Neuropilin-2 Is Associated With Increased Hepatoblastoma Cell Viability and Motility.

Katja Eloranta,Stefano Cairo,Mikko P Pakarinen,Markku Heikinheimo,Ruth Nousiainen,Marjut Pihlajoki,David B Wilson

doi:10.3389/fped.2021.660482

Abstract

The neuropilins NRP1 and NRP2 are multifunctional glycoproteins that have been implicated in several cancer-related processes including cell survival, migration, and invasion in various tumor types. Here, we examine the role of neuropilins in hepatoblastoma (HB), the most common pediatric liver malignancy. Using a combination of immunohistochemistry, RNA analysis and western blotting, we observed high level expression of NRP1 and NRP2 in 19 of 20 HB specimens and in a majority of human HB cell lines (HUH6 and five cell lines established from patient-derived xenografts) studied but not in normal hepatocytes. Silencing of NRP2 expression in HUH6 and HB-282 HB cells resulted in decreased cell viability, impaired cytoskeleton remodeling, and reduced cell motility, suggesting that NRP2 contributes to the malignant phenotype. We propose that neuropilins warrant further investigation as biomarkers of HB and potential therapeutic targets.

Highlights

Hepatoblastoma (HB) is the most common primary liver malignancy in the pediatric population with an incidence of 1.9 cases per million [1, 2]
We assessed NRP1 and NRP2 expression in formalin-fixed paraffin-embedded (FFPE) samples collected from 20 HB patients treated at Helsinki University Hospital between January 1, 1990 and December 31, 2016
Consistent with prior reports [28, 38], NRP1 and NRP2 expression was limited to liver sinusoidal endothelial cell (LSEC) in healthy liver; hepatocytes did not display specific immunoreactivity (Figures 1A–D)

Summary

INTRODUCTION

Hepatoblastoma (HB) is the most common primary liver malignancy in the pediatric population with an incidence of 1.9 cases per million [1, 2]. Primary antibody incubations were performed either at +4◦C for overnight (NRP2 at dilution 1:2,000; sc-13117, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or at room temperature for 1 h (NRP1 at dilution 1:3,000; ab81321, Abcam, Cambridge, MA). NRP2 expression was inhibited in HUH6 and HB-282 cells via small interfering RNA (siRNA) transfection. Cells were seeded into 96-well plates and transfected with NRP2 or NT siRNA. Cell viability was measured utilizing clonogenic assay and ATPlite assay (PerkinElmer, Waltham, MA, USA) at 72 h post-transfection. NRP2 or NT siRNA transfected cells (at density of 50 × 103/insert for HUH6 cells and 20 × 103/insert for HB-282 cells) were seeded to upper side of membrane in serum-free medium. NRP2 or NT siRNA transfected HUH6 cells were grown in 2-well chamber slides coated with collagen I for 72 h. RNA-sequencing data was analyzed with edgeR-package and significance level was set at adjusted p-value < 0.05

RESULTS

DISCUSSION

ETHICS STATEMENT