m6A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation.

N6­methyladenosine (m6A) RNA modification maintained by N6­methyltransferases and demethylases is involved in multiple biological functions. Methyltransferase like 3 (METTL3) is a major N6­methyltransferase. However, the role of METTL3 and its installed m6A modification in colorectal tumorigenesis remains to be fully elucidated. METTL3 is highly expressed as indicated in colorectal cancer samples in the TCGA and Oncomine databases, implying its potential role in colon tumorigenesis. SW480 cell line with stable METTL3 knockout (METTL3­KO) was generated using CRISPR/Cas9 and were confirmed by the loss of METTL3 expression and suppression of m6A modification. The proliferation of METTL3­KO cells was significantly inhibited compared with that of control cells. METTL3­KO decreased the decay rate of suppressor of cytokine signaling 2 (SOCS2) RNA, resulting in elevated SOCS2 protein expression. m6A­RNA immunoprecipitation­qPCR (MeRIP­qPCR) revealed that SOCS2 mRNA was targeted by METTL3 for m6A modification. Similar to METTL3­KO SW480 cells, SW480 cells treated with 3­deazaadenosine, an RNA methylation inhibitor, exhibited elevated SOCS2 protein expression. Increased levels of SOCS2 in METTL3­KO SW480 cells were associated with decreased expression of leucine­rich repeat­containing G protein­coupled receptor 5 (LGR5), contributing to the inhibition of cell proliferation. The underlying associations among METTL3, SOCS2, and LGR5 were further confirmed in SW480 cells transfected with si­METTL3 and in tumor samples from patients with colorectal cancer. Taken together, our data demonstrate that an increased level of METTL3 may maintain the tumorigenicity of colon cancer cells by suppressing SOCS2.

Faecalibacterium prausnitzii-derived microbial anti-inflammatory molecule regulates intestinal integrity in diabetes mellitus mice via modulating tight junction protein expression.

BACKGROUND: Impaired intestinal barrier structure and function have been validated as an important pathogenic process in type 2 diabetes mellitus (T2DM). Gut dysbiosis is thought to be the critical factor in diabetic intestinal pathogenesis. As the most abundant commensal bacteria, Faecalibacterium prausnitzii (F. prausnitzii) play important roles in gut homeostasis. The microbial anti-inflammatory molecule (MAM), an F. prausnitzii metabolite, has anti-inflammatory potential in inflammatory bowel disease (IBD). Thus, we aimed to explore the function and mechanism of MAM on the diabetic intestinal epithelium. METHODS: 16S high-throughput sequencing was used to analyze the gut microbiota of db/db mice (T2DM mouse model). We transfected a FLAG-tagged MAM plasmid into human colonic cells to explore the protein-protein interactions and observe cell monolayer permeability. For in vivo experiments, db/db mice were supplemented with recombinant His-tagged MAM protein from E. coli BL21 (DE3). RESULTS: The abundance of F. prausnitzii was downregulated in the gut microbiota of db/db mice. Immunoprecipitation (IP) and mass spectroscopy (MS) analyses revealed that MAM potentially interacts with proteins in the tight junction pathway, including zona occludens 1 (ZO-1). FLAG-tagged MAM plasmid transfection stabilized the cell permeability and increased ZO-1 expression in NCM460, Caco2, and HT-29 cells. The db/db mice supplemented with recombinant His-tagged MAM protein showed restored intestinal barrier function and elevated ZO-1 expression. CONCLUSIONS: Our study shows that MAM from F. prausnitzii can restore the intestinal barrier structure and function in DM conditions via the regulation of the tight junction pathway and ZO-1 expression.