Knockdown of LGR5 suppresses the proliferation of glioma cells in vitro and in vivo

Abstract

Leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), one of the target genes of the Wnt signaling pathway, has recently been identified as a marker for brain cancer stem-like cells. However, the role of LGR5 in glioma is poorly understood. The aim of the present study was to investigate the relationship between LGR5 expression and pathological grade in glioma, and the impact of LGR5 on the proliferation of glioma cells in vitro and in vivo. Firstly, LGR5 expression was immunohistochemically evaluated in 54 resected gliomas of different pathologic grades, and its association with Ki-67 was evaluated. Subsequently, using western blotting and qRT-PCR, the expression of LGR5 was assessed in three glioma cell lines U87, U118 and U251. Moreover, the effects of LGR5 knockdown by siRNA on glioma cell proliferation, cell cycle, clone formation and tumorsphere formation in vitro and gliomagenesis in vivo were assessed. The results revealed that i) LGR5 was positively expressed in all glioma specimens and its expression increased with pathologic grade and Ki-67 expression; ii) LGR5 was highly expressed in three glioma cell lines and its expression was reduced significantly by siRNA; and iii) RNAi-mediated downregulation of endogenous LGR5 in U87 cells resulted in the suppression of cell proliferation, arrest of the cell cycle, and reduction in clone and tumorsphere formation in vitro. In addition, LGR5 depletion significantly inhibited tumor orthotopic xenograft growth in nude mice. These findings indicate that LGR5 plays a major role in gliomagenesis by promoting neoplastic cell proliferation, suggesting LGR5 as a molecular marker for pathology and a novel therapeutic target for malignant glioma.