HMGA2 overexpression activates IGF2BP2 to stabilize APLP2 via m6A modification and promote pancreatic cancer progression
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ABSTRACT:
SUBMITTER: Liu K
PROVIDER: S-EPMC10915557 | biostudies-literature | 2024 Feb
REPOSITORIES: biostudies-literature
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Project description:N6-methyladenosine (m6A) is capable of mediating circRNA generation in carcinoma biology. Nevertheless, the posttranscriptional systems of m6A and circRNA in hepatocellular carcinoma (HCC) development are still unclear. The present study identified a circRNA with m6A modification, circHPS5, which was increased in neoplasm HCC tissues and indicated poor patient survival. Silencing of circHPS5 inhibited epithelial-mesenchymal transition (EMT) and cancer stem-like cell (CSC) phenotypes. Notably, METTL3 could direct the formation of circHPS5, and specific m6A controlled the accumulation of circHPS5. YTHDC1 facilitated the cytoplasmic output of circHPS5 under m6A modification. In addition, we demonstrated that circHPS5 can act as a miR-370 sponge to regulate the expression of HMGA2 and further accelerate HCC cell tumorigenesis. Accordingly, the m6A modification of circHPS5 was found to modulate cytoplasmic output and increase HMGA2 expression to facilitate HCC development. The new regulatory model of "circHPS5-HMGA2" provides a new perspective for circHPS5 as an important prognostic marker and therapeutic target in HCC and provides mechanistic insight for exploring the carcinogenic mechanism of circHPS5 in HCC.
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Project description:Both N6-methyladenosine (m6A) RNA modification and microRNAs (miRNAs) are common regulatory mechanisms for gene post-transcription by modulating mRNA stability and translation. They also share the same 3'-untranslated regions (UTRs) regions for their target gene. However, little is known about their potential interaction in cell development and biology. Here, we aimed to investigate how m6A regulates the specific miRNA repression during cardiac development and hypertrophy. Our multiple lines of bioinformatic and molecular biological evidence have shown that m6A modification on cardiac miR-133a target sequence promotes miR-133a repressive effect via AGO2-IGF2BP2 (Argonaute 2-Insulin-like growth factor 2 mRNA binding protein 2) complex. Among 139 cardiac miRNAs, only the seed sequence of miR-133a was inversely complement to m6A consensus motif "GGACH" by sequence alignment analysis. Immunofluorescence staining, luciferase reporter, and m6A-RIP (RNA immunoprecipitation) assays revealed that m6A modification facilitated miR-133a binding to and repressing their targets. The inhibition of the miR-133a on cardiac proliferation and hypertrophy could be prevented by silencing of Fto (FTO alpha-ketoglutarate dependent dioxygenase) which induced m6A modification. IGF2BP2, an m6A binding protein, physically interacted with AGO2 and increased more miR-133a accumulation on its target site, which was modified by m6A. In conclusion, our study revealed a novel and precise regulatory mechanism that the m6A modification promoted the repression of specific miRNA during heart development and hypertrophy. Targeting m6A modification might provide a strategy to repair hypertrophic gene expression induced by miR-133a.
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Project description:The process of post-transcriptional regulation has been recognized to be significantly impacted by the presence of N6-methyladenosine (m6A) modification. As an m6A demethylase, ALKBH5 has been shown to contribute to the progression of different cancers by increasing expression of several oncogenes. Hence, a better understanding of the key targets of ALKBH5 in cancer cells could potentially lead to the development of new therapeutic targets. However, the specific role of ALKBH5 in pancreatic neuroendocrine neoplasms (pNENs) remains largely unknown. Here, we demonstrated that ALKBH5 was up-regulated in pNENs and played a critical role in tumor growth and lipid metabolism. Mechanistically, ALKBH5 over-expression was found to increase the expression of FABP5 in an m6A-IGF2BP2 dependent manner, leading to disorders in lipid metabolism. Additionally, ALKBH5 was found to activate PI3K/Akt/mTOR signaling pathway, resulting in enhanced lipid metabolism and proliferation abilities. In conclusion, our study uncovers the ALKBH5/IGF2BP2/FABP5/mTOR axis as a mechanism for aberrant m6A modification in lipid metabolism and highlights a new molecular basis for the development of therapeutic strategies for pNENs treatment.
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Project description:Subretinal fibrosis is a major cause of the poor visual prognosis for patients with neovascular age-related macular degeneration (nAMD). Myofibroblasts originated from retinal pigment epithelial (RPE) cells through epithelial-mesenchymal transition (EMT) contribute to the fibrosis formation. N6-Methyladenosine (m6A) modification has been implicated in the EMT process and multiple fibrotic diseases. The role of m6A modification in EMT-related subretinal fibrosis has not yet been elucidated. In this study, we found that during subretinal fibrosis in the mouse model of laser-induced choroidal neovascularization, METTL3 was upregulated in RPE cells. Through m6A epitranscriptomic microarray and further verification, high-mobility group AT-hook 2 (HMGA2) was identified as the key downstream target of METTL3, subsequently activating potent EMT-inducing transcription factor SNAIL. Finally, by subretinal injections of adeno-associated virus vectors, we confirmed that METTL3 deficiency in RPE cells could efficiently attenuate subretinal fibrosis in vivo. In conclusion, our present research identified an epigenetic mechanism of METTL3-m6A-HMGA2 in subretinal fibrosis and EMT of RPE cells, providing a novel therapeutic target for subretinal fibrosis secondary to nAMD.
| S-EPMC10603769 | biostudies-literature
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.
| S-EPMC10570365 | biostudies-literature
Project description:BackgroundN6-methyladenosine (m6A) modification is the most abundant reversible methylation modification in eukaryotes, and it is reportedly closely associated with a variety of cancers progression, including colorectal cancer (CRC). This study showed that activated lipid metabolism and glycolysis play vital roles in the occurrence and development of CRC. However, only a few studies have reported the biological mechanisms underlying this connection.MethodsProtein and mRNA levels of FTO and ALKBH5 were measured using western blot and qRT-PCR. The effects of FTO and ALKBH5 on cell proliferation were examined using CCK-8, colony formation, and EdU assays, and the effects on cell migration and invasion were tested using a transwell assay. m6A RNA immunoprecipitation (MeRIP) and RNA-seq was used to explore downstream target gene. RIP was performed to verify the interaction between m6A and HK2. The function of FTO and ALKBH5 in vivo was determined by xenograft in nude mice.ResultsIn this study, FTO and ALKBH5 were significantly down-regulated in CRC patients and cells both in vivo and in vitro in a high-fat environment. Moreover, FTO and ALKBH5 over-expression hampered cell proliferation both in vitro and in vivo. Conversely, FTO and ALKBH5 knockdown accelerated the malignant biological behaviors of CRC cells. The mechanism of action of FTO and ALKBH5 involves joint regulation of HK2, a key enzyme in glycolysis, which was identified by RNA sequencing and MeRIP-seq. Furthermore, reduced expression of FTO and ALKBH5 jointly activated the FOXO signaling pathway, which led to enhanced proliferation ability in CRC cells. IGF2BP2, as a m6A reader, positively regulated HK2 mRNA in m6A dependent manner. Additionally, down-regulation of FTO/ALKBH5 increased METTL3 and decreased METTL14 levels, further promoting CRC progression.ConclusionIn conclusion, our study revealed the FTO-ALKBH5/IGF2BP2/HK2/FOXO1 axis as a mechanism of aberrant m6A modification and glycolysis regulation in CRC.
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Project description:Laryngeal squamous cell carcinoma (LSCC) is a common malignancy of the head and neck. Recently, circular RNA (circRNA) has been studied extensively in multisystem diseases. However, there are few research on biological functions and molecular mechanisms of circRNAs in LSCC. CircRNA array was used to detect the differentially expressed circRNAs. Kaplan-Meier and cox regression analysis were used to identify survival based on circMMP9. The qRT-PCR, RNase R treatment, sanger sequencing and in situ hybridization were used to verify circMMP9 expression, characteristics and localization in LSCC tissues and cells. Functionally, colony formation, MTS, transwell and in vivo assays were proceeded to detect the biological function of circMMP9 in LSCC progression. The RNA-seq was conducted to identify the molecular targets of circMMP9. Mechanically, MeRIP, RNA Immunoprecipitation (RIP), RNA pulldown, Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were carried on to verify the regulatory mechanism of circMMP9. CircMMP9 was discovered upregulated in LSCC tissues and cells, and high level of circMMP9 was associated with poor prognosis, low degree of pathological grading, high TNM stage and lymph node metastasis of LSCC. CircMMP9 knockdown prevented LSCC progression both in vitro and in vivo, whereas, circMMP9 overexpression had the opposite effect. CircMMP9 was stabilized by IGF2BP2 in m6A-dependent manner. TRIM59 was identified as downstream target of circMMP9. CircMMP9 recruited ETS1 to stimulate TRIM59 transcription. Moreover, TRIM59 accelerated LSCC progression via activating the PI3K/AKT signal pathway. Our findings offered a unique regulatory mechanism for circMMP9 in LSCC, as well as a novel proof that circMMP9 may be utilize as a diagnostic marker and therapeutic target for LSCC patients.
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Project description:Aerobic glycolysis has been shown to play a key role in tumor cell proliferation and metastasis. However, how it is directly regulated is largely unknown. Here, we found that HES1 expression was significantly higher in CRC tissues than that in adjacent normal tissues. Moreover, high HES1 expression is associated with poor survival in CRC patients. HES1 knockdown markedly inhibited cell growth and metastasis both in vitro and in vivo. Additionally, silencing of HES1 suppressed aerobic glycolysis of CRC cells. Mechanistic studies revealed that HES1 knockdown decreased the expression of GLUT1, a key gene of aerobic glycolysis, in CRC cells. GLUT1 overexpression abolished the effects of HES1 knockdown on cell aerobic glycolysis, proliferation, migration and invasion. ChIP-PCR and dual-luciferase reporter gene assay showed that HES1 directly bound the promoter of IGF2BP2 and promoted IGF2BP2 expression. Furthermore, our data indicated that IGF2BP2 recognized and bound the m6A site in the GLUT1 mRNA and enhanced its stability. Taken together, our findings suggest that HES1 has a significant promotion effect on CRC aerobic glycolysis and progression by enhancing the stability of m6A-modified GLUT1 mRNA in an IGF2BP2-dependent manner, which may become a viable therapeutic target for the treatment of CRC in humans. The mechanism of HES1 regulating glycolysis in CRC.
| S-EPMC10643658 | biostudies-literature
Project description:BackgroundPancreatic carcinoma (PC) is a highly lethal cancer with an increasing mortality rate, its five-year survival rate is only approximately 4%. N6-methyladenosine (m6A) modification is the most common posttranscriptional modification of RNA, it could affect tumor formation by regulating m6A modifications in the mRNA of key oncogenes or tumor suppressor genes. However, its role in PC remains unclear.MethodsWe combined bioinformatic analysis with in vitro and in vivo experiments to investigate the expression profile of methylation modulators and identify key m6A regulators in the progression of PC. Further study focused on exploring the target genes binding to the regulators through RIP and immunofluorescence staining experiment.ResultsTCGA and Gene Expression Omnibus (GEO) analyses revealed an overall increasing trend in the expression of m6A regulators in PC, and consensus clustering analysis of m6A modification showed that the expression of regulators was negatively correlated with the survival rate. LASSO-Cox regression analysis revealed that IGF2BP2, METTL3, ALKBH5 and KIAA1429 were associated with hazard ratios (HR), but only IGF2BP2 was sufficiently appropriate for the m6A survival prognosis model. The IHC and WB results verified high protein expression of IGF2BP2 in PC, and IGF2BP2 knockdown inhibited the proliferation and migration of PC cells. We predicted and verified B3GNT6 was observably regulated by IGF2BP2 via RIP assays. In addition, IF staining confirmed the co-expression of IGF2BP2 and B3GNT6. The tumor-promoting effect of IGF2BP2 and its co-expression with B3GNT6 were verified in an animal model.ConclusionsElevated m6A levels promote PC progression. IGF2BP2 is a credible marker and modulates B3GNT6 mRNA stability, indicating that IGF2BP2 is a potential prognostic marker and therapeutic target in PC progression.
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Project description:A group of genes that are highly and specifically expressed in proliferating skeletal myoblasts during myogenesis was identified. Expression of one of these genes, Hmga2, increases coincident with satellite cell activation, and later its expression significantly declines correlating with fusion of myoblasts into myotubes. Hmga2 knockout mice exhibit impaired muscle development and reduced myoblast proliferation, while overexpression of HMGA2 promotes myoblast growth. This perturbation in proliferation can be explained by the finding that HMGA2 directly regulates the RNA-binding protein IGF2BP2. Add-back of IGF2BP2 rescues the phenotype. IGF2BP2 in turn binds to and controls the translation of a set of mRNAs, including c-myc, Sp1, and Igf1r. These data demonstrate that the HMGA2-IGF2BP2 axis functions as a key regulator of satellite cell activation and therefore skeletal muscle development.
| S-EPMC3645921 | biostudies-literature