Cross talk between RNA N6-methyladenosine methyltransferase-like 3 and miR-186 regulates hepatoblastoma progression through Wnt/?-catenin signalling pathway.
ABSTRACT: OBJECTIVES:N6-methyladenosine (m6A) is a ubiquitous epigenetic RNA modification that plays a pivotal role in tumour development and metastasis. In this study, we aimed to investigate the expression profiling, clinical significance, biological function and the regulation of m6A-related genes in hepatoblastoma (HB). MATERIALS AND METHODS:The mRNA and protein expression levels of m6A-related genes were analysed using Gene Expression Omnibus (GEO) and tissue microarray (TMA) cohort. Kaplan-Meier analysis was performed to evaluate the prognostic value of m6A-related genes in HB. Knockdown of m6A-related genes was conducted to analyse its function on cell proliferation, migration and invasion. Furthermore, bioinformatics analysis and experimental verification were used to explore the potential molecular mechanism and signalling pathway. RESULTS:We found that most m6A-related genes were significantly upregulated in HB tumour tissues. High levels of methyltransferase-like 3 (METTL3, P = .013), YTHDF2 (P = .037) and FTO (P = .032) indicated poor clinical outcomes, and the upregulation of METTL3 was an independent prognostic factor in HB patients. Functional assays showed that knockdown of METTL3 could dramatically suppress the proliferation, migration and invasion of HB cells. In addition, METTL3 was identified to be a direct target of microRNA-186 (miR-186). Consistently, miR-186 was low expressed in HB tumour tissues. Moreover, overexpression of miR-186 significantly inhibited cell aggressive phenotype both in vitro and in vivo, while the inhibitory effect could be reversed by METTL3 overexpression. Mechanism study indicated that miR-186/METTL3 axis contributed to the progression of HB via the Wnt/?-catenin signalling pathway. CONCLUSIONS:M6A-related genes were frequently dysregulated in HB. miR-186/METTL3/Wnt/?-catenin axis might serve as novel therapeutic targets and prognostic biomarkers in HB.
Project description:BACKGROUND:N6-Methyladenosine (m6A) modification has been implicated in many biological processes. It is important for the regulation of messenger RNA (mRNA) stability, splicing, and translation. However, its role in cancer has not been studied in detail. Here we investigated the biological role and underlying mechanism of m6A modification in hepatoblastoma (HB). METHODS:We used Reverse transcription quantitative real-time PCR (RT-qPCR) and Western blotting to determine the expression of m6A related factors. And we clarified the effects of these factors on HB cells using cell proliferation assay, colony formation, apoptotic assay. Then we investigated of methyltransferase-like 13 (METTL3) and its correlation with clinicopathological features and used xenograft experiment to check METTL3 effect in vivo. m6A-Seq was used to profiled m6A transcriptome-wide in hepatoblastoma tumor tissue and normal tissue. Finally, methylated RNA immunoprecipitation (MeRIP) assay, RNA remaining assay to perform the regulator mechanism of MEETL3 on the target CTNNB1 in HB. RESULTS:In this research, we discovered that m6A modifications are increased in hepatoblastoma, and METTL3 is the main factor involved with aberrant m6A modification. We also profiled m6A across the whole transcriptome in hepatoblastoma tumor tissues and normal tissues. Our findings suggest that m6A is highly expressed in hepatoblastoma tumors. Also, m6A is enriched not only around the stop codon, but also around the coding sequence (CDS) region. Gene ontology analysis indicates that m6A mRNA methylation contributes significantly to regulate the Wnt/?-catenin pathway. Reduced m6A methylation can lead to a decrease in expression and stability of the CTNNB1. CONCLUSION:Overall our findings suggest enhanced m6A mRNA methylation as an oncogenic mechanism in hepatoblastoma, METTL3 is significantly up-regulated in HB and promotes HB development. And identify CTNNB1 as a regulator of METTL3 guided m6A modification in HB.
Project description:Background: N6-methyladenosine (m6A) is the most prevalent modification of mammalian RNA. Emerging evidence suggest that m6A has critical roles in multiple biological activities, but little is known about its roles in cancer pathogenesis. Herein, we report the expression profiles and prognostic relevance of twelve m6A-related genes in hepatocellular carcinoma (HCC) by analyzing four independent datasets. Materials and methods: RNA levels of twelve m6A-related genes were detected in samples of 162 HCC patients who underwent curative resection (the Guangdong General Hospital dataset). We additionally analyzed the expression profiles of m6A-related genes in The Cancer Genome Atlas liver HCC dataset and two Gene Expression Omnibus datasets (GSE14520, GSE63898). Prognostic value of genes was evaluated by Kaplan-Meier curves of overall survival (OS) with the log-rank test and multivariate Cox regression analysis. Gene set enrichment analysis (GSEA) was conducted to identify associated KEGG pathways. Results: Five genes (METTL3, YTHDF1, YTHDF2, YTHDF3, and EIF3) showed consistent upregulation in all four datasets. Abnormal expressions of either METTL3 or YTHDF1 but not the other ten genes were associated with OS. Protein expression of METTL3 and YTHDF1 were confirmed in HCC tissues by immunohistochemical staining. Multivariate Cox regression analysis confirmed the independent predictive value of both METTL3 and YTHDF1 on OS. We further divided patients into three groups based on the median expression values of METTL3 and YTHDF1. In all datasets, the low METTL3/low YTHDF1 group showed a consistent better prognosis than other groups. GSEA revealed that both METTL3 and YTHDF1 regulate HCC cell cycle, RNA splicing, DNA replication, base excision repair, and RNA degradation. Conclusion: Both METTL3 and YTHDF1 were upregulated in HCC, and they were independent poor prognostic factors. Combination of METTL3 and YTHDF1 can be regarded as the biological marker that reflect malignant degree and evaluate prognosis in HCC.
Project description:BACKGROUND:Methyltransferase-like 3 (METTL3) is a member of the m6A methyltransferase family and acts as an oncogene in cancers. Recent studies suggest that host innate immunity is regulated by the enzymes controlling m6A epitranscriptomic changes. Here, we aim to explore the associations between the levels of METTL3 and CD33+ myeloid-derived suppressor cells (MDSCs) in tumour tissues and the survival of patients with cervical cancer (CC). METHODS:Specimens of paraffin embedded tumour from 197 CC patients were collected. The expression levels of METTL3 and CD33 were measured by immunohistochemical (IHC) staining. The clinical associations of the IHC variants were analysed by Pearson's or Spearman's chi-square tests. Overall survival (OS) and disease-free survival (DFS) were estimated by the Kaplan-Meier method and log-rank test. Hazard ratios (HRs) and independent significance were obtained via Cox proportional hazards models for multivariate analyses. METTL3 in CD33+ cells or CC-derived cells was knocked down by METTL3-specific siRNA, and MDSC induction in vitro was performed in a co-culture system in the presence of METTL3-siRNA and METTL3-knockdown-CC-derived cells compared with that of the corresponding controls. RESULTS:We found that tumour tissues displayed increased levels of METTL3 and CD33+ MDSCs compared with tumour-adjacent tissues from the same CC patients. Importantly, METTL3 expression was positively related to the density of CD33+ cells in tumour tissues (P?=?0.011). We further found that the direct CD33+CD11b+HLA-DR- MDSC induction and tumour-derived MDSC induction in vitro were decreased in the absence of METTL3. The level of METTL3 in tumour microenvironments was significantly related to advanced tumour stage. The levels of METTL3 and CD33+ MDSCs in tumour tissues were notably associated with reduced DFS or OS. Cox model analysis revealed that the level of METTL3 in tumour cells was an independent factor for patient survival, specifically for DFS (HR?=?3.157, P?=?0.022) and OS (HR?=?3.271, P?=?0.012), while the CD33+ MDSC number was an independent predictor for DFS (HR: 3.958, P?=?0.031). Interestingly, in patients with advanced-disease stages (II-IV), METTL3 in tumour cells was an independent factor for DFS (HR?=?6.725, P?=?0.010) and OS (HR?=?5.140, P?=?0.021), while CD33+ MDSC density was an independent factor for OS (HR?=?8.802, P?=?0.037). CONCLUSION:Our findings suggest that CD33+ MDSC expansion is linked to high levels of METTL3 and that METTL3 and CD33+ MDSCs are independent prognostic factors in CC.
Project description:Methyltransferase-like 3 (METTL3) is the main enzyme for N6-methyladenosine (m6A)-based methylation of RNAs and it has been implicated in many biological and pathophysiological processes. In this study, we aimed to explore the potential involvement of METTL3 in osteoblast differentiation and decipher the underlying cellular and molecular mechanisms. We demonstrated that METTL3 is downregulated in human osteoporosis and the ovariectomized (OVX) mouse model, as well as during the osteogenic differentiation. Silence of METTL3 by short interfering RNA (siRNA) decreased m6A methylation levels and inhibited osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and reduced bone mass, and similar effects were observed in METTL3+/- knockout mice. In contrast, adenovirus-mediated overexpression of METTL3 produced the opposite effects. In addition, METTL3 enhanced, whereas METTL3 silence or knockout suppressed, the m6A methylations of runt-related transcription factor 2 (RUNX2; a key transcription factor for osteoblast differentiation and bone formation) and precursor (pre-)miR-320. Moreover, downregulation of mature miR-320 rescued the decreased bone mass caused by METTL3 silence or METTL3+/- knockout. Therefore, METTL3-based m6A modification favors osteogenic differentiation of BMSCs through m6A-based direct and indirect regulation of RUNX2, and abnormal downregulation of METTL3 is likely one of the mechanisms underlying osteoporosis in patients and mice. Thus, METTL3 overexpression might be considered a new approach of replacement therapy for the treatment of human osteoporosis.
Project description:N6-methyladenosine (m6A) is the most common epigenetic RNA modification with essential roles in cancer progression. However, roles of m6A and its regulator METTL3 on non-coding RNA in gastric cancer are unknown. In this study, we found elevated levels of m6A and METTL3 in gastric cancer. Increased METTL3 expression indicated poor outcomes of patients and high malignancy in vitro and in vivo. Mechanically, m6A facilitated processing of pri-miR-17-92 into the miR-17-92 cluster through an m6A/DGCR8-dependent mechanism. The m6A modification that mediated this process occurred on the A879 locus of pri-miR-17-92. The miR-17-92 cluster activated the AKT/mTOR pathway by targeting PTEN or TMEM127. Compared with those with low levels of METTL3, METTL3-high tumors showed preferred sensitivity to an mTOR inhibitor, everolimus. These results reveal a perspective on epigenetic regulations of non-coding RNA in gastric cancer progression and provide a theoretical rationale for use of everolimus in the treatment of m6A/METTL3-high gastric cancer.
Project description:N6-methyladenosine (m6A) is the richest modification in mammalian messenger RNAs (mRNAs), and exerts key roles in many biological processes, including cancer development, whereas its roles in prostate carcinoma (PCa) remain to be unclear. Here, we found that m6A modifications are increased in PCa and methyltransferase-like 3 (METTL3), but not other major m6A modification genes including METTL14, fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5), was the major dysregulated gene associated with abnormal m6A modification. In addition, METTL3 up-regulation acted as a poor prognostic factor for overall survival and disease-free survival in PCa patients. Knockdown of METTL3 significantly inhibited PCa cells proliferation, migration, and invasion. In addition, over-expression of METTL3, but not its catalytic mutant form, significantly promoted PCa cells growth and progression. Mechanistically, we revealed that METTL3 enhanced MYC(c-myc) expression by increasing m6A levels of MYC mRNA transcript, leading to oncogenic functions in PCa. Importantly, PCa cells growth and progression inhibition by METTL3 knockdown were restored through over-expression of MYC. Our results uncovered a METTL3/m6A/MYC axis and provided insight into the mechanisms of PCa progression.
Project description:Objectives: This study aims to explore the roles of 13 m6A RNA methylation regulators in clear cell renal cell carcinoma (ccRCC), and identify a risk signature and prognostic values of m6A RNA methylation regulators in ccRCC. Materials and Methods: RNA sequence data of ccRCC was obtained from The Cancer Genome Atlas (TCGA) database. Differentially expressed of 13 m6A RNA methylation regulators in ccRCC stratified by different clinicopathological characteristics were unveiled using "limma" package in R version 3.6.0. Cox regression and LASSO analyses were conducted to identify the powerful independent prognostic factors in ccRCC associated with overall survival (OS). Protein-protein interaction (PPI) network and correlation analyses of the 13 m6A RNA methylation regulators were performed using "STRING" and R package, respectively. Principal component analysis (PCA) was also done using R. In addition, gene ontology (GO), GSEA and Kyoto Encyclopedia of Genes and Genomes pathways were used to functionally annotate the differentially expressed genes in different subgroups. Results: Most of the 13 m6A RNA methylation regulators are differentially expressed in ccRCC tissue samples stratified by different clinicopathological characteristics in 537 patients. Next, a risk signature for predicting prognosis of ccRCC patients, was established based on two powerful independent prognostic m6A RNA methylation regulators (METTL14 and METTL3). Then, two subgroups (cluster1 and 2) were identified by consensus clustering to the two powerful independent factors and the cluster1 had a poorer prognosis than cluster2. Furthermore, the genes in cluster1 were significantly enriched in cancer-related pathways, biological process, and hallmarks, including "cell adhesion molecules (CAMs)," "leukocyte migration," "Wnt/?-catenin signaling," and so on. Conclusion: M6A RNA methylation regulators play important roles in the initiation and progression of ccRCC and provide a novel sight to understand m6A RNA modification in ccRCC.
Project description:Nephrotoxicity of colistin is the major factor limiting its clinical application. However, the exact mechanism of colistin-induced nephrotoxicity is still elusive. N6-Methyladenosine (m6A) modification has been implicated in many biological processes, however, its role in colistin-induced nephrotoxicity needs to be elucidated. Mouse renal tubular epithelial cells (mRTECs) were treated with 200 ?M colistin with or without METTL3 overexpression. Cells injury, m6A assay, oxidative stress and apoptosis were examined. Levels of m6A are decreased after colistin treatment in mRTECs. METTL3 is the major factor involved in abnormal m6A modification. METTL3 overexpression plays a protective role against colistin-induced oxidative stress and apoptosis. Moreover, METTL3 interacts with the microprocessor protein DGCR8 and positively modulates miR-873-5p mature process in an m6A-dependent manner. Further experiments show that miR-873-5p could regulate Keap1-Nrf2 pathway against colistin-induced oxidative stress and apoptosis. These studies revealed an important role of METTL3/m6A in colistin-induced nephrotoxicity and provide a new insight on m6A modification in drug induced toxicity.
Project description:N6-methyladenosine (m6A) modification has been reported in various diseases and implicated in increasing numbers of biological processes. However, previous studies have not focused on the role of m6A modification in fracture healing. Here, we demonstrated that m6A modifications are decreased during fracture healing and that methyltransferase-like 3 (METTL3) is the main factor involved in the abnormal changes in m6A modifications. Down-regulation of METTL3 promotes osteogenic processes both in vitro and in vivo, and this effect is recapitulated by the suppression of miR-7212-5p maturation. Further studies have shown that miR-7212-5p inhibits osteoblast differentiation in MC3T3-E1 cells by targeting FGFR3. The present study demonstrated an important role of the METTL3/miR-7212-5p/FGFR3 axis and provided new insights on m6A modification in fracture healing.
Project description:BACKGROUND:METTL3 is an RNA methyltransferase that mediates m6A modification and is implicated in mRNA biogenesis, decay, and translation. However, the biomechanism through which METTL3 regulates MALAT1-miR-1914-3p-YAP axis activity to induce NSCLC drug resistance and metastasis is not very clear. METHODS:The expression of mRNA was analyzed by qPCR assays. Protein levels were analyzed by western blotting and immunofluorescent staining. Cellular proliferation was detected by CCK8 assays. Cell migration and invasion were analyzed by wound healing and transwell assays, respectively. Promoter activities and gene transcription were analyzed by luciferase reporter assays. Finally, m6A modification was analyzed by MeRIP. RESULTS:METTL3 increased the m6A modification of YAP. METTL3, YTHDF3, YTHDF1, and eIF3b directly promoted YAP translation through an interaction with the translation initiation machinery. Moreover, the RNA level of MALAT1 was increased due to a higher level of m6A modification mediated by METTL3. Meanwhile, the stability of MALAT1 was increased by METTL3/YTHDF3 complex. Additionally, MALAT1 functions as a competing endogenous RNA that sponges miR-1914-3p to promote the invasion and metastasis of NSCLC via YAP. Furthermore, the reduction of YAP m6A modification by METTL3 knockdown inhibits tumor growth and enhances sensitivity to DDP in vivo. CONCLUSION:Results indicated that the m6A mRNA methylation initiated by METTL3 promotes YAP mRNA translation via recruiting YTHDF1/3 and eIF3b to the translation initiation complex and increases YAP mRNA stability through regulating the MALAT1-miR-1914-3p-YAP axis. The increased YAP expression and activity induce NSCLC drug resistance and metastasis.