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.

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