Proteomics analysis of LGR5 knockdown in neuroblastoma cells

Abstract

Background: Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been reported to play a critical role in the proliferation of neuroblastoma cells. However, the specific intracellular signaling proteins directly associated with LGR5 remain unknown in neuroblastoma.

Purpose: To identify the signaling pathways downstream of LGR5, we performed small-interfering RNA (siRNA)-mediated knockdowns of LGR5 in the SH-SY5Y neuroblastoma cell line.

Material and Methods: Proteomics analysis of neuroblastoma cell line was performed with 2D-polyacrylamide gel electrophoresis (PAGE) and multiple landmarks were selected for the classification of protein spots that exhibited at least a two-fold change in expression level compared to control samples. Based on the landmarks, 12 protein spots were selected and subsequently identified by peptide mass fingerprinting.

Result: Among the 12 proteins identified, the expression of four proteins was increased while the expression of eight proteins was decreased in neuroblastoma cells with LGR5 knockdown. The proteins with increased expression comprised tubulin-beta, heat shock protein (HSP) 90-beta, 78 kDa glucose-regulated protein precursor (GRP78), and internexin neuronal intermediate filament protein-alpha (α-internexin). On the contrary, proteins with decreased expression comprised alpha-tubulin, beta-actin, prohibitin, heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNPA2B1), elongation factor Tu (EF-Tu), heterogeneous nuclear ribonucleoprotein H3 (hnRNPH3), peroxiredoxin 3, and protein disulfide isomerase family A, member 3 (PDIA3).

Conclusions: Taken together, our findings may be helpful for enhancing our understanding of LGR5 and its role in neuroblastoma, as well as for developing new drugs targeting neuroblastomas based on LGR5 and its related proteins.