Metformin inhibits oral squamous cell carcinoma progression through regulating RNA alternative splicing

Aims: Oral squamous cell carcinoma (OSCC) is considered as the sixth most common cancer worldwide char-acterized by high invasiveness, high metastasis rate and high mortality. It is urgent to explore novel therapeutic strategies to overcome this feature. Metformin is currently a strong candidate anti-tumor drug in multiple cancers. However, whether metformin could inhibit cancer progression by regulating RNA alternative splicing remains largely unknown.Main methods: Cell proliferation and growth ability of CAL-27 and UM-SCC6 were analyzed by CCK8 and colony formation assays. Cell migration was judged by wound healing assay. Mechanistically, RNA-seq was applied to systematically identify genes that are regulated by metformin. The expression of metformin-regulated genes was determined by real-time quantitative PCR (RT-qPCR). Metformin-regulated alternative splicing events were confirmed by RT-PCR.Key findings: We demonstrated that metformin could significantly inhibit the proliferation and migration of oral squamous cell carcinoma cells. Mechanistically, in addition to transcriptional regulation, metformin induces a wide range of alternative splicing alteration, including genes involved in centrosome, cellular response to DNA damage stimulus, GTPase binding, histone modification, catalytic activity, regulation of cell cycle process and ATPase complex. Notably, metformin specifically modulates the splicing of NUBP2, a component of the cytosolic iron-sulfur (Fe/S) protein assembly (CIA). Briefly, metformin favors the production of NUBP2-L, the long splicing isoform of NUBP2, thereby inhibiting cancer cell proliferation.Significance: Our findings provide mechanistic insights of metformin on RNA alternative splicing regulation, thus to offer a potential novel route for metformin to inhibit cancer progression.