Introduction

During the past a few years, RNA modifications have been found to play an important role in the occurrence and development of many tumors. More than 100 types of chemical modifications have been identified in various types of RNAs, with methylation being the most common1. Methylation is a prevalent post-transcriptional modification that occurs in almost all RNA species. N6-methyladenosine (m6A) is the most abundant internal modification in mammalian messenger RNA (mRNAs) and widely involved in various biological processes of mRNAs2,3,4. Recently, many studies revealed that aberrant m6A modification is closely related to tumorigenesis, including acute myeloid leukemia5, hepatocellular carcinoma6,7, breast cancer8,9, bladder cancer10,11, cervical cancer12, and lung cancer13.

Another important RNA modification, 5-methylcytosine(m5C), was first identified in stable and highly abundant transfer RNAs (tRNAs) and ribosome RNAs (rRNAs)14. Recently, m5C modification and related m5C sites have been found in mRNA by advanced high-throughput techniques combined with next-generation sequencing in mRNAs. Yang et al.15 found that NSUN2 (NOP2/Sun domain family, member 2; MYC-induced SUN domain–containing protein, Misu) was the main enzyme catalyzing m5C formation, while Aly/REF export factor (ALYREF, an mRNA transport adaptor, also named THOC4) functioned as a specific mRNA m5C-binding protein regulating mRNA export. It was found that m5C could promote the pathogenesis of bladder cancer through stabilizing mRNAs16. Recent studies showed that NSUN2 was linked to cell proliferation, stem cell differentiation and testis differentiation17,18. Wang and colleagues19 found that NSUN2 could delay the replicative senescence by repressing Cyclin-dependent kinase inhibitor 1B (CDKN1B, p27Kip1) translation and promote cell proliferation by elevating Cyclin-dependent kinase 1 (CDK1) translation20. Moreover, elevated protein expression of NSUN2 was found in various types cancers, including the esophageal, stomach, liver, pancreas, uterine cervix, prostate, kidney, bladder, thyroid, and breast cancers by immunohistochemistry (IHC) analysis21. Indeed, Wang and colleagues22 found that NSUN2 was associated with metastatic progression by affecting DNA hypomethylation in human breast cancer. Gao et al.23 also found NSUN2 could promote tumor progression via its interacting partner RPL6 in gallbladder carcinoma. However, the role and related mechanisms of NSUN2 in gastric cancer has not been investigated.

In the present study, we firstly showed that NSUN2 was significantly upregulated in gastric cancers, compared to adjacent normal gastric tissues. Moreover, NSUN2 could promote the gastric cancer cells proliferation both in vitro and in vivo. Further study demonstrated that p57Kip2 was the potential downstream gene regulated by NSUN2 in gastric cancer. Furthermore, NSUN2 could promote gastric cancer cell proliferation by repressing p57Kip2 in an m5C-dependent manner. This study suggested that NSUN2-mediated m5C methylation of p57Kip2 mRNA may serve as novel mechanism for gastric cancer development and progression.

Results

NSUN2 was upregulated in human gastric cancers compared to adjacent normal gastric tissues

Firstly, TCGA database analysis showed that NSUN2 was upregulated in tumors compared to adjacent normal gastric tissues (Fig. 1a, b). Meanwhile, to determine NSUN2 expression in gastric cancer tissues, we examined expression of NSUN2 in gastric cancer patients’ tissues by performing quantitative real-time PCR (qRT-PCR) and western blot assay. As shown in Fig. 1c–e, both the mRNA and protein expressions of NSUN2 was significantly upregulated in gastric tissues, compared to corresponding adjacent normal gastric tissues. These findings implied that NSUN2 was upregulated in human gastric cancer, compared to adjacent normal gastric tissues.

Fig. 1: NSUN2 was upregulated in human gastric cancer tissues, compared to adjacent gastric normal tissues.
figure 1

a, b NSUN2 was significantly upregulated in gastric cancer tissues, compared with adjacent gastric normal tissues from the TCGA database, *p < 0.05; c Relative expression of NSUN2 mRNA in gastric cancer tissues and compared with corresponding adjacent normal gastric tissues. NSUN2 expression was examined using qRT-PCR and normalized to β-actin expression. The horizontal lines and numbers represent the median values of the distribution. *p < 0.05. d Expression of NSUN2 at protein level in eight paired gastric cancer tissues and adjacent normal gastric tissues by western blot. T: Gastric tumor tissues, N: Adjacent normal tissues. e The protein levels of NSUN2 were quantified by densitometry and the relative gray value of NSUN2 protein (normalized to β-actin) in gastric cancer tissues and adjacent gastric normal tissues was statistically significant. *p < 0.05.

NSUN2 promoted the human gastric cancer proliferation in vitro

Firstly, qRT-PCR and western blot assay revealed that NSUN2 was stably knockdown or overexpression in SGC 7901 and MGC 803 cells (Fig. 2a). Subsequently, the CCK-8 assay and colony formation assay showed that NSUN2 knockdown significantly inhibited cell proliferation and colony formation, whereas NSUN2 overexpression promoted cell proliferation and colony formation (Fig. 2b, c). Flow cytometry analysis further revealed that NSUN2 knockdown induced at the G1/G0 cell cycle arrest and NSUN2 overexpression decreased the percentage of G1/G0 phase, compared with wild-type cells, both in SGC 7901 and MGC 803 cells (Additional file: Supplementary Fig. S1a, b). Taken together, our results suggested that NSUN2 could promote the gastric cancer cell proliferation in vitro.

Fig. 2: NSUN2 promoted the human gastric cancer proliferation in vitro.
figure 2

a qRT-PCR analysis of overexpress and knockdown of NSUN2 by lentiviral constructs in MGC 803 and SGC 7901 cells. NSUN2, pLOV-NSUN2; Ctrl, pLOV-control vector shNSUN2#2, pLKD-shNSUN2-1; shNSUN2#3, pLKD-shNSUN2-2; shCtrl, pLKD-control vector. Data were presented as the mean ± SD; *p < 0.05. b, c CCK-8 and colony formation assays were performed to determine the growth ability of overexpress and knockdown of NSUN2. Data were presented as the mean ± SD; *p < 0.05.

NSUN2 promoted human gastric cancer tumorigenesis in vivo

MGC 803 cell line was used in this experiment. Firstly, stable NSUN2 overexpress cells, NSUN2 knockdown cells and their corresponding wild-type cells were injected into female nude mice. Up to 3 weeks after injection, we found that stable knockdown of NSUN2 suppressed tumor growth in nude mice effectively. The average tumor weight and volume of NSUN2 knockdown cells group were also significantly lower than those in the wild-type group (Fig. 3a, b). Moreover, IHC analysis confirmed that the tumors formed from the wild-type cells group displayed stronger Ki-67 staining than those from NSUN2 knockdown cells group (Fig. 3c). whereas, overexpression of NSUN2 significantly promoted tumor growth in nude mice (Fig. 3d–f). Our results indicated that NSUN2 could promote human gastric cancer tumorigenesis in vivo.

Fig. 3: NSUN2 promoted human gastric cancer tumorigenesis in vivo.
figure 3

NSUN2 overexpress cells, NSUN2 knockdown cells and corresponding wild-type cells were injected into female nude mice subcutaneously. a, d Tumor-bearing mice and tumors were shown. Data were showed as the mean ± SD; *p < 0.05. b, e Tumor size and weight were measured after 3 weeks. Data were showed as the mean ± SD; *p < 0.05. c, f HE and IHC staining of NSUN2, p57Kip2, and Ki-67 expression in tumor sections at ×200 magnification. Scale bars indicated 10 μm.

p57KIP2 was identified as potential target regulated by NSUN2 by RNA sequencing (RNA-seq)

To identify the downstream targets regulated by NSUN2 in gastric cancer, we performed RNA-Seq assay to determine the mRNA expression changes in NSUN2 knockdown and corresponding wild-type cells. In this study, Kyoto Encyclopedia of Genes and Genome (KEGG) analysis revealed that differentially expressed genes were significantly changed in ribosome, endocytosis, cell cycle, protein processing in endoplasmic reticulum and apoptosis, suggesting that NSUN2 may play important roles in protein modification and cell proliferation (Table 1). As cell proliferation plays an important role in tumor development, we selected cell cycle related pathways as a candidate targets of NSUN2 for further study. In total, we found 86 differential genes expression from cell cycle related pathways in NSUN2 stable knockdown cells, compare with corresponding wild-type cells (Table 2). We then found that p57Kip2 was obviously upregulated among these genes and was identified as potential target regulated by NSUN2.

Table 1 Enrichment list of differential gene in KEGG, which indicated that cell cycle pathways were significantly associated with NSUN2.
Table 2 Ten of 86 differential genes enriched in cell cycle pathway of RNA-Seq.

NSUN2 destabilized the p57Kip2 transcript and it may be involved in the oncogenic function of NSUN2 in gastric cancer

The level of p57Kip2 was remarkably upregulated in NSUN2 knockdown cells identified by qRT-PCR and Western blot (Fig. 4a, b). In addition, we found the relative half-life of p57Kip2 mRNA increased from 3.44 to 6.26 h in MGC 803 cells, and from 4.93 to 7.14 h in SGC 7901 cells, following the NSUN2 knockdown (Fig. 4c). We silenced p57Kip2 via transfecting with siRNA in NSUN2 knockdown cells and wild-type cells (Fig. 4d, e). Upon silencing of p57Kip2 expression by siRNAs, the ability of cell proliferation in both NSUN2 knockdown cells and wild-type cells was enhanced (Fig. 4f, g). These results suggested that NSUN2 might exert its oncogenic effects in gastric cancer cells by repressing p57Kip2 expression.

Fig. 4: p57KIP2 was identified as potential target regulated by NSUN2 by RNA sequencing.
figure 4

a RNA was isolated from SGC 7901 and MGC 803 cells with stable knockdown of NSUN2 and p57Kip2 mRNAs expression was determined by qRT-PCR. Data were showed as the mean ± SD; *p < 0.05. b The protein levels of p57Kip2 and β-actin was assessed by western blot. c The mRNA half-life (t1/2) of p57Kip2 in SGC 7901 and MGC 803 cells with stable knockdown of NSUN2 or corresponding wild-type cells. d, e The relative mRNA and protein expression of p57Kip2 in NSUN2 knockdown cells or corresponding wild-type cells, transfected with small-interfering RNAs (siRNAs), was tested using qRT-PCR and western blot. siNC: negative control siRNA; sip57Kip2: siRNA against p57Kip2. Data were showed as the mean ± SD; *p < 0.05. f, g CCK-8 and colony formation assays were performed to determine the growth ability of NSUN2 knockdown cells after transfection of siRNA against p57Kip2. Data were showed as the mean ± SD; *p < 0.05.

NSUN2 destabilized the p57Kip2 mRNA relies on its methyltransferase activity and m5C modifications in the 3′- untranslated region (UTR) of p57Kip2 mRNA

The Dot blot assay showed that NSUN2 knockdown significantly decreased the m5C levels, whereas NSUN2 overexpression increased the m5C levels (Fig. 5a). To assess the role of the m5C modifications in p57Kip2 mRNA regulated by NSUN2, we conducted wild-type and mutant 3′-UTR of p57Kip2 reporter plasmids for the luciferase reporter assays (Fig. 5b). Relative luciferase activity of the wild-type and mutant 3′-UTR of p57Kip2 reporter genes was measured in MGC 803 and SGC 7901 cells. As expected, the luciferase activity of the wild-type 3′-UTR of p57Kip2 reporter gene was significantly enhanced after NSUN2 silencing. However, knockdown of NSUN2 had no effect on the expression of the mutated 3′-UTR of p57Kip2 reporter gene (Fig. 5c). More importantly, by m5C RNA immunoprecipitation (RIP) assay and qPCR assay, we found that m5C antibody significantly enriched 3′-UTR of p57Kip2 mRNA and knockdown of NSUN2 reduced the m5C levels on 3′-UTR of p57Kip2 mRNA (Fig. 5d). Altogether, our data indicated that NSUN2 destabilized the p57Kip2 mRNA relies on its m5C methyltransferase activity in its 3′-UTR.

Fig. 5: NSUN2 destabilized the p57Kip2 mRNA relies on its methyltransferase activity and m5C modifications in the 3′- UTR of p57Kip2 mRNA.
figure 5

a m5C dot blot assay of overexpress or knockdown of NSUN2 in MGC 803 and SGC 7901 cells, methylene blue staining (as control). b, c Pezx-FR02- p57Kip2 -3′-UTR plasmid with either wild-type or mutant (CCT mutation) m5C sites were constructed. The pattern diagram was shown. Above constructed plasmid was transfected into stable knockdown of NSUN2 or corresponding wild-type cells. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data were presented as the mean ± SD; *p < 0.05. d m5C RIP and qRT-PCR analysis of m5C level in mRNA of p57Kip2 in MGC 803 cells transduced with stable knockdown of NSUN2 or corresponding wild-type cells. Data were showed as the mean ± SD; *p < 0.05.

Discussion

Gastric cancer is the most common gastrointestinal tumors, representing one of the leading causes of cancer-related deaths worldwide24,25. Despite the improvement in surgical techniques and patient management, there has been unsatisfactory improvement in the 5-year overall survival rate. Many patients were diagnosed with advanced stages that limited the successful therapeutic strategies. Furthermore, the molecular mechanisms underlying gastric cancer progression is still poorly understood. Therefore, better understanding of the tumor formation and diagnostic markers will improve the diagnosis and treatment of gastric cancer.

m5C modification is another important post-transcriptional RNAs modification beside m6A modification. As a main m5C methyltransferase, NSUN2 was reported to promote cell proliferation, mobility, invasion in breast cancer22, gallbladder carcinoma23, and associated with poor prognosis in head and neck squamous carcinoma by bioinformatics analysis26. However, NSUN2 was few studied in tumors formation related its m5C modification activity, especially gastrointestinal cancers. In this study, we found NSUN2 was upregulated in gastric cancer tissues, compared to adjacent normal gastric tissues in mRNA and protein levels. Interestingly, there is much more heterogeneity at protein than mRNA levels both in gastric cancer tissues and normal adjacent tissues. There may be multiple reasons for inconsistent changes in proteins and mRNAs27,28. Post-translational regulation of NSUN2 may be one of the important reasons, which may be different in various gastric cancer cases. Its detailed mechanism needs further investigation. Subsequently. We also found NSUN2 could increase gastric cancer cells proliferation both in vitro and in vivo significantly.

Subsequent RNA-seq and KEGG analysis found that cell cycle was the main pathway regulated by NSUN2 in gastric cancer. Cell cycle dysregulation is a hallmark of cancer due to uncontrolled proliferative signaling29. The affected transitions in the cell cycle are regulated by the balanced activities of cyclin-dependent kinases (CDKs) and CDK inhibitors. Although p57Kip2 might not be the only targeted gene of NSUN2, our results confirmed that p57Kip2 was an important downstream gene regulated by NSUN2 in gastric cancer. p57Kip2 is the recently found CDK inhibitors of the Cip/Kip family, and has been involved in many biological processes, including cell cycle control, differentiation, apoptosis, tumorigenesis and development30,31. Recent studies indicated that p57Kip2 was frequently downregulated in multiple types of human cancers such as breast cancer, hepatocellular carcinoma, colorectal cancer, and ovarian cancer32,33. Importantly, De and colleagues34 found that p57Kip2 could serve as a tumor suppressor in gastric cancer. In our study, we found that the expression level of NSUN2 was negatively correlated with p57Kip2 and the ability of NSUN2 knockdown cells proliferation was enhanced after p57Kip2 silencing in gastric cancer. It revealed another regulatory mechanism that NSUN2 play an oncogenic role by repressing p57Kip2 expression in gastric cancer.

Previous studies showed that RNA methylase play potential role in regulating mRNA decay, translation, and processing35. In our study, we demonstrated that relative half-life of p57Kip2 mRNA increased in NSUN2 knockdown cells. These results indicated that RNA methyltransferase NSUN2 could affect p57Kip2 mRNA stability. Wang et al. found that different functional mechanisms of RNA methyltransferase NSUN2 depended on the location of methylation by m5C modification19,20,36,37,38. Our dual-luciferase reporter assay also found that the luciferase activity of the wild-type 3′-UTR of p57Kip2 reporter gene was significantly enhanced, compared with the mutated 3′-UTR of p57Kip2 reporter gene. Subsequently, our m5C RIP and qRT-PCR assays found that m5C antibody significantly enriched 3′-UTR of p57Kip2 mRNA and knockdown of NSUN2 reduced the m5C levels on 3′-UTR of p57Kip2 mRNA, indicating that RNA methyltransferase NSUN2 regulated p57Kip2 expression by m5C modification in 3′-UTR of its mRNA. Based on the above results, we elaborated a novel mechanism indicating that NSUN2 mainly methylated the 3′-UTR of p57Kip2 mRNA, which led to the downregulation of p57Kip2 at the RNA level and related protein levels. In the present study, we explored the effects of NSUN2 on gastric cancer cell cycle progression and demonstrated that NSUN2 could repress p57Kip2 by an m5C-dependent manner.

In summary, we found that NSUN2 acted as an oncogene through promoting gastric cancer development by repressing p57Kip2 in an m5C-dependent manner, which may provide a novel therapeutic target against gastric cancer.

Materials and methods

Bioinformatics analysis

Clinical data for Bioinformatics analysis were downloaded from The Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov/), and applied for analyzing the expression of NSUN2 in 415 gastric cancer tissues and 34 normal tissues.

Clinical sample

Twenty pairs of gastric tumor and adjacent normal tissues were obtained from the First Affiliated Yijishan Hospital with Wannan Medical College from 2017 to 2018. All samples were obtained with written informed consent from patients and the ethics committee of Wannan Medical College approved these tissues for research use.

Cell culture and transfection

Human gastric cancer cell lines MGC 803 and SGC 7901 were obtained from the Chinese Academy of Sciences Committee on Type Culture Collection Cell Bank (shanghai, China), and has recently been tested for mycoplasma contamination. The cells were maintained in RPMI 1640 medium (GIBCO, USA) supplemented with 10% fetal bovine serum (GIBCO, USA), 100 U/ml penicillin–streptomycin (GIBCO, USA). All the cell lines were maintained at 37 °C, 5% CO2.

Lentivirus constructs for NSUN2 overexpression and knockdown were obtained from Obio Technology (Shanghai, China), and generated as described previously39. Briefly, the gastric cancer cells were stably transfected with NSUN2 overexpression lentivirus (termed as NSUN2) and negative control (termed as Ctrl) using polybrene (Obio Technology, China). Similarly, cells were stably transfected with negative control (termed as shCtrl) and NSUN2 knockdown lentivirus (termed as shNSUN2#2 and #3). Subsequently, stably transfected gastric cancer cells were used for further studies by selection using puromycin (5 μg/ml) for 1–2 weeks.

To explore the further relationship between NSUN2 and p57Kip2 in gastric cancer cells, small-interfering RNAs (siRNAs) against p57Kip2 or negative control RNAs were purchased from GenePharma (Shanghai, China), and transfected into the stably transfected gastric cancer cells using EndoFectinTM-Max (GeneCopoeia, China) according to the manufacturer’s instructions.

RNA isolation, reverse transcription, and qRT-PCR

Total RNA was isolated from tissues and cell lines using Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol. cDNA was synthesized by reverse transcription by 1 or 2 μg RNA using PrimescriptTMreverse transcription reagent (Takara, Japan). qRT-PCR was performed with SYBR Green PCR Kit (Takara, Japan) in the StepOnePlus system (Applied Biosystems, USA). Primer pairs used were as follows: NSUN2, forward primer: 5′-GAACTTGCCTGGCACACAAAT-3′, reverse primer: 5′-TGCTAACAGCTTCTTGACGACTA-3′.

p57Kip2, forward primer: 5′-GCGGCGATCAAGAAGCTGT-3′, reverse primer: 5′-GCTTGGCGAAGAAATCGGAGA-3′.

β-actin, forward primer: 5′-GAACTTGCCTGGCACACAAAT-3′, reverse primer: 5′-TGCTAACAGCTTCTTGACGACTA-3′. The fold change of gene expression was presented by the 2–ΔΔCt method and normalize base on β-actin.

Western blot

Cells were lysed completely in RIPA lysis buffer supplemented with 1% phenylmethanesulfonyl fluoride (PMSF) and 0.1% protease inhibitor cocktail (Beyotime, China) at 4 °C. The protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Total protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The membranes were incubated with primary antibodies and then with specific secondary antibodies after washed three times with 0.1% Tris-HCl with Tween-20 (TBST). The membranes were probed using immobilon Western chemiluminescent horseradish peroxidase substrate (Millipore, USA) and autoradiographed (TANON 5200, China). β-actin antibody was used as internal control. anti-NSUN2 (3H24L11) antibody was provided by ThermoFisher Scientific (USA). anti-p57Kip2 (ab75974) antibody was purchased from Abcam (USA). β-actin (8H10D10), anti-rabbit (#7074), and anti-mouse (#7076) secondary antibodies were obtained from Cell Signaling technology (USA).

RNA m5C dot blot assay

Total RNA was first isolated from stable NSUN2 overexpression and knockdown cells and their corresponding negative control cells, and then treated with deoxyribonuclease I (DNase) according to the manufacturer’s protocol. RNA quality was analyzed by NanoDrop 2000 (Thermo Scientific, USA). Different amounts of RNA (400,600,800 ng) were loaded onto the Amersham Hybond N+ membrane (GE Healthcare) fixed on the Bio-Dot apparatus (Bio-Rad). After ultraviolet rays crosslinking for 5 min at 254 nm, the membrane was blocked with 5% non-fat dried milk in phosphate buffer solution with Tween-20 (PBST) followed by incubation with the primary mouse anti-m5C antibody (ab10805, Abcam, USA) and corresponding anti-mouse secondary antibody. After, the membranes were washed three times with 0.1% PBST, the intensity of the dot blot was determined by autoradiographed and analyzed by image J.

Cell proliferation assay

The transfected gastric cells were monitored in 96 wells at ~2000 cells per well incubated at 37 °C in an atmosphere of humidified air 5% CO2 incubator, and the cell proliferation assay was carried out with cell counting kit-8 (CCK-8, Obio Technology, China) following the manufacturer’s protocol after 5 days of culture. Optical densities (ODs) were measured at 490 nm with a microplate reader (BioTek, USA). For colony formation assay, a certain number of cells were placed into each well of 6-well plates and cultured for 14 days before stained 0.5% crystal violet (Beyotime, China). The colonies with more than 50 cells was manually counted.

Flow cytometry analysis

The transfected gastric cells were seeded in 96-well plates and harvested by trypsinization with 0.25% EDTA (Sigma, USA), when the cells were grown to 80% confluence. Cells for cell cycle were stained with propidium iodide (BD Biosciences, USA) and the percentages of cells in G0/G1, S, and G2/M phase were analyzed by flow cytometry (CtytoFLEX, Beckman,USA) and ModFit LT 5.0software.

Tumor xenograft

Four- to eight-week-old female BALB/c nude mice were purchased from model animal research center of Nanjing University, and randomly divided into four groups (five per group). Five mice/group were subjected to the experiment to have statistical importance. Sable NSUN2 overexpression cells, NSUN2 knockdown cells and their corresponding wild-type cells (6 × 106 cells in 100 ml PBS) were injected into the upper left flank region of each nude mice. Mice were sacrificed and tumor tissues were collected after 3 weeks. Tumor volumes (1/2 × length × width2) and weight were measured in mice. Hematoxylin-eosin (HE) staining and IHC were performed on processed and sectioned tissues from Serviebio company (Wuhan, China). The investigators were not blinded to the mice group during experiments. All procedures were approved by the animal care and use committee of Nanjing Medical University (acceptance no.: IACUC1804027).

RNA-seq

RNA samples were isolated from stable NSUN2 knockdown cells and corresponding negative control cells in MGC 803 cells. The RNA samples were sequenced by the Allwegene Technology Inc (Beijing, China). Three biological replicates for each sample were included in this experiment. The threshold for screening differential genes (DEG) is generally as follows: |log2(Foldchange)| > 1, q-value < 0.05 and read count ≥100. DEG function analysis was by Gene Ontology (GO) and KEGG.

RNA stability assay

To analyze RNA stability, stable NSUN2 knockdown cells and corresponding wild-type cells were treated with actinomycin D (1 μg/ml). Cells were collected at different time points (0, 2, 4, 6, 8 h), and RNA was extracted using Trizol reagent. Reverse transcription was performed using oligo primers and mRNA levels were measured using qRT-PCR.

Dual-luciferase reporter assay

Dual-luciferase reporter assay was performed using Luc-PairTM Duo-luciferase HS assay kit (GeneCopoeia, USA) according to the manufacturer’s instructions. Wild-type and mutant 3′-UTR of p57Kip2 reporter plasmid were constructed from GenePharma (USA). Briefly, wild-type 3′-UTR of p57Kip2 reporter plasmid was made by inserting the 3′-UTR of p57Kip2 transcript after the Fluc coding sequence, and mutant 3′-UTR of p57Kip2 reporter plasmid was made by inserting the 3′-UTR of p57Kip2 transcript without “CCG” sequence according to Yang et al.15. Stable NSUN2 knockdown and corresponding negative control cells were plated in 96 wells dishes and infected with 100 ng of wild-type and mutated 3′-UTR of p57Kip2 transcript reporter plasmid using EndoFectinTM-Max (GeneCopoeia, China). After 24 h, cells were collected and assayed with NanoDrop 2000 (Thermo Scientific, USA). Firefly luciferase (F-luc) was used to assess the effect of m5C modification on p57Kip2 expression. Renilla Luciferase (R-luc) was used to standardize the transfection efficiency of the reporter plasmid.

m5C RNA RIP assay

For m5C RIP, the standard procedure was described as previous study with some modifications37,38. Briefly, total RNAs were firstly isolated and treated with DNase. Then, total RNAs were chemically fragmented (~100 nucleotide) with 1×fragmentation buffer (100 mM Tris-HCl, 100 mM ZnCl2) and incubated with the m5C antibody (Abcam, USA) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% (vol/vol) Igepal CA-630). The IP samples were washed with elution buffer [1×IP buffer, 6.7 mM 5-methylcytosine hydrochloride (Sigma, USA)]. Enrichment of m5C containing mRNA was analyzed by RT-PCR.

Statistical analysis

All experiments in this study were repeated in triplicate, unless otherwise specified. All dates were presented as the mean ± SD, and student’s t-tests (unpaired, two-tailed) were performed using the SPSS 19.0 software (SPSS, Chicago, IL, USA) and graphical presentations were conducted with GraphPad Prism 7.0 software (San Diego, CA). p-value < 0.05 was statistically significant.