Project description:N6-methyladenosine (m6A) is a type of nucleotide modification abundant in mRNA, which regulates mRNA stability, splicing and translation. However, its physiological role in intratumoral microenvironment and drug resistancehave not been fully understood. We demonstrated that METTL3,a primary m6A methyltransferase, was significantly down-regulated in human sorafenib-resistant hepatocellular carcinoma (HCC). Depletion of METTL3 under hypoxia promoted sorafenib-resistance and angiogenesis and exacerbated progression by activating autophagy-associated pathway. Mechanistically, we identified FOXO3 as a key downstream target of the METTL3-mediated m6A modification. The m6A modification of FOXO3 at the 3'-untranslated region increased FOXO3 mRNA stability. Analysis of clinical samples showed that METTL3levels aretightly correlated with FOXO3levels in patients with HCC, and suppression of FOXO3 predicted poor clinical outcomes. Importantly, METTL3-depletion significantly enhanced sorafenib-resistance of HCC via a METTL3-FOXO3 axis, whereasoverexpression of FOXO3 restored the m6A-dependent sorafenib-sensitivity. Collectively, our work revealedthe critical function of the METTL3-mediated m6A modification in HCC in hypoxic tumor microenvironment, and provided insights into the molecular mechanism of the m6A modification in the resistance of HCC to sorafenib therapy.
Project description:N6-methyladenosine (m6A) is a type of nucleotide modification abundant in mRNA, which regulates mRNA stability, splicing and translation. However, its physiological role in intratumoral microenvironment and drug resistancehave not been fully understood. We demonstrated that METTL3,a primary m6A methyltransferase, was significantly down-regulated in human sorafenib-resistant hepatocellular carcinoma (HCC). Depletion of METTL3 under hypoxia promoted sorafenib-resistance and angiogenesis and exacerbated progression by activating autophagy-associated pathway. Mechanistically, we identified FOXO3 as a key downstream target of the METTL3-mediated m6A modification. The m6A modification of FOXO3 at the 3'-untranslated region increased FOXO3 mRNA stability. Analysis of clinical samples showed that METTL3levels aretightly correlated with FOXO3levels in patients with HCC, and suppression of FOXO3 predicted poor clinical outcomes. Importantly, METTL3-depletion significantly enhanced sorafenib-resistance of HCC via a METTL3-FOXO3 axis, whereasoverexpression of FOXO3 restored the m6A-dependent sorafenib-sensitivity. Collectively, our work revealedthe critical function of the METTL3-mediated m6A modification in HCC in hypoxic tumor microenvironment, and provided insights into the molecular mechanism of the m6A modification in the resistance of HCC to sorafenib therapy.
Project description:We performed m6A-RIPs in Ascl1-induced neurons (iNeurons) to investigate the neuronal m6A epitranscriptome. Immunoprecipitation was done twice using two different antibodies, acquired from Abcam and Synaptic Systems (SySy), allowing for a more robust detection of m6A modification marks. Additionally, RIP-seq was performed separately with intact and fragmented RNA. The former approach allowed to identify proportions of m6A-modified transcripts among the total number, while the latter approach provided the information to identify genomic coordinates of m6A peaks.
Project description:N6-methyladenosine (m6A) is an abundant nucleotide modification in mRNA, known to regulate mRNA stability, splicing, and translation, but it is unclear whether it is also has a physiological role in the intratumoral microenvironment and cancer drug resistance. Here, we find that METTL3, a primary m6A methyltransferase, is significantly down-regulated in human sorafenib-resistant hepatocellular carcinoma (HCC). Depletion of METTL3 under hypoxia promotes sorafenib resistance and expression of angiogenesis genes in cultured HCC cells, and activates autophagy-associated pathways. Mechanistically, we have identified FOXO3 as a key downstream target of METTL3, with m6A modification of the FOXO3 mRNA 3'-untranslated region increasing its stability through a YTHDF1-dependent mechanism. Analysis of clinical samples furthermore showed that METTL3 and FOXO3 levels are tightly correlated in HCC patients. In mouse xenograft models, METTL3 depletion significantly enhances sorafenib resistance of HCC by abolishing the identified METTL3-mediated FOXO3 mRNA stabilization, and overexpression of FOXO3 restores m6A-dependent sorafenib sensitivity. Collectively, our work reveals a critical function for METTL3-mediated m6A modification in the hypoxic tumor microenvironment, and identifies FOXO3 as an important target of m6A modification in the resistance of HCC to sorafenib therapy.
Project description:To investigate the effect of HSATIII lncRNA on m6A modification, we performed m6A-RIP(RNA immuno precipitation) RNA-seq from heat shock-exposed HeLa cells upon HSATIII knockdown.
Project description:m6A-RIP sequencing of primary hepatic stellate cells (HSCs) isolated from Control and HSC-specific Mettl3-knockout (Mettl3 cKO) mouse liver tissues.