Project description:Spermatogenesis is a differentiation process during which diploid spermatogonial stem cells (SSCs) produce haploid spermatozoa. This highly specialized process is precisely controlled at the transcriptional, posttranscriptional, and translational levels. Here we report that N6-methyladenosine (m6A), an epitranscriptomic mark regulating gene expression, plays essential roles during spermatogenesis. We present comprehensive m6A mRNA methylomes of mouse spermatogenic cells from five developmental stages: undifferentiated spermatogonia, type A1 spermatogonia, preleptotene spermatocytes, pachytene/diplotene spermatocytes, and round spermatids. Germ cell-specific inactivation of the m6A RNA methyltransferase Mettl3 or Mettl14 with Vasa-Cre causes loss of m6A and depletion of SSCs. m6A depletion dysregulates translation of transcripts that are required for SSC proliferation/differentiation. Combined deletion of Mettl3 and Mettl14 in advanced germ cells with Stra8-GFPCre disrupts spermiogenesis, whereas mice with single deletion of either Mettl3 or Mettl14 in advanced germ cells show normal spermatogenesis. The spermatids from double-mutant mice exhibit impaired translation of haploid-specific genes that are essential for spermiogenesis. This study highlights crucial roles of mRNA m6A modification in germline development, potentially ensuring coordinated translation at different stages of spermatogenesis.
Project description:Metastasis remains the major obstacle to improved survival for colorectal cancer (CRC) patients. Dysregulation of N6-methyladenosine (m6A) is causally associated with the development of metastasis through poorly understood mechanisms. Here, we report that METTL14, a key component of m6A methylation, is functionally related to the inhibition of ARRDC4/ZEB1 signaling and to the consequent suppression of CRC metastasis. We unveil METTL14-mediated m6A modification profile and identify ARRDC4 as a direct downstream target of METTL14. Knockdown of METTL14 significantly enhanced ARRDC4 mRNA stability relying on the "reader" protein YHTDF2 dependent manner. Moreover, we demonstrate that TCF4 can induce METTL14 protein expression, and HuR suppress METTL14 expression by directly binding to its promoter. Clinically, our results show that decreased METTL14 is correlated with poor prognosis and acts as an independent predictor of CRC survival. Collectively, our findings propose that METTL14 functions as a metastasis suppressor, and define a novel signaling axis of TCF4/HuR-METTL14-YHTDF2-ARRDC4-ZEB1 in CRC, which might be potential therapeutic targets for CRC.
Project description:Rheumatoid arthritis (RA) is largely caused by the inflammatory response triggered by macrophage polarization. Through epigenetic reprogramming, the inflammatory state of macrophages can be modified. Macrophage polarization is associated with the RNA epigenetic alteration N6-methyladenosine (m6A) RNA methylation. However, the specific function and underlying mechanisms of m6A methylation in the role of macrophage polarization in RA remain to be elucidated. The mRNA expression levels of m6A methylase genes and signaling pathway components associated with RA macrophages were determined in the present study using reverse-transcription quantitative PCR. Methyltransferase 14 (METTL14) protein expression levels were determined using western blot analysis, and the levels of specific cellular secretion factors were determined using ELISA and flow cytometry. The results of the present study demonstrated that elevated METTL14 expression was associated with joint tenderness, and METTL14 expression was positively correlated with both C-reactive protein and rheumatoid factor expression levels. Moreover, METTL14 exhibited potential in the prediction of visual analogue scale. Pro-inflammatory cytokines (TNF-α) and M1 macrophage markers (CD68+CD86+) were also positively associated with METTL14 expression. The results of the Kyoto Encyclopedia of Genes and Genomes analysis revealed that METTL14 was strongly associated with the MAPK signaling pathway. Notably, JNK and ERK2 exhibited a positive correlation with the M1 macrophage marker, CD68+CD86+, which was positively associated with the pro-inflammatory factor, TNF-α. JNK and ERK2 expression levels were markedly increased in the METTL14 high-expression group, compared with in the low-expression group; however, p38 and ERK1 expression levels were not significantly different between these groups. Collectively, the results of the present study demonstrated that METTL14 expression was significantly increased in the peripheral blood and synovial tissue of patients with RA, highlighting the potential association with both immunoinflammatory markers and clinical symptoms. In addition, it was suggested that METTL14 may exacerbate the downstream inflammatory response, through mediating macrophage polarization via the MAPK pathway.
Project description:The first step in the biogenesis of microRNAs is the processing of primary microRNAs (pri-miRNAs) by the microprocessor complex, composed of the RNA-binding protein DGCR8 and the type III RNase DROSHA. This initial event requires recognition of the junction between the stem and the flanking single-stranded RNA of the pri-miRNA hairpin by DGCR8 followed by recruitment of DROSHA, which cleaves the RNA duplex to yield the pre-miRNA product. While the mechanisms underlying pri-miRNA processing have been determined, the mechanism by which DGCR8 recognizes and binds pri-miRNAs, as opposed to other secondary structures present in transcripts, is not understood. Here we find in mammalian cells that methyltransferase-like 3 (METTL3) methylates pri-miRNAs, marking them for recognition and processing by DGCR8. Consistent with this, METTL3 depletion reduced the binding of DGCR8 to pri-miRNAs and resulted in the global reduction of mature miRNAs and concomitant accumulation of unprocessed pri-miRNAs. In vitro processing reactions confirmed the sufficiency of the N(6)-methyladenosine (m(6)A) mark in promoting pri-miRNA processing. Finally, gain-of-function experiments revealed that METTL3 is sufficient to enhance miRNA maturation in a global and non-cell-type-specific manner. Our findings reveal that the m(6)A mark acts as a key post-transcriptional modification that promotes the initiation of miRNA biogenesis.
Project description:Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer?related death among women worldwide. Evidence indicates that posttranscriptional N6?methyladenosine (m6A) modification modulates BC development. In the present study, we assessed BC and normal tissues to investigate this connection. RNA m6A levels were determined by methylation quantification assay. The effects of methyltransferase?like 14 (METTL14) gain?of?expression or co?transfection with an m6A inhibitor on cell migration and invasion abilities were determined by Transwell assays. The levels of differentially expressed (DE) miRNAs were verified by real?time quantitative PCR (RT?qPCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG) were performed to analyze potential function of target genes of the DE miRNAs. The effects of candidate miRNAs modulated by METTL14 on cell migration and invasion abilities were confirmed by Transwell assays. We demonstrated that m6A methyltransferase METTL14 was significantly upregulated in BC tissues compared with normal tissues. METTL14 gain? and loss?of?expression regulated m6A levels in MCF?7 and MDA?MB?231 cells. The cell function assays revealed that METTL14 overexpression enhanced the migration and invasion capacities of BC cells. Moreover, treatment with the m6A inhibitor suppressed this enhanced cell migration and invasion. Additionally, aberrant expression of METTL14 reshaped the miRNA profile in BC cell lines. The remodeled DE miRNA/mRNA network was found to be most enriched in cancer pathways, and DE miRNAs were enriched in cell adhesion terms. hsa?miR?146a?5p modulated by METTL14 promoted cell migration and invasion. METTL14 modulates m6A modification and hsa?miR?146a?5p expression, thereby affecting the migration and invasion of breast cancer cells.
Project description:BACKGROUND:Colorectal cancer (CRC) is one of the leading causes of tumor-related death worldwide, and its main cause of death is distant metastasis. Methyltransferase-like 14(METTL14), a major RNA N6-adenosine methyltransferase, is involved in tumor progression via regulating RNA function. The goal of the study is to uncover the biological function and molecular mechanism of METTL14 in CRC. METHODS:Quantitative real-time PCR (qRT-PCR), western blot and immunohistochemical (IHC) assays were employed to detect METTL14 and SOX4 in CRC cell lines and tissues. The biological functions of METTL14 were demonstrated using in vitro and in vivo experiments. Chromatin immunoprecipitation (ChIP), Transcrptomic RNA sequencing (RNA-Seq), m6A-RNA immunoprecipitation sequencing (MeRIP-Seq), RNA immunoprecipitation and luciferase reporter assays were used to explore the mechanism of METTL14 action. RESULTS:METTL14 expression was significantly downregulated in CRC and decreased METTL14 was associated with poor overall survival (OS). Both the univariate and multivariate Cox regression analysis indicated that METTL14 was an independent prognostic factor in CRC. Moreover, lysine-specific histone demethylase 5C(KDM5C)-mediated demethylation of histone H3 lysine 4 tri-methylation(H3K4me3) in the promoter of METTL14 inhibited METTL14 transcription. Functionally, we verified that METTL14 inhibited CRC cells migration, invasion and metastasis through in vitro and in vivo assays, respectively. Furthermore, we identified SRY-related high-mobility-group box 4(SOX4) as a target of METTL14-mediated m6A modification. Knockdown of METTL14 markedly abolished SOX4 mRNA m6A modification and elevated SOX4 mRNA expression. We also revealed that METTL14-mediated SOX4 mRNA degradation relied on the YTHDF2-dependent pathway. Lastly, we demonstrated that METTL14 might inhibit CRC malignant process partly through SOX4-mediated EMT process and PI3K/Akt signals. CONCLUSIONS:Decreased METTL14 facilitates tumor metastasis in CRC, suggesting that METTL14 might be a potential prognostic biomarker and effective therapeutic target for CRC.
Project description:N6-methyladenosine (m6A) is the most abundant internal mRNA modification in eukaryotes and is related to stability, localization, or translation efficiency in tumorigenesis. Autophagy plays an important role in the occurrence and development of tumours. However, the relationship between m6A and autophagy remains unclear. In this study, we used a rapamycin-induced autophagy model of oral squamous cell carcinoma (OSCC) cells, and observed increased m6A RNA methylation. When autophagy was activated, the methyltransferase-like 14 (METTL14) expression was upregulated and influenced the proliferation, migration, and invasiveness of OSCC cells. Through meRIP-seq and RNA-seq analysis, we found that METTL14 directly combined with eukaryotic translation initiation factor gamma 1 (eIF4G1) mRNA and decreased its RNA stability. According to the dual-luciferase reporter and mutagenesis assay, the mutated site 1 of exon 11 of eIF4G1 is the key target of METTL14. Knockdown of the main m6A binding protein YTHDF2 may rescue the shortened half-life of eIF4G1 mRNA induced by METTL14 overexpression. Furthermore, an in vivo tumour xenograft model confirmed that a high METTL14 expression can effectively reduce OSCC growth. Additionally, using clinical samples, we found that patients with advanced or moderately/poorly differentiated tumours exhibited lower METTL14 levels. Taken together, our results revealed that METTL14 mediated eIF4G1 expression via m6A modification and regulated autophagy levels and biological functions in OSCC. Our findings not only expand our understanding of the correlation between autophagy and RNA methylation in tumorigenesis but also present an opportunity to develop new therapeutic options.
Project description:ObjectivesRecently, an increasing number of studies have revealed that N6-methyladenosine (m6A) functions as a significant post-transcriptional modification which plays a critical role in the occurrence and progression of enriched tumors by regulating coding and non-coding RNA biogenesis. However, the biological function of m6A in breast cancer remains largely unclear.Materials and methodsIn this study, we used a series of bioinformatic databases and tools to jointly analyze the expression of m6A methylation transferases (METTL3, METTL14, WTAP, RBM15, RBM15B and ZC3H13) and investigate the prognostic value of METTL14 and ZC3H13 in breast cancer. Besides, we analyzed the downstream carcinogenic molecular mechanisms related to METTL14 and ZC3H13 and their relationship with immune infiltration in breast tumor tissues.ResultsThe results showed that METTL14 and ZC3H13 were the down-regulated m6A methylation transferases in breast cancer. Survival outcome analysis suggested that abnormally low expression of METTL14 and ZC3H13 could predict unfavorable prognosis in four breast cancer subtypes. Moreover, their down-regulation was associated with ER-, PR- and triple-negative breast cancer patients, as well as tumor progression (increased Scarff, Bloom and Richardson grade status and Nottingham Prognostic Index classification). Co-expression analysis revealed that METTL14 and ZC3H13 had a strong positive correlation with APC, an antagonist of the Wnt signaling pathway, indicating they might cooperate in regulating proliferation, invasion, and metastasis of tumor cells. METTL14, ZC3H13, and APC expression levels had significant positive correlation with infiltrating levels of CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells, and negative correlation with Treg cells in breast cancer.ConclusionsThis study demonstrated that down-regulation of METTL14 and ZC3H13 which act as two tumor suppressor genes was found in breast cancer and predicted poor prognosis. Their abnormal expression promoted breast cancer invasion by affecting pathways related to tumor progression and mediating immunosuppression.
Project description:BackgroundStomach adenocarcinoma (STAD) is a common reason for tumor-related fatalities globally, as it results in distant metastasis. Methyltransferase-like 14 (METTL14), a notable RNA N6-adenosine methyltransferase (m6A), plays a significant role in the growth of tumor through controlling the RNA working. This study aims to highlight METTL14 in STAD's biological function and molecular mechanism.MethodsBioinformatics and immunohistochemical (IHC) assays have been utilized for the detection of METTL14 expression in the STAD. METTL14's biological function has been shown while making use of HGC-27 and AGS cells in vitro experiments. MeRIP-qPCR and luciferase reporter assays were employed for the exploration of METTL14's mechanism modifying the target of phosphatase and tensin homologue (PTEN). Subcutaneous xeno transplantation model and STAD liver metastasis orthotopic tumor model were used to study METTL14 in STAD in vivo.ResultsMETTL14 expression was substantially downregulated in STAD reflecting contribution to major tumors, progressed TNM stage as well as poor overall survival (OS) in STAD. Moreover, METTL14's inhibition of STAD cells proliferation, migration and invasion has been verified in vitro assays. Furthermore, an identification of PTEN being METTL14-mediated m6A modification's substrate has been made. METTL14's overexpression highly enhanced PTEN mRNA m6A variation, stabilized PTEN mRNA and increased protein expression. Further, it has been found out that METTL14-mediated STAD cells inhibition of proliferation and invasion dependent on PTEN. At last, we demonstrated that METTL14 inhibit STAD growth and metastasis in vivo models.ConclusionsMETTL14 inhibits tumor growth and metastasis of STAD via stabilization of PTEN mRNA expression. Therefore, METTL14 is a potential biomarker of prognosis and therapeutic targets for STAD.
Project description:Renal fibrosis induced by urinary tract obstruction is a common clinical occurrence; however, effective treatment is lacking, and a deeper understanding of the mechanism of renal fibrosis is needed. Previous studies have revealed that miR-21 impacts liver and lung fibrosis progression by activating the SPRY1/ERK/NF-kB signalling pathway. However, whether miR-21 mediates obstructive renal fibrosis through the same signalling pathway has not been determined. Additionally, studies have shown that N6-methyladenosine (m6 A) modification-dependent primary microRNA (pri-microRNA) processing is essential for maturation of microRNAs, but its role in the maturation of miR-21 in obstructive renal fibrosis has not yet been investigated in detail. To address these issues, we employed a mouse model of unilateral ureteral obstruction (UUO) in which the left ureters were ligated for 3, 7 and 14 days to simulate the fibrotic process. In vitro, human renal proximal tubular epithelial (HK-2) cells were transfected with plasmids containing the corresponding sequence of METTL3, miR-21-5p mimic or miR-21-5p inhibitor. We found that the levels of miR-21-5p and m6 A modification in the UUO model groups increased significantly, and as predicted, the SPRY1/ERK/NF-kB pathway was activated by miR-21-5p, confirming that miR-21-5p plays an important role in obstructive renal fibrosis by enhancing inflammation. METTL3 was found to play a major catalytic role in m6 A modification in UUO mice and drove obstructive renal fibrosis development by promoting miR-21-5p maturation. Our research is the first to demonstrate the role of the METTL3-m6 A-miR-21-5p-SPRY1/ERK/NF-kB axis in obstructive renal fibrosis and provides a deeper understanding of renal fibrosis.