Dataset Information

The m6A Methyltransferase METTL3 Is Functionally Implicated in DLBCL Development by Regulating m6A Modification in PEDF.

ABSTRACT: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma, whose treatment still has a major challenge of achieving a satisfactory curative effect. The underlying mechanisms also have not been fully illustrated. N 6-Methyladenosine (m6A) has been identified as the most prevalent internal modification of mRNAs present in eukaryotes, which is involved in the pathogenesis of cancers. It remains unclear how m6A mRNA methylation is functionally linked to the pathogenesis of DLBCL. In this study, we sought to explore the roles of METTL3 on DLBCL development. The results showed that m6A level for RNA methylation and the expression level of METTL3 were upregulated in DLBCL tissues and cell lines. Functionally, downregulated METTL3 expression in DLBCL cells inhibited the cell proliferation ability. Further mechanism analysis indicated that METTL3 knockdown abates the m6A methylation and total mRNA level of pigment epithelium-derived factor (PEDF). However, Wnt/?-catenin signaling was not thus activated. Overexpressed PEDF abrogates the inhibition of cell proliferation in DLBCL cells that is caused by METTL3 silence. In summary, the above-mentioned results demonstrated that the METTL3 promotes DLBCL progression by regulating the m6A level of PEDF.

SUBMITTER: Cheng Y

PROVIDER: S-EPMC7481464 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The m6A Methyltransferase METTL3 Is Functionally Implicated in DLBCL Development by Regulating m6A Modification in PEDF.

Cheng Yingying Y Fu Yuanyuan Y Wang Ying Y Wang Jinbi J

Frontiers in genetics 20200827

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma, whose treatment still has a major challenge of achieving a satisfactory curative effect. The underlying mechanisms also have not been fully illustrated. <i>N</i> <sup>6</sup>-Methyladenosine (m6A) has been identified as the most prevalent internal modification of mRNAs present in eukaryotes, which is involved in the pathogenesis of cancers. It remains unclear how m6A mRNA methylation is functionally linked to the patho ...[more]

PMID: 33061938

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Project description:BackgroundAs an important N6-methyladenosine (m6A) regulator, abnormal expression of methyltransferase-like protein 3 (METTL3) has been reported in certain human cancers. Although some data have shown that METTL3 plays an essential role in the progression of clear-cell renal cell carcinoma RCC (ccRCC), the detailed mechanism still remains largely undetermined.MethodsImmunohistochemistry (IHC) assay was used to examine the expression of METTL3 and its clinical implications in human ccRCC by using tissue-microarray (TMA). The cellular models based on ccRCC cell lines such as 786-O and ACHN, were established by operating METTL3 and HHLA2 via knockdown or overexpression, followed by in vitro cellular function studies and in vivo subcutaneous transplantation tumor model.ResultsWe found that METTL3 expression in ccRCC tissues was significantly higher compared with adjacent normal tissues. We also found the overall survival (OS) of the patients with low METTL3 expression was significantly better compared with the patients with high METTL3 expression. Furthermore, HHLA2highMETTL3high could serve as a better prognostic predictor for ccRCC patients. Depletion of METTL3 could significantly inhibit the cell viability, migration, and invasion abilities in ccRCC cell lines. Cellular studies further revealed that METTL3 could regulate HHLA2 expression via m6A modification of HHLA2 mRNA. In vitro studies revealed that HHLA2 overexpression could reverse the inhibition of cellular functions mediated by METTL3 depletion. The subcutaneous transplantation tumor model confirmed that HHLA2 overexpression could reverse the inhibition of tumor growth mediated by METTL3 depletion.ConclusionOur study indicated that METTL3 served as an important prognostic predictor for ccRCC patients, and we demonstrated a novel regulatory mechanism of HHLA2 by mRNA epigenetic modification via METTL3. Moreover, we found that the METTL3/HHLA2 axis could promote tumorigenesis of ccRCC. Collectively, our current findings provided new insights into the therapeutic strategy against this malignancy targeting METTL3.

Project description:Colorectal cancer (CRC) is among the commonest malignant tumors of humans. Existing evidence has linked the poor prognosis of CRC with high expression of stromal antigen 3 (STAG3), but, the exact biological effect of STAG3 in CRC is still unclear. The aim of this research is to reveal the biological function and molecular mechanism of STAG3 in CRC. To investigate the differential expression of STAG3 in CRC tissues and cell lines compared to normal colon tissues and cell lines, Western blot (WB) and quantitative real-time PCR (qRT-PCR) techniques were utilized. STAG3 N6-methyladenosine (m6A) modification level were identified using m6A RNA immunoprecipitation (MeRIP). Additionally, the functional roles of methyltransferase-like protein 3 (METTL3) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) in CRC were explored by manipulating their levels via knockdown or overexpression. Cell proliferation was evaluated through Cell Counting Kit 8 (CCK-8) and clone formation experiments, while cell migration was assessed through wound healing experiments. Furthermore, cell apoptosis was detected using flow cytometry, and the protein expressions associated with proliferation and apoptosis were detected using WB. To identify the specific binding of target genes, RIP and pull-down assays were employed. Finally, the biological function of STAG3 in vivo was investigated through a xenotransplantation mouse tumor model. In CRC tissues and cell lines, STAG3 was up-regulated and accompanied by m6A methylation. Additionally, the expression of METTL3 was found to be upregulated in CRC tissues. Knocking down METTL3 resulted in a decrease in both the m6A level and protein expression of STAG3, inhibited cell proliferation and migration while promoting apoptosis, which were restored through STAG3 overexpression. Furthermore, online prediction indicated the interaction between STAG3 mRNA and IGF2BP2 protein, which was further verified by RIP experiments. IGF2BP2 downregulation led to decreased STAG3 protein expression, cell proliferation, and migration, but increased apoptosis. However, these impacts were reversed by STAG3 overexpression. Finally, subcutaneous tumor experiments conducted in nude mice also confirmed that METTL3 regulated CRC progression through STAG3 in vivo. The METTL3/IGF2BP2/STAG3 axis affects CRC progression in an m6A modification-dependent manner. This may guide targeted therapy in CRC patients.

Project description:Gastric cancer (GC) is the fifth most common cancer and the third deadliest cancer in the world, and the occurrence and development of GC are influenced by epigenetics. Methyltransferase-like 3 (METTL3) is a prominent RNA n6-adenosine methyltransferase (m6A) that plays an important role in tumor growth by controlling the work of RNA. This study aimed to reveal the biological function and molecular mechanism of METTL3 in GC. The expression level of METTL3 in GC tissues and cells was detected by qPCR, Western blot and immunohistochemistry, and the expression level and prognosis of METTL3 were predicted in public databases. CCK-8, colony formation, transwell and wound healing assays were used to study the effect of METTL3 on GC cell proliferation and migration. In addition, the enrichment effect of METTL3 on DEK mRNA was detected by the RIP experiment, the m6A modification effect of METTL3 on DEK was verified by the MeRIP experiment and the mRNA half-life of DEK when METTL3 was overexpressed was detected. The dot blot assay detects m6A modification at the mRNA level. The effect of METTL3 on cell migration ability in vivo was examined by tail vein injection of luciferase-labeled cells. The experimental results showed that METTL3 was highly expressed in GC tissues and cells, and the high expression of METTL3 was associated with a poor prognosis. In addition, the m6A modification level of mRNA was higher in GC tissues and GC cell lines. Overexpression of METTL3 in MGC80-3 cells and AGS promoted cell proliferation and migration, while the knockdown of METTL3 inhibited cell proliferation and migration. The results of in vitro rescue experiments showed that the knockdown of DEK reversed the promoting effects of METTL3 on cell proliferation and migration. In vivo experiments showed that the knockdown of DEK reversed the increase in lung metastases caused by the overexpression of METTL3 in mice. Mechanistically, the results of the RIP experiment showed that METTL3 could enrich DEK mRNA, and the results of the MePIP and RNA half-life experiments indicated that METTL3 binds to the 3'UTR of DEK, participates in the m6A modification of DEK and promotes the stability of DEK mRNA. Ultimately, we concluded that METTL3 promotes GC cell proliferation and migration by stabilizing DEK mRNA expression. Therefore, METTL3 is a potential biomarker for GC prognosis and a therapeutic target.

Dataset Information

The m6A Methyltransferase METTL3 Is Functionally Implicated in DLBCL Development by Regulating m6A Modification in PEDF.

Publications

The m6A Methyltransferase METTL3 Is Functionally Implicated in DLBCL Development by Regulating m6A Modification in PEDF.

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