (CWBIO, China, CW0101, 1:5000) antibodies were used. For the details of western blot, see the Supplementary of Materials and methods.
Chromatin isolation by RNA purification and mass spectrometry analysis (ChIRP-MS)
CHIRP was performed as previously described with slight modifications [49]. For the details, see the Supplementary of Materials and methods.
RNA-binding protein immunoprecipitation (RIP)
The RIP experiment was performed as described by Tenenbaum et al. [50] For the details, see the Supplementary of Materials and methods.
RNA electrophoretic mobility shift assay
rEMSA was performed as previously described with some modifications [9]. For the details, see the Supplementary of Materials and methods.
Statistical analysis
All statistical results were analyzed using IBM SPSS Statistics 21 software. For the details, see the Supplementary of Materials and methods.
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Acknowledgements
This research was supported by the the National Key Research and Development Program in China (grant nos. 2016YFC1303604 and 2018YFA0208902), the National Natural Science Foundation of China (grant nos. 81773013, 91540117, and U1604281) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (Animal Science and Veterinary Medicine).
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Sun, Z., Xue, S., Zhang, M. et al. Aberrant NSUN2-mediated m5C modification of H19 lncRNA is associated with poor differentiation of hepatocellular carcinoma. Oncogene 39, 6906–6919 (2020). https://doi.org/10.1038/s41388-020-01475-w
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DOI: https://doi.org/10.1038/s41388-020-01475-w
