A newly defined risk signature, consisting of three m6A RNA methylation regulators, predicts the prognosis of ovarian cancer.
ABSTRACT: N6-methyladenosine (m6A) RNA methylation, involved in cancer initiation and progression, is dynamically regulated by the m6A RNA methylation regulators. However, the expression of m6A RNA methylation regulators in ovarian cancer and their correlation with prognosis remain elusive. Here, we demonstrated that the 18 central m6A RNA methylation regulators were expressed differently between ovarian cancer (OC) and normal tissues. By applying consensus clustering, all ovarian cancer patient cases can be divided into three subgroups (cluster1/2/3) based on overall expression levels of all 18 m6A RNA methylation regulators. We systematically analyzed the prognostic value of transcription levels of 18 m6A RNA methylation regulators in ovarian cancer and found that insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), vir like m6A methyltransferase associated (VIRMA), and zinc finger CCCH-type containing 13 (ZC3H13) yield the highest scores for predicting the prognosis of ovarian cancer. Accordingly, we derived a risk signature consisting of transcription levels of these three selected m6A RNA methylation regulators as an independent prognostic marker for OC and validated our findings with data derived from a different ovarian cancer cohort. Moreover, by the Gene Set Enrichment Analysis (GSEA), we demonstrated that the three selected regulators were all correlated with pathways in cancer and WNT signaling pathways. In conclusion, m6A RNA methylation regulators are vital participants in ovarian cancer pathology; and IGF2BP1, VIRMA, and ZC3H13 mRNA levels are valuable factors for prognosis prediction and treatment strategy development.
Project description:N6-methyladenosine (m6A) is enriched in 3'untranslated region (3'UTR) and near stop codon of mature polyadenylated mRNAs in mammalian systems and has regulatory roles in eukaryotic mRNA transcriptome switch. Significantly, the mechanism for this modification preference remains unknown, however. Herein we report a characterization of the full m6A methyltransferase complex in HeLa cells identifying METTL3/METTL14/WTAP/VIRMA/HAKAI/ZC3H13 as the key components, and we show that VIRMA mediates preferential mRNA methylation in 3'UTR and near stop codon. Biochemical studies reveal that VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide region-selective methylations. Around 60% of VIRMA mRNA immunoprecipitation targets manifest strong m6A enrichment in 3'UTR. Depletions of VIRMA and METTL3 induce 3'UTR lengthening of several hundred mRNAs with over 50% targets in common. VIRMA associates with polyadenylation cleavage factors CPSF5 and CPSF6 in an RNA-dependent manner. Depletion of CPSF5 leads to significant shortening of 3'UTR of over 2800 mRNAs, 84% of which are modified with m6A and have increased m6A peak density in 3'UTR and near stop codon after CPSF5 knockdown. Together, our studies provide insights into m6A deposition specificity in 3'UTR and its correlation with alternative polyadenylation.
Project description:RNA methylation at position N6 in adenosine (m6A) and its associated methyltransferase complex (MTC) are involved in tumorigenesis. We aimed to explore m6A biological function for long non-coding RNAs (lncRNAs) in prostate cancer (PCa) and its clinical significance. m6A and MTC levels in PCa cells were characterized by ELISA and western blot. Putative m6A-regulated lncRNAs were identified and validated by lncRNA profiler qPCR array and bioinformatics analysis, followed by m6A/RNA co-immunoprecipitation. Impact of m6A depletion on RNA stability was assessed by Actinomycin D assay. The association of m6A-levels with PCa prognosis was examined in clinical samples. Higher m6A-levels and VIRMA overexpression were detected in metastatic castration-resistant PCa (mCRPC) cells (p < 0.05). VIRMA knockdown in PC-3 cells significantly decreased m6A-levels (p = 0.0317), attenuated malignant phenotype and suppressed the expression of oncogenic lncRNAs CCAT1 and CCAT2 (p < 0.00001). VIRMA depletion and m6A reduction decreased the stability and abundance of CCAT1/2 transcripts. Higher expression of VIRMA, CCAT1, and CCAT2 as a group variable was an independent predictor of poor prognosis (HR = 9.083, CI95% 1.911-43.183, p = 0.006). VIRMA is a critical factor sustaining m6A-levels in PCa cells. VIRMA downregulation attenuates the aggressive phenotype of PCa by overall reduction of m6A-levels decreasing stability and abundance of oncogenic lncRNAs.
Project description:N6-methyladenosine (m6A) is an abundant modification in eukaryotic mRNA, regulating mRNA dynamics by influencing mRNA stability, splicing, export, and translation. However, the precise m6A regulating machinery still remains incompletely understood. Here we demonstrate that ZC3H13, a zinc-finger protein, plays an important role in modulating RNA m6A methylation in the nucleus. We show that knockdown of Zc3h13 in mouse embryonic stem cell significantly decreases global m6A level on mRNA. Upon Zc3h13 knockdown, a great majority of WTAP, Virilizer, and Hakai translocate to the cytoplasm, suggesting that Zc3h13 is required for nuclear localization of the Zc3h13-WTAP-Virilizer-Hakai complex, which is important for RNA m6A methylation. Finally, Zc3h13 depletion, as does WTAP, Virilizer, or Hakai, impairs self-renewal and triggers mESC differentiation. Taken together, our findings demonstrate that Zc3h13 plays a critical role in anchoring WTAP, Virilizer, and Hakai in the nucleus to facilitate m6A methylation and to regulate mESC self-renewal.
Project description:Background: Cervical squamous cell carcinoma (CESC) is one of the most common causes of cancer-related death worldwide. N6-methyladenosine (m6A) plays an important role in various cellular responses by regulating mRNA biology. This study aimed to develop and validate an m6A RNA methylation regulator-based signature for prognostic prediction in CESC. Methods: Clinical and survival data as well as RNA sequencing data of 13 m6A RNA methylation regulators were obtained from The Cancer Genome Atlas (TCGA) CESC database. Consensus clustering was performed to identify different CESC clusters based on the differential expression of the regulators. LASSO Cox regression analysis was used to generate a prognostic signature based on m6A RNA methylation regulator expression. The effect of the signature was further explored by univariate and multivariate Cox analyses. Results: Four regulators (RBM15, METTL3, FTO, and YTHDF2) were identified to be aberrantly expressed in CESC tissues. A prognostic signature that includes ZC3H13, YTHDC1, and YTHDF1 was developed, which can act as an independent prognostic indicator. Significant differences of survival rate and clinicopathological features were found between the high- and low-risk groups. The results of bioinformatics analysis were then validated in the clinical CESC cohort by qRT-PCR and immunohistochemistry staining. Conclusion: In the present study, we developed and validated an m6A RNA methylation regulator-based prognostic signature, which might provide useful insights regarding the development and prognosis of CESC.
Project description:Adenomyosis is a prevalent, estrogen-dependent uterine disorder wherein endometrial cells are abnormally present in the myometrium and are surrounded by hyperplastic/hypertrophic smooth muscle. Its etiology is unclear, although endometrial cell invasion into the myometrium has been postulated. RNA methylation, particularly N6-methyladenosine (m6A), plays an important role in regulating various physiological processes and invasive disorders. The goal of this in silico and lab-based experimental study was to explore a possible role for m6A in adenomyosis. Gene expression profiles of both the endometrium and myometrium of women with adenomyosis (cases) and without disease (controls) were obtained from the publicly available Gene Expression Omnibus (GEO) database. In the endometrium, STRING database analysis revealed that METTL3 functions as a "hub" gene of m6A RNA methylation regulators, and the genes involved in m6A regulation, including METTL3, FTO, ZC3H13, and YTHDC1 expression, were significantly decreased in cases versus controls. Functional, co-expression, and correlational analyses of endometrium from cases versus controls revealed decreased total m6A levels, induced by METTL3, and the downstream elevated insulin-like growth factor-1(IGF1) and D-Dopachrome Tautomerase (DDT), with the latter two having known functions in epithelial proliferation and cell migration, which are important processes in the pathogenesis of adenomyosis in endometrium. m6A RNA methylation regulators, including RBM15/15B, ALKBH5, FTO, YTHDF1/2, KIAA1429, HNRNPC, METTL3, ZC3H13, and YTHDC2, were also differentially expressed in the myometrium from cases versus controls. We validated decreased total m6A levels and differential expression of m6A RNA methylation regulators in the myometrium of patients with adenomyosis using qRT-PCR, immunohistochemistry and tissues available from our biorepository. Possible target genes, including cadherin 3(CDH3), sodium channel?-subunit 4 (SCN4B), and placenta-specific protein 8 (PLAC8), which are involved in cell adhesion, muscle contraction and immune response in the myometrium of adenomyosis patients were also validated. Thus, through extensive public database mining and validation of select genes, this study, for the first time, implicates m6A and its methylation regulators in the pathogenesis of adenomyosis. Follow on functional studies are anticipated to elucidate mechanisms involving m6A and its regulators and down-stream effectors in the pathogenesis of this enigmatic reproductive disorder and potentially identify druggable targets to control its associated symptoms.
Project description:Pancreatic cancer (PC) has a very poor prognosis and is usually diagnosed only at an advanced stage. The discovery of new biomarkers for PC will help in early diagnosis and a better prognosis for patients. Recently, N6-methyladenosine (m6A) RNA modifications and their regulators have been implicated in the development of many cancers. To investigate the functions and mechanisms of m6A modifications in the development of PC, 19 m6A regulators, including m6A-methyltransferases (ZC3H13, RBM15/15B, WTAP, KIAA1429, and METTL3/14), demethylases (FTO and ALKBH5), and binding proteins (YTHDF1/2/3, YTHDC1/2, IGF2BP1/2/3, HNRNPC, and HNRNPA2B1) were analyzed in 178 PC tissues from the cancer genome atlas (TCGA) database. The results were verified in PC cell lines Mia-PaCa-2, BXPC-3, and the control cell line HDE-CT. The m6A regulators-based sample clusters were significantly related to overall survival (OS). Further, lasso regression identified a six-m6A-regulator-signature prognostic model (KIAA1429, HNRNPC, METTL3, YTHDF1, IGF2BP2, and IGF2BP3). Model-based high-risk and low-risk groups were significantly correlated with OS and clinical traits (pathologic M, N, and clinical stages and vital status). The risk signature was verified as an independent prognostic marker for patients with PC. Finally, gene set enrichment analysis revealed m6A regulators (KIAA1429, HNRNPC, and IGF2BP2) were related to multiple biological behaviors in PC, including adipocytokine signaling, the well vs. poorly differentiated tumor pathway, tumor metastasis pathway, epithelial mesenchymal transition pathway, gemcitabine resistance pathway, and stemness pathway. In summary, the m6A regulatory factors which related to clinical characteristics can be involved in the malignant progression of PC, and the constructed risk markers may be a promising prognostic biomarker that can guide the individualized treatment of PC patients.
Project description:BACKGROUND:Covalent RNA modifications, such as N-6-methyladenosine (m6A), have been associated with various biological processes, but their role in cancer remains largely unexplored. m6A dynamics depends on specific enzymes whose deregulation may also impact in tumorigenesis. Herein, we assessed the differential abundance of m6A, its writer VIRMA and its reader YTHDF3, in testicular germ cell tumors (TGCTs), looking for clinicopathological correlates. METHODS:In silico analysis of TCGA data disclosed altered expression of VIRMA (52%) and YTHDF3 (48%), prompting subsequent validation. Formalin-fixed paraffin-embedded tissues from 122 TGCTs (2005-2016) were selected. RNA extraction, cDNA synthesis and real-time qPCR (Taqman assays) for VIRMA and YTHDF3 were performed, as well as immunohistochemistry for VIRMA, YTHDF3 and m6A, for staining intensity assessment. Associations between categorical variables were assessed using Chi square and Fisher's exact test. Distribution of continuous variables between groups was compared using the nonparametric Mann-Whitney and Kruskal-Wallis tests. Biomarker performance was assessed through receiver operating characteristics (ROC) curve construction and a cut-off was established by Youden's index method. Statistical significance was set at p < 0.05. RESULTS:In our cohort, VIRMA and YTHDF3 mRNA expression levels differed among TGCT subtypes, with Seminomas (SEs) depicting higher levels than Non-Seminomatous tumors (NSTs) (p < 0.01 for both). A positive correlation was found between VIRMA and YTHDF3 expression levels. VIRMA discriminated SEs from NSTs with AUC = 0.85 (Sensitivity 77.3%, Specificity 81.1%, PPV 71.6%, NPV 85.3%, Accuracy 79.7%). Immunohistochemistry paralleled transcript findings, as patients with strong m6A immunostaining intensity depicted significantly higher VIRMA mRNA expression levels and stronger VIRMA immunoexpression intensity (p < 0.001 and p < 0.01, respectively). CONCLUSION:Abundance of m6A and expression of VIRMA/YTHDF3 were different among TGCT subtypes, with higher levels in SEs, suggesting a contribution to SE phenotype maintenance. VIRMA and YTHDF3 might cooperate in m6A establishment in TGCTs, and their transcript levels accurately discriminate between SEs and NSTs, constituting novel candidate biomarkers for patient management.
Project description:Background:The mortality rate of clear cell renal cell carcinoma (ccRCC) remains high. The aim of this study was to identify novel prognostic biomarkers by using m6A RNA methylation regulators capable of improving the risk-stratification criteria of survival for ccRCC patients. Methods:The gene expression data of 16 m6A methylation regulators and its relevant clinical information were extracted from The Cancer Genome Atlas (TCGA) database. The expression pattern of these m6A methylation regulators were evaluated. Consensus clustering analysis was conducted to identify clusters of ccRCC patients with different prognosis. Univariate, least absolute shrinkage and selection operator (LASSO), and multivariate Cox regression analysis were performed to construct multiple-gene risk signature. A survival analysis was carried out to determine the independent prognostic significance of the signature. Results:Five m6A-related genes (ZC3H13, METTL14, YTHDF2, YTHDF3 and HNRNPA2B1) showed significantly downregulated in tumor tissue, while seven regulators (YTHDC2, FTO, WTAP, METTL3, ALKBH5, RBM15 and KIAA1429) was remarkably upregulated in ccRCC. Consensus clustering analysis identified two clusters of ccRCC with significant differences in overall survival (OS) and tumor stage between them. We also constructed a two-gene signature, METTL3 and METTL14, serving as an independent prognostic indicator for distinguishing ccRCC patients with different prognosis both in training, validation and our own clinical datasets. The receiver operator characteristic (ROC) curve indicated the area under the curve (AUC) in these three datasets were 0.721, 0.684 and 0.828, respectively, demonstrated that the prognostic signature had a good prediction efficiency. Conclusions:m6A methylation regulators exert as potential biomarkers for prognostic stratification of ccRCC patients and may assist clinicians achieving individualized treatment for this patient population.
Project description:The IGF2 mRNA-binding protein 1 (IGF2BP1) is a non-catalytic post-transcriptional enhancer of tumor growth upregulated and associated with adverse prognosis in solid cancers. However, conserved effector pathway(s) and the feasibility of targeting IGF2BP1 in cancer remained elusive. We reveal that IGF2BP1 is a post-transcriptional enhancer of the E2F-driven hallmark in solid cancers. IGF2BP1 promotes G1/S cell cycle transition by stabilizing mRNAs encoding positive regulators of this checkpoint like E2F1. This IGF2BP1-driven shortening of the G1 cell cycle phase relies on 3'UTR-, miRNA- and m6A-dependent regulation and suggests enhancement of cell cycle progression by m6A-modifications across cancers. In addition to E2F transcription factors, IGF2BP1 also stabilizes E2F-driven transcripts directly indicating post-transcriptional 'super'-enhancer role of the protein in E2F-driven gene expression in cancer. The small molecule BTYNB disrupts this enhancer function by impairing IGF2BP1-RNA association. Consistently, BTYNB interferes with E2F-driven gene expression and tumor growth in experimental mouse tumor models.
Project description:Uterine corpus endometrial carcinoma (UCEC) is the most common type of gynecologic malignancy worldwide. Despite advances in the treatments of UCEC, its incidence and mortality rates are still increasing. N6-methyladenosine (m6A) is the most common form of RNA modification and has attracted increasing interest in cancer pathogenesis and progression. Thus, we aimed to identify the landscape of m6A regulators and build a prognostic gene signature in UCEC. In this study, we first analyzed copy number variations (CNVs), single nucleotide variations (SNVs) and gene expression profiles as well as matched clinical information of UCEC patients from The Cancer Genome Atlas (TCGA) database. Next, we determined that CNVs in m6A regulatory genes had a significant negative impact on patient survival. The mRNA expression levels of a total of 16 m6A regulators were significantly correlated with different CNV patterns. Using univariate Cox regression analysis, IGF2BP1, KIAA1429, IGF2BP3, YTHDF3, and IGF2BP2 were found to be closely associated with UCEC patient survival outcomes. Based on the least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression models, we built a 3-gene (IGF2BP3, KIAA1429 and IGF2BP1) signature of m6A regulators with prognostic value in UCEC that could effectively predict patient prognosis (log-rank test p-value < 0.0001). In addition, risk scores were significantly different between patients stratified by tumor stage, SNV, and CNV. Multivariate Cox regression analysis suggested that risk score might be an independent prognostic indicator for the overall survival of patients with UCEC (p-value < 0.05). Gene enrichment analysis indicated that high IGF2BP1 gene expression is associated with cytoplasmic stress granules. KIAA1429 gene expression is associated with cellular nucleic acid metabolism. The expression of the IGF2BP3 gene is associated with RNA binding processes. In conclusion, we determined that genetic alterations in m6A regulatory genes could be effective and reliable biomarkers for UCEC prognosis prediction.