Metformin Suppresses Hepatocellular Carcinoma through Regulating Alternative Splicing of LGR4

Abstract

Background and aims. Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related deaths worldwide. The role of leucine-rich repeats containing G protein-coupled receptor 4 (LGR4) in HCC remains unclear. Metformin can prevent and control the development of a variety of cancers, especially in HCC. Alternative splicing (AS) can produce many cancer-driving genes. We aim to uncover that metformin regulates HCC development through alternative splicing of LGR4. Methods. First, the expression of LGR4 in HCC tumor tissues and cell lines was detected by western blotting and immunofluorescence. The ability of cell proliferation, migration, and invasion was detected with CCK8, wound-healing, and transwell assays when overexpressing LGR4 or treating with metformin. The β-catenin expression was detected by immunofluorescence. In order to investigate novel AS-associated LGR4, we discarded LGR4 isoforms from GSO databases. We used siRNA to knock down the specific isoform to check the cell proliferation, migration, and invasion when treated with metformin. Results. The level of LGR4 expression was higher in HCC cell lines and tumor tissues. The HCC cell proliferation, migration, and invasion were increased when overexpressing LGR4, which could be reduced by metformin treatment. The GEO database (GSE190076) showed that LGR4 had switching properties in HCC cell lines treated with metformin. We used siRNA to knock down the specific isoform, and the result showed that the specific isoform siRNA could promote the inhibition of cell invasion caused by metformin treatment. Conclusions. LGR4 could promote the ability of cell proliferation, migration, and invasion in HCC, which could be reduced by metformin through alternative splicing.