Studies on biological functions of N 6-methyladenosine (m 6 A) modification in mRNA have sprung up in recent years. We find m 6 A can positively regulate the glycolysis of cancer cells. Specifically, m 6 A-sequencing and functional studies confirm that pyruvate dehydrogenase kinase 4 (PDK4) is involved in m 6 A regulated glycolysis and ATP generation. The m 6 A modified 5′UTR of PDK4 positively regulates its translation elongation and mRNA stability via binding with YTHDF1/eEF-2 complex and IGF2BP3, respectively. Targeted specific demethylation of PDK4 m 6 A by dm 6 ACRISPR system can significantly decrease the expression of PDK4 and glycolysis of cancer cells. Further, TATA-binding protein (TBP) can transcriptionally increase the expression of Mettl3 in cervical cancer cells via binding to its promoter. In vivo and clinical data confirm the positive roles of m 6 A/PDK4 in tumor growth and progression of cervical and liver cancer. Our study reveals that m 6 A regulates glycolysis of cancer cells through PDK4.

BackgroundBrain metastasis (BM) is one of the principal causes of mortality for lung cancer patients. While the molecular events that govern BM of lung cancer remain frustrating cloudy.MethodsThe miRNA expression profiles are checked in the paired human BM and primary lung cancer tissues. The effect of miR-143-3p on BM of lung cancer cells and its related mechanisms are investigated.ResultsmiR-143-3p is upregulated in the paired BM tissues as compared with that in primary cancer tissues. It can increase the invasion capability of in vitro blood brain barrier (BBB) model and angiogenesis of lung cancer by targeting the three binding sites of 3’UTR of vasohibin-1 (VASH1) to inhibit its expression. Mechanistically, VASH1 can increase the ubiquitylation of VEGFA to trigger the proteasome mediated degradation, further, it can endow the tubulin depolymerization through detyrosination to increase the cell motility. m6A methyltransferase Mettl3 can increase the splicing of precursor miR-143-3p to facilitate its biogenesis. Moreover, miR-143-3p/VASH1 axis acts as adverse prognosis factors for in vivo progression and overall survival (OS) rate of lung cancer.ConclusionsOur work implicates a causal role of the miR-143-3p/VASH1 axis in BM of lung cancers and suggests their critical roles in lung cancer pathogenesis.

Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have drawn significant attention in recent years. Here we describe the construction and characterization of a CRISPR–Cas13b-based tool for targeted demethylation of specific mRNA. A fusion protein, named dm6ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5–125 (dPspCas13b) to m6A demethylase AlkB homolog 5 (ALKBH5). dm6ACRISPR specifically demethylates m6A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate β-catenin-encoding CTNNB1 mRNA that contains multiple m6A sites to trigger its translation. In addition, the dm6ACRISPR system incurs efficient demethylation of targeted epitranscriptome transcripts with limited off-target effects. Targeted demethylation of transcripts coding for oncoproteins such as epidermal growth factor receptor (EGFR) and MYC can suppress proliferation of cancer cells. Together, we provide a programmable and in vivo manipulation tool to study mRNA modification of specific genes and their related biological functions.

N6-methyladenosine (m 6 A) is the most abundant modification on eukaryotic mRNA, which regulates all steps of the mRNA life cycle. An increasing number of studies have shown that m 6 A methylation plays essential roles in tumor development. However, the relationship between m 6 A and the progression of cancers remains to be explored. Here, we reported that transforming growth factor-β (TGFβ1)-induced epithelial-mesenchymal transition (EMT) was inhibited in methyltransferase-like 3 (METTL3) knockdown (Mettl3 Mut/− ) cells. The expression of TGFβ1 was up-regulated, while self-stimulated expression of TGFβ1 was suppressed in Mettl3 Mut/− cells. We further revealed that m 6 A promoted TGFB1 mRNA decay, but impaired TGFB1 translation progress. Besides this, the autocrine of TGFβ1 was disrupted in Mettl3 Mut/− cells via interrupting TGFβ1 dimer formation. Lastly, we found that Snail, which was down-regulated in Mettl3 Mut/− cells, was a key factor responding to TGFβ1-induced EMT. Together, our research demonstrated that m 6 A performed multi-functional roles in TGFβ1 expression and EMT modulation, suggesting the critical roles of m 6 A in cancer progression regulation.structural supports for METTL3 [15][16][17]. Acting as the executor of m 6 A modification, METTL3 plays crucial roles in various biological processes, including tumor development [18,19]. For instance, METTL3 is necessary for the development and maintenance of mouse and human myeloid leukemia [20]. Our recent study indicated that METTL3 regulated the epithelial-mesenchymal transition (EMT) of cancer cells via Snail translation [21]. Although associations between m 6 A methylation and tumorigenesis, especially EMT process, have arisen for the last decade, the detailed mechanisms remained to be elucidated.EMT of cancer can be induced by a plethora of signaling pathways, and transforming growth factor β (TGFβ) is the prominent EMT inducer in cancer cells [22]. TGFβ, which contains three isoforms, TGFβ1, TGFβ2, and TGFβ3, is synthesized as a pro-protein monomer. During the maturation, the TGFβ dimer forms a complex with latent TGFβ binding proteins (LTBPs), called a latent complex [23,24], which is crucial for the secretion of TGFβ and the activation of TGFβ receptor (TGFR)-mediated cell signaling [25,26]. TGFβ induces the expression of many other growth factors and cytokines to initiate EMT, while also cooperating with the initial stimulus of TGFβ to stimulate self-expression, which is necessary for sustained signaling, which continually supports the long process of EMT [22,27].In this study, we investigate the potential effects of m 6 A on the TGFβ1-induced EMT of cancer cells. Our data reveal that TGFβ1-induced EMT is suppressed in METTL3 knockdown cells (Mettl3 Mut/− ). However, the expression of TGFβ1 is enhanced in Mettl3 Mut/− cells but decreased in TGFβ1-treated Mettl3 Mut/− cells. We demonstrate that m 6 A regulates the stability and translation of TGFB1 mRNA. In addition, METTL3 modulates the secretion and activation of TGFβ1. Besides this, we furth...

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Brain-derived neurotrophic factor (BDNF) is a master regulator of synaptic plasticity in various neural circuits of the mammalian central nervous system. Neuron activity-induced BDNF gene expression is regulated through the Ca/CREB pathway, but other regulatory factors may also be involved in controlling BDNF levels. We report here that Wnt/β-catenin signaling plays a key role in controlling neuron activity-regulated BDNF expression. Using primary cortical cultures, we show that blockade of Wnt/β-catenin signaling inhibits the BDNF up-regulation that is induced by activation of the -methyl-d-aspartic acid (NMDA) receptor and that activation of the Wnt/β-catenin signaling pathway stimulates BDNF expression., Wnt/β-catenin signaling activated BDNF expression and was required for peripheral pain-induced up-regulation of BDNF in the mouse spine. We also found that conditional deletion of one copy of either Wntless (Wls) or β-catenin by Nestin-Cre-mediated recombination is sufficient to inhibit the pain-induced up-regulation of BDNF. We further show that the Wnt/β-catenin/BDNF axis in the spinal neural circuit plays an important role in regulating capsaicin-induced pain. These results indicate that neuron activity-induced Wnt signaling stimulates BDNF expression in the pain neural circuits. We propose that pain-induced Wnt secretion may provide an additional mechanism for intercellular coordination of BDNF expression in the neural circuit.

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Mammalian target of rapamycin (mTOR) signaling plays a critical role in the regulation of activity-dependent protein synthesis in neurons. It is well established that the GTPase-activating protein tuberous sclerosis complex proteins (2TSC2) is an upstream inhibitor of mTOR. In this study, we show that glutamate stimulation down-regulates TSC2 protein in cortical cultures via NMDA receptor (NMDAR) activation. Interestingly, the mTOR-specific inhibitor rapamycin blocks the glutamate-induced TSC2 down-regulation. This finding suggests that NMDAR activation evokes an mTOR-mediated negative regulation of TSC2. In addition, we also show that the glutamate-induced down-regulation of TSC2 protein is blocked by proteasome inhibitor MG132, indicating the involvement of proteasome-mediated protein degradation. We propose that the NMDAR activation stimulates an mTOR-proteasome pathway to degrade TSC2 protein.