NXPH4 mediated by m5C contributes to the malignant characteristics of colorectal cancer via inhibiting HIF1A degradation.

OBJECTIVE: Colorectal cancer (CRC) is a form of malignancy that exhibits a comparatively elevated occurrence and fatality rate. Given the relatively slower progress in diagnostic and therapeutic approaches for CRC, there is a need to investigate more accurate and efficient biomarkers. METHODS: Core regulatory genes were screened using the TCGA database, and the expression of neurexophilin 4 (NXPH4) and its prognostic implications were validated using tissue microarray staining. The assessment of NXPH4 functions involved a range of experiments, including cellular, organoid, and murine models. Furthermore, a regulatory network between m5C, NXPH4, and HIF1A was established through several in vitro experiments. RESULTS: The overexpression of NXPH4 is associated with unfavorable prognoses in patients with CRC and hepatocellular carcinoma. Additionally, it facilitates the progression of malignant tumors both in laboratory settings and in living organisms of colorectal carcinoma. Our research also reveals that NXPH4 mRNA can avoid degradation through RNautophagy, relying on an m5C-dependent mechanism. Moreover, NXPH4 amplifies the HIF signaling pathway and stabilizes HIF1A by competitively binding to PHD4. CONCLUSIONS: NXPH4, regulated by m5C, promotes malignant tumor progression and regulates the HIF pathway. Consequently, targeting NXPH4 through molecular therapies could potentially serve as an efficacious therapeutic strategy for the management of CRC exhibiting elevated NXPH4 expression.

NSUN6-mediated 5-methylcytosine modification of NDRG1 mRNA promotes radioresistance in cervical cancer.

BACKGROUND: Radioresistance is the leading cause of death in advanced cervical cancer (CC). Dysregulation of RNA modification has recently emerged as a regulatory mechanism in radiation and drug resistance. We aimed to explore the biological function and clinical significance of 5-methylcytosine (m5C) in cervical cancer radiosensitivity. METHODS: The abundance of RNA modification in radiotherapy-resistant and sensitive CC specimens was quantified by liquid chromatography-tandem mass spectrometry. The essential RNA modification-related genes involved in CC radiosensitivity were screened via RNA sequencing. The effect of NSUN6 on radiosensitivity was verified in CC cell lines, cell-derived xenograft (CDX), and 3D bioprinted patient-derived organoid (PDO). The mechanisms of NSUN6 in regulating CC radiosensitivity were investigated by integrative m5C sequencing, mRNA sequencing, and RNA immunoprecipitation. RESULTS: We found a higher abundance of m5C modification in resistant CC samples, and NSUN6 was the essential m5C-regulating gene concerning radiosensitivity. NSUN6 overexpression was clinically correlated with radioresistance and poor prognosis in cervical cancer. Functionally, higher NSUN6 expression was associated with radioresistance in the 3D PDO model of cervical cancer. Moreover, silencing NSUN6 increased CC radiosensitivity in vivo and in vitro. Mechanistically, NDRG1 was one of the downstream target genes of NSUN6 identified by integrated m5C-seq, mRNA-seq, and functional validation. NSUN6 promoted the m5C modification of NDRG1 mRNA, and the m5C reader ALYREF bound explicitly to the m5C-labeled NDRG1 mRNA and enhanced NDRG1 mRNA stability. NDRG1 overexpression promoted homologous recombination-mediated DNA repair, which in turn led to radioresistance in cervical cancer. CONCLUSIONS: Aberrant m5C hypermethylation and NSUN6 overexpression drive resistance to radiotherapy in cervical cancer. Elevated NSUN6 expression promotes radioresistance in cervical cancer by activating the NSUN6/ALYREF-m5C-NDRG1 pathway. The low expression of NSUN6 in cervical cancer indicates sensitivity to radiotherapy and a better prognosis.

m6A- and m5C- modified lncRNAs orchestrate the prognosis in cutaneous melanoma and m6A- modified LINC00893 regulates cutaneous melanoma cell metastasis.

BACKGROUND: As the most important modifications on the RNA level, N6-methyladenosine (m6A-) and 5-methylcytosine (m5C-) modification could have a direct influence on the RNAs. Long non-coding RNAs (lncRNAs) could also be modified by methylcytosine modification. Compared with mRNAs, the function of lncRNAs could be more potent to some extent in biological processes like tumorigenesis. Until now, rare reports have been done associated with cutaneous melanoma. Herein, we wonder if the m6A- and m5C- modified lncRNAs could influence the immune landscape and prognosis in melanoma, and we also want to find some lncRNAs which could directly affect the malignant behaviors of melanoma. METHODS: Systematically, we explored the expression pattern of m6A- and m5C- modified lncRNAs in melanoma from datasets including UCSC Xena and NCBI GEO, and the prognostic lncRNAs were selected. Then, according to the expression pattern of lncRNAs, melanoma samples from these datasets were divided into several subtypes. Prognostic model, nomogram survival model, drug sensitivity, GO, and KEGG pathway analysis were performed. Furthermore, among several selected lncRNAs, we identified one lncRNA named LINC00893 and investigated its expression pattern and its biological function in melanoma cell lines. RESULTS: We identified 27 m6A- and m5C- related lncRNAs which were significantly associated with survival, and we made a subtype analysis of melanoma samples based on these 27 lncRNAs. Among the two subtypes, we found differences of immune cells infiltration between these two subtypes. Then, LASSO algorithm was used to screen the optimized lncRNAs combination including ZNF252P-AS1, MIAT, FAM13A-AS1, LINC-PINT, LINC00893, AGAP2-AS1, OIP5-AS1, and SEMA6A-AS1. We also found that there was a significant correlation between the different risk groups predicted based on RS model and the actual prognosis. The nomogram survival model based on independent survival prognostic factors was also constructed. Besides, sensitivity to chemotherapeutic agents, GO and KEGG analysis were performed. In different risk groups, a total of 14 drug molecules with different distributions were obtained, which included AZD6482, AZD7762, AZD8055, camptothecin, dasatinib, erlotinib, gefitinib, gemcitabine, GSK269962A, nilotinib, rapamycin, and sorafenib. A total of 55 significantly related biological processes and 17 KEGG signaling pathways were screened. At last, we noticed that LINC00893 had a relatively lower expression in melanoma tissue and cell lines compared with adjacent tissues and epidermal melanocyte, and down-regulation of LINC00893 could promote the malignant behavior of melanoma cells in A875 and MV3. In these two melanoma cell lines, down-regulation of m6A-related molecules like YTHDF3 and METTL3 could promote the expression of LINC00893. CONCLUSION: We made an analysis of m6A- and m5C- related lncRNAs in melanoma samples and a prediction of these lncRNAs' role in prognosis, tumor microenvironment, immune infiltration, and clinicopathological features. We also found that LINC00893, which is potentially regulated by m6A modification, could serve as a tumor-suppressor in melanoma and play an inhibitory role in melanoma metastasis.

A novel prognostic risk-scoring system based on m5C methylation regulator-mediated patterns for glioma patients.

N5-methylcytosine (m5C) methylation modification plays a crucial role in the epigenetic mechanisms underlying tumorigenesis, aggressiveness, and malignancy in diffuse glioma. Our study aimed to develop a novel prognostic risk-scoring system to assess the impact of m5C modification in glioma patients. Initially, we identified two distinct m5C clusters based on the expression level of m5C regulators in The Cancer Genome Atlas glioblastoma (TCGA-GBM) dataset. Differentially expressed genes (DEGs) between the two m5C cluster groups were determined. Utilizing these m5C regulation-related DEGs, we classified glioma patients into three gene cluster groups: A, B, and C. Subsequently, an m5C scoring system was developed through a univariate Cox regression model, quantifying the m5C modification patterns utilizing six DEGs associated with disease prognosis. The resulting scoring system allowed us to categorize patients into high- or low-risk groups based on their m5C scores. In test (TCGA-GBM) and validation (Chinese Glioma Genome Atlas [CGGA]-1018 and CGGA-301) datasets, glioma patients with a higher m5C score consistently exhibited shorter survival durations, fewer isocitrate dehydrogenase (IDH) mutations, less 1p/19q codeletion and higher World Health Organization (WHO) grades. Additionally, distinct immune cell infiltration characteristics were observed among different m5C cluster groups and risk groups. Our study developed a novel prognostic scoring system based on m5C modification patterns for glioma patients, complementing existing molecular classifications and providing valuable insights into prognosis for glioma patients.

Genetic variants in m5C modification genes are associated with survival of patients with HBV-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with a high mortality rate. The 5-methylcytosine (m5C), a type of RNA modification, plays crucial regulatory roles in HCC carcinogenesis, metastasis, and prognosis. However, a few studies have investigated the effect of genetic variants in m5C modification genes on survival of patients with hepatitis B virus (HBV)-related HCC. In the present study, we evaluated associations between 144 SNPs in 15 m5C modification genes and overall survival (OS) in 866 patients with the HBV-related HCC. Expression quantitative trait loci (eQTL) analysis and differential expression analysis were conducted to investigate biological mechanisms. As a result, we identified that two SNPs (NSUN7 rs2437325 A > G and TRDMT1 rs34434809 G > C) were significantly associated with HBV-related HCC OS with adjusted allelic hazards ratios of 1.25 (95% confidence interval = 1.05-1.48 and P = 0.011) and 1.19 (1.02-1.38 and P = 0.027), respectively, with a trend of combined risk genotypes (Ptrend < 0.001). Moreover, the results of eQTL analyses showed that both NSUN7 rs2437325 G and TRDMT1 rs34434809 C alleles were associated with a reduced mRNA expression level in 208 normal liver tissues (P = 0.007 and P < 0.001, respectively). Taken together, genetic variants in the m5C modification genes may be potential prognostic biomarkers of HBV-related HCC after hepatectomy, likely through mediating the mRNA expression of corresponding genes.

TET3 gene rs828867 G>A polymorphism reduces neuroblastoma risk in Chinese children.

Objective: Neuroblastoma (NB) is a prevalent pediatric tumor originating from primordial neural crest cells. As one of the latest epigenetics investigations focuses, RNA 5-methylcytosine (m5C) is closely related to cancer risk. TET methylcytosine dioxygenase 3 (TET3) is a demethylase for m5C modification. Whether there is an association between TET3 gene polymorphisms and neuroblastoma risk remains unclear. Methods: We conducted an epidemiological study in 402 patients and 473 controls to evaluate the relationship between TET3 gene SNPs (rs7560668 T > C, rs828867 G > A, and rs6546891 A > G) and NB susceptibility. Results: Our results showed that rs828867 G > A significantly reduced NB risk in Chinese children [GA vs. GG, adjusted odds ratio (OR) = 0.72, 95% confidence interval (CI) = 0.52-0.98, P=0.040; GA/AA vs. GG, adjusted OR = 0.74, 95% CI = 0.55-0.998, P=0.048]. Individuals with 2-3 risk genotypes had a significantly higher NB risk than those with 0-1 risk genotypes (adjusted OR = 1.40, 95% CI = 1.04-1.88, P=0.027). The stratified analysis showed that the rs828867 G > A associated with decreased NB risk is remarkable among children aged >18 months (adjusted OR = 0.67, 95% CI = 0.46-0.96, P=0.029) and patients at clinical III + IV stages (adjusted OR = 0.67, 95% CI = 0.45-0.98, P=0.040). Compared with the 0-1 risk genotype, the concurrence of 2-3 risk genotypes significantly increased NB risk in the following subgroups: children aged >18 months and patients at clinical III + IV stages. GTEx analysis suggested that rs828867 G > A was significantly associated with RP11-287D1.4 and POLE4 mRNA expression. Conclusions: Overall, our results revealed that rs828867 G > A in the TET3 gene is significantly associated with predisposition to NB.

5-Methylcytosine transferase NSUN2 drives NRF2-mediated ferroptosis resistance in non-small cell lung cancer.

RNA 5-methylcytosine (m5C) is an abundant chemical modification in mammalian RNAs and plays crucial roles in regulating vital physiological and pathological processes, especially in cancer. However, the dysregulation of m5C and its underlying mechanisms in non-small cell lung cancer (NSCLC) remain unclear. Here we identified that NSUN2, a key RNA m5C methyltransferase, is highly expressed in NSCLC tumor tissue. We found elevated NSUN2 expression levels strongly correlate with tumor grade and size, predicting poor outcomes for NSCLC patients. Furthermore, RNA-seq and subsequent confirmation studies revealed the antioxidant-promoting transcription factor NRF2 is a target of NSUN2, and depleting NSUN2 decreases the expression of NRF2 and increases the sensitivity of NSCLC cells to ferroptosis activators both in vitro and in vivo. Intriguingly, the methylated-RIP-qPCR assay results indicated that NRF2 mRNA has a higher m5C level when NSUN2 is overexpressed in NSCLC cells but shows no significant changes in the NSUN2 methyltransferase-deficient group. Mechanistically, we confirmed that NSUN2 upregulates the expression of NRF2 by enhancing the stability of NRF2 mRNA through the m5C modification within its 5'UTR region recognized by the specific m5C reader protein YBX1, rather than influencing its translation. In subsequent rescue experiments, we show knocking down NRF2 diminished the proliferation, migration, and ferroptosis tolerance mediated by NSUN2 overexpression. In conclusion, our study unveils a novel regulatory mechanism in which NSUN2 sustains NRF2 expression through an m5C-YBX1-axis, suggesting that targeting NSUN2 and its regulated ferroptosis pathway might offer promising therapeutic strategies for NSCLC patients.

5-methylcytosine-mediated upregulation of circular RNA 0102913 augments malignant properties of colorectal cancer cells through a microRNA-571/Rac family small GTPase 2 axis.

Circular RNAs (circRNAs) are a class of stable non-coding RNAs that have emerged as key regulators in human diseases including cancer. This study investigates the role of circRNA_0102913 (circ_0102913) in malignant behavior of colorectal cancer (CRC) cells and the underpinning mechanisms. By analyzing CRC-related GSE197991, GSE159669, and GSE223001 datasets, we obtained circ_0102913 as an aberrantly upregulated circRNA in CRC. Increased circ_0102913 expression was detected in CRC tissues and cells. By querying multiple bioinformatics systems (circBank, Circular RNA Interactome, TargetScan, miRDIP, miRwalk, and miRDB), we identified microRNA-571 (miR-571) as a target of circ_0102913 and Rac family small GTPase 2 (RAC2) mRNA as a target of miR-571. Biotinylated-RNA pull-down and/or luciferase assays showed that circ_0102913 bound to miR-571 to restore the expression of RAC2 mRNA. Circ_0102913 silencing or miR-571 overexpression repressed proliferation, migration and invasion, and in vivo tumorigenesis abilities of CRC cells. However, the malignant properties of cells were restored by RAC2 overexpression. The increased circ_0102913 expression in CRC cells was attributed to increased 5-methylcytosine (m5C) modification levels. Silencing of NOP2/Sun RNA methyltransferase 5 reduced the m5C level and therefore reduced stability and expression of circ_0102913 expression in CRC cells. In conclusion, this study demonstrates that m5C-mediated upregulation of circ_0102913 augments malignant properties of CRC cells through a miR-571/RAC2 axis.

Functional TET2 gene polymorphisms increase the risk of neuroblastoma in Chinese children.

The 5-methylcytosine (m5C) is the key chemical modification in RNAs. As one of the demethylases in m5C, TET2 has been shown as a tumor suppressor. However, the impact of TET2 gene polymorphisms on neuroblastoma has not been elucidated. 402 neuroblastoma patients and 473 controls were genotyped for TET2 gene polymorphisms using the TaqMan method. The impact of TET2 gene polymorphisms on neuroblastoma susceptibility was determined using multivariate logistic regression analysis. We also adopted genotype-tissue expression database to explore the impact of TET2 gene polymorphisms on the expression of host and nearby genes. We used the R2 platform and Sangerbox tool to analyze the relationship between gene expression and neuroblastoma risk and prognosis through non-parametric testing and Kaplan-Meier analysis, respectively. We found the TET2 gene polymorphisms (rs10007915 G > C and rs7670522 A > C) and the combined 2-5 risk genotypes can significantly increase neuroblastoma risk. Stratification analysis showed that these significant associations were more prominent in certain subgroups. TET2 rs10007915 G > C and rs7670522 A > C are significantly associated with reduced expression of TET2 mRNA. Moreover, lower expression of TET2 gene is associated with high risk, MYCN amplification, and poor prognosis of neuroblastoma. The rs10007915 G > C and rs7670522 A > C are significantly related to the increased expression of inorganic pyrophosphatase 2 mRNA, and higher expression of PPA2 gene is associated with high risk, MYCN amplification, and poor prognosis of neuroblastomas. In summary, TET2 rs10007915 G > C and rs7670522 A > C significantly confer neuroblastoma susceptibility, and further research is needed to investigate the underlying mechanisms.

LINC02159 promotes non-small cell lung cancer progression via ALYREF/YAP1 signaling.

Lung cancer is the leading cause of cancer-related deaths worldwide. Long non-coding RNAs (lncRNAs) have emerged as key regulators of cancer development and progression, and as promising biomarkers for the diagnosis and prognosis of cancer. In this study, we identified a new lncRNA (LINC02159) that was upregulated in the tumor tissues and serum of non-small cell lung cancer (NSCLC) patients. We demonstrated that knockdown of LINC02159 inhibited NSCLC cell proliferation, migration, and invasion, but induced cell apoptosis and cell cycle arrest in vitro and retarded tumor growth in vivo, while overexpression of LINC02159 led to the opposite effect. We discovered that LINC02159 was highly correlated with cancer growth and metastasis-related pathways by using transcriptomic analysis and that YAP1 was a potential target gene of LINC02159. Mechanistically, LINC02159 bound to the Aly/REF export factor (ALYREF) to enhance the stability of YAP1 messenger RNA (mRNA) via m5C modification, which led to the overexpression of YAP1 and the activation of the Hippo and ß-catenin signaling pathways in NSCLC cells. Rescue experiments showed that LINC01259 promoted NSCLC progression in a YAP1- and ALYREF-dependent manner. In conclusion, LINC02159 plays an oncogenic role in NSCLC progression by regulating ALYREF/YAP1 signaling, and it has the potential to be utilized as a diagnostic marker and therapeutic target for NSCLC.

Functional polymorphisms of the TET1 gene increase the risk of neuroblastoma in Chinese children.

Common genetic mutations are absent in neuroblastoma, one of the most common childhood tumours. As a demethylase of 5-methylcytosine (m5C) modification, TET1 plays an important role in tumourigenesis and differentiation. However, the association between TET1 gene polymorphisms and susceptibility to neuroblastoma has not been reported. Three TET1 gene polymorphisms (rs16925541 A > G, rs3998860 G > A and rs12781492 A > C) in 402 Chinese patients with neuroblastoma and 473 cancer-free controls were assessed using TaqMan. Multivariate logistic regression analysis was used to evaluate the association between TET1 gene polymorphisms and susceptibility to neuroblastoma. The GTEx database was used to analyse the impact of these polymorphisms on peripheral gene expression. The relationship between gene expression and prognosis was analysed using Kaplan-Meier analysis with the R2 platform. We found that both rs3998860 G > A and rs12781492 A > C were significantly associated with increased neuroblastoma risk. Stratified analysis further showed that rs3998860 G > A and rs12781492 A > C significantly increased neuroblastoma risk in certain subgroups. In the combined risk genotype model, 1-3 risk genotypes significantly increased risk of neuroblastoma compared with the 0 risk genotype. rs3998860 G > A and rs12781492 A > C were significantly associated with increased STOX1 mRNA expression in adrenal and whole blood, and high expression of STOX1 mRNA in adrenal and whole blood was significantly associated with worse prognosis. In summary, TET1 gene polymorphisms are significantly associated with increased neuroblastoma risk; further research is required for the potential mechanism and therapeutic prospects in neuroblastoma.

Transcriptome-wide 5-methylcytosine modification profiling of long non-coding RNAs in A549 cells infected with H1N1 influenza A virus.

BACKGROUND: In recent years, accumulating evidences have revealed that influenza A virus (IAV) infections induce significant differential expression of host long noncoding RNAs (lncRNAs), some of which play important roles in the regulation of virus-host interactions and determining the virus pathogenesis. However, whether these lncRNAs bear post-translational modifications and how their differential expression is regulated remain largely unknown. In this study, the transcriptome-wide 5-methylcytosine (m5C) modification of lncRNAs in A549 cells infected with an H1N1 influenza A virus was analyzed and compared with uninfected cells by Methylated RNA immunoprecipitation sequencing (MeRIP-Seq). RESULTS: Our data identified 1317 upregulated m5C peaks and 1667 downregulated peaks in the H1N1 infected group. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the differentially modified lncRNAs were associated with protein modification, organelle localization, nuclear export and other biological processes. Furthermore, conjoint analysis of the differentially modified (DM) and differentially expressed (DE) lncRNAs identified 143 'hyper-up', 81 'hypo-up', 6 'hypo-down' and 4 'hyper-down' lncRNAs. GO and KEGG analyses revealed that these DM and DE lncRNAs were predominantly associated with pathogen recognition and disease pathogenesis pathways, indicating that m5C modifications could play an important role in the regulation of host response to IAV replication by modulating the expression and/or stability of lncRNAs. CONCLUSION: This study presented the first m5C modification profile of lncRNAs in A549 cells infected with IAV and demonstrated a significant alteration of m5C modifications on host lncRNAs upon IAV infection. These data could give a reference to future researches on the roles of m5C methylation in virus infection.

NSUN2-mediated m5 C RNA methylation dictates retinoblastoma progression through promoting PFAS mRNA stability and expression.

BACKGROUND: The precise temporal and spatial regulation of N5 -methylcytosine (m5 C) RNA modification plays essential roles in RNA metabolism, and is necessary for the maintenance of epigenome homeostasis. Howbeit, the mechanism underlying the m5 C modification in carcinogenesis remains to be fully addressed. METHODS: Global and mRNA m5 C levels were determined by mRNA isolation and anti-m5 C dot blot in both retinoblastoma (RB) cells and clinical samples. Orthotopic intraocular xenografts were established to examine the oncogenic behaviours of RB. Genome-wide multiomics analyses were performed to identify the functional target of NSUN2, including proteomic analysis, transcriptome screening and m5 C-methylated RNA immunoprecipitation sequencing (m5 C-meRIP-seq). Organoid-based single-cell analysis and gene-correlation analysis were performed to verify the NSUN2/ALYREF/m5 C-PFAS oncogenic cascade. RESULTS: Herein, we report that NSUN2-mediated m5 C RNA methylation fuels purine biosynthesis during the oncogenic progression of RB. First, we discovered that global and mRNA m5 C levels were significantly enriched in RBs compared to normal retinas. In addition, tumour-specific NSUN2 expression was noted in RB samples and cell lines. Therapeutically, targeted correction of NSUN2 exhibited efficient therapeutic efficacy in RB both in vitro and in vivo. Through multiomics analyses, we subsequently identified phosphoribosylformylglycinamidine synthase (PFAS), a vital enzyme in purine biosynthesis, as a downstream candidate target of NSUN2. The reintroduction of PFAS largely reversed the inhibitory phenotypes in NSUN2-deficient RB cells, indicating that PFAS was a functional downstream target of NSUN2. Mechanistically, we found that the m5 C reader protein ALYREF was responsible for the recognition of the m5 C modification of PFAS, increasing its expression by enhancing its RNA stability. CONCLUSIONS: Conclusively, we initially demonstrated that NSUN2 is necessary for oncogenic gene activation in RB, expanding the current understanding of dynamic m5 C function during tumour progression. As the NSUN2/ALYREF/m5 C-PFAS oncogenic cascade is an important RB trigger, our study suggests that a targeted m5 C reprogramming therapeutic strategy may be a novel and efficient anti-tumour therapy approach.

NSUN2 gene rs13181449 C>T polymorphism reduces neuroblastoma risk.

Neuroblastoma is the most common tumor in infants. RNA m5C modification regulates the survival, differentiation, and migration of cells affecting RNA function. However, the effects of the m5C modification methyltransferase gene NSUN2 polymorphism on neuroblastoma susceptibility have not been reported. TaqMan method was used to determine genotypes of four NSUN2 polymorphisms (rs4702373 C>T, rs13181449 C>T, rs166049 T>G, and rs8192120 A>C) in 402 patients with neuroblastoma and 473 cancer-free controls from Jiangsu province, China. Odds ratio (OR) and 95% confidence interval (CI) were used to evaluate the association of NSUN2 polymorphisms with neuroblastoma susceptibility. The association was also further assessed in subgroups stratified by age, sex, tumor origin, and stage. GTEx was used to analyze the effect of these polymorphisms on NSUN2 expression. We found the rs13181449 C>T was significantly associated with reduced neuroblastoma risk (CT vs. CC: adjusted OR = 0.68, 95% CI = 0.51-0.92, P = 0.012; CT/TT vs. CC: adjusted OR = 0.70, 95% CI = 0.53-0.92, P = 0.010). Compared with 0-2 protective genotypes, those with 3-4 protective genotypes could significantly reduce the neuroblastoma risk (adjusted OR = 0.68, 95% CI = 0.52 to 0.90, P = 0.006). Stratification analysis showed that the protective effect of rs13181449 polymorphism remained significant in children with age >18 months, boys, and those with early INSS stages. Moreover, children with more protective genotypes in the same subgroups also exhibited significantly reduced neuroblastoma risk. GTEx analysis showed that the rs13181449 T genotype was related with decreased NSUN2 gene expression. In conclusions, NSUN2 rs13181449 polymorphism is associated with decreased neuroblastoma risk, and the underlying mechanism in neuroblastoma needs further study.

Emerging Role of RNA m5C Modification in Cardiovascular Diseases.

Epitranscriptomics is the emerging field of research that comprises the study of epigenetics changes in RNAs. Progressing development in the field of epigenetics has helped to manage and comprehend human diseases. RNA methylation regulates all aspects of RNA functions, which are involved in the pathogenesis of human diseases. Interestingly, RNA m5C methylation is significantly linked to various types of human disease, including cardiovascular diseases (CVD). The m5C methylation is controlled by m5C regulatory proteins, which act as methyltransferase, demethyltransferase, and RNA-binding protein. Dysregulated expression in m5C regulatory proteins is significantly associated with cardiovascular disease, and these regulatory proteins have crucial roles in biological and cellular functions. This review is mainly focused on the role of RNA m5C modification in CVD and mitochondrial dysfunction. Thus, m5C will contribute to discovering the new diagnostic marker and therapeutic target for CVD.

Deciphering the vital roles and mechanism of m5C modification on RNA in cancers.

5-methylcytosine (m5C modification) plays an essential role in tumors, which affects different types of RNA, the expression of downstream target genes, and downstream pathways, thus participating in the tumor process. However, the effect of m5C modification on RNA in tumors and the exact mechanism have not been systematically reviewed. Therefore, we reviewed the status and sites of m5C modification, as well as the expression pattern and biological functions of m5C regulators in tumors, and further summarized the effects and regulation mechanism of m5C modification on messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), long non-coding RNA (lncRNA) and other RNA in tumors. Finally, we summed up the interaction network, potential application, and value in clinical diagnosis and treatment of tumors. Taken together, this review benefits revealing the mechanism of m5C modification in tumor progression and provides new strategies for tumor diagnosis and treatment.

Low expression of PEBP1P2 promotes metastasis of clear cell renal cell carcinoma by post-transcriptional regulation of PEBP1 and KLF13 mRNA.

BACKGROUND: Pseudogenes play an essential role in tumor occurrence and progression. However, the functions and mechanisms of pseudogenes in clear cell renal cell carcinoma (ccRCC) remain largely elusive. METHODS: We quantified PEBP1P2 expression in ccRCC tissues and cells using fluorescence in situ hybridization and real-time PCR. Besides, we evaluated the role of PEBP1P2 in ccRCC using a lung metastasis model and a transwell assay. Finally, we documented the interactions between PEBP1P2, PEBP1, and KLF13 by performing luciferase, RNA immunoprecipitation, RNA pulldown, and targeted RNA demethylation assays. RESULTS: Low PEBP1P2 expression correlates significantly with advanced stages and poor prognosis in ccRCC patients. Besides, PEBP1P2 overexpression inhibits ccRCC metastasis formation in vivo and in vitro. Interestingly, PEBP1P2 directly interacted with 5-methylcytosine (m5C)-containing PEBP1 mRNA and recruited the YBX1/ELAVL1 complex, stabilizing PEBP1 mRNA. In addition, PEBP1P2 increased KLF13 mRNA levels by acting as a sponge for miR-296, miR-616, and miR-3194. CONCLUSIONS: PEBP1P2 inhibits ccRCC metastasis formation and regulates both PEBP1 and KLF13. Therefore, molecular therapies targeting PEBP1P2 might be an effective treatment strategy against ccRCC and other cancers with low PEBP1P2 levels.

mRNA m5C inhibits adipogenesis and promotes myogenesis by respectively facilitating YBX2 and SMO mRNA export in ALYREF-m5C manner.

Although 5-methylcytosine (m5C) has been identified as a novel and abundant mRNA modification and associated with energy metabolism, its regulation function in adipose tissue and skeletal muscle is still limited. This study aimed at investigating the effect of mRNA m5C on adipogenesis and myogenesis using Jinhua pigs (J), Yorkshire pigs (Y) and their hybrids Yorkshire-Jinhua pigs (YJ). We found that Y grow faster than J and YJ, while fatness-related characteristics observed in Y were lower than those of J and YJ. Besides, total mRNA m5C levels and expression rates of NSUN2 were higher both in backfat layer (BL) and longissimus dorsi muscle (LDM) of Y compared to J and YJ, suggesting that higher mRNA m5C levels positively correlate with lower fat and higher muscle mass. RNA bisulfite sequencing profiling of m5C revealed tissue-specific and dynamic features in pigs. Functionally, hyper-methylated m5C-containing genes were enriched in pathways linked to impaired adipogenesis and enhanced myogenesis. In in vitro, m5C inhibited lipid accumulation and promoted myogenic differentiation. Furthermore, YBX2 and SMO were identified as m5C targets. Mechanistically, YBX2 and SMO mRNAs with m5C modification were recognized and exported into the cytoplasm from the nucleus by ALYREF, thus leading to increased YBX2 and SMO protein expression and thereby inhibiting adipogenesis and promoting myogenesis, respectively. Our work uncovered the critical role of mRNA m5C in regulating adipogenesis and myogenesis via ALYREF-m5C-YBX2 and ALYREF-m5C-SMO manners, providing a potential therapeutic target in the prevention and treatment of obesity, skeletal muscle dysfunction and metabolic disorder diseases.

m5C Regulator-Mediated Methylation Modification Patterns and Tumor Microenvironment Infiltration Characterization in Papillary Thyroid Carcinoma.

Recently, immune response modulation at the epigenetic level is illustrated in studies, but the possible function of RNA 5-methylcytosine (m5C) modification in cell infiltration within the tumor microenvironment (TME) is still unclear. Three different m5C modification patterns were identified, and high differentiation degree was observed in the cell infiltration features within TME under the above three identified patterns. A low m5C-score, which was reflected in the activated immunity, predicted the relatively favorable prognostic outcome. A small amount of effective immune infiltration was seen in the high m5C-score subtype, indicating the dismal patient survival. Our study constructed a diagnostic model using the 10 signature genes highly related to the m5C-score, discovered that the model exhibited high diagnostic accuracy for PTC, and screened out five potential drugs for PTC based on this m5C-score model. m5C modification exerts an important part in forming the TME complexity and diversity. It is valuable to evaluate the m5C modification patterns in single tumors, so as to enhance our understanding towards the infiltration characterization in TME.

Development and Validation of a Novel Gene Signature for Predicting the Prognosis by Identifying m5C Modification Subtypes of Cervical Cancer.

Background: 5-Methylcytidine (m5C) is the most common RNA modification and plays an important role in multiple tumors including cervical cancer (CC). We aimed to develop a novel gene signature by identifying m5C modification subtypes of CC to better predict the prognosis of patients. Methods: We obtained the expression of 13 m5C regulatory factors from The Cancer Genome Atlas (TCGA all set, 257 patients) to determine m5C modification subtypes by the "nonnegative matrix factorization" (NMF). Then the "limma" package was used to identify differentially expressed genes (DEGs) between different subtypes. According to these DEGs, we performed Cox regression and Kaplan-Meier (KM) survival analysis to establish a novel gene signature in TCGA training set (128 patients). We also verified the risk prediction effect of gene signature in TCGA test set (129 patients), TCGA all set (257 patients) and GSE44001 (300 patients). Furthermore, a nomogram including this gene signature and clinicopathological parameters was established to predict the individual survival rate. Finally, the expression and function of these signature genes were explored by qRT-PCR, immunohistochemistry (IHC) and proliferation, colony formation, migration and invasion assays. Results: Based on consistent clustering of 13 m5C-modified genes, CC was divided into two subtypes (C1 and C2) and the C1 subtype had a worse prognosis. The 4-gene signature comprising FNDC3A, VEGFA, OPN3 and CPE was constructed. In TCGA training set and three validation sets, we found the prognosis of patients in the low-risk group was much better than that in the high-risk group. A nomogram incorporating the gene signature and T stage was constructed, and the calibration plot suggested that it could accurately predict the survival rate. The expression levels of FNDC3A, VEGFA, OPN3 and CPE were all high in cervical cancer tissues. Downregulation of FNDC3A, VEGFA or CPE expression suppressed the proliferation, migration and invasion of SiHa cells. Conclusions: Two m5C modification subtypes of CC were identified and then a 4-gene signature was established, which provide new feasible methods for clinical risk assessment and targeted therapies for CC.