CRISPR/Cas9 nickase mediated targeting of urokinase receptor gene inhibits neuroblastoma cell proliferation.

Karina D Rysenkova,Ekaterina V Semina,Kseniya A Rubina,Petr A Vasiluev,Vsevolod A Tkachuk,Anna A Shmakova,Daniyar T Dyikanov,Maxim N Karagyaur,Yury P Rubtsov

doi:10.18632/oncotarget.25647

Abstract

Neuroblastoma is a tumor arising from pluripotent sympathoadrenal precursor cells of neural cell origin. Neuroblastoma is one of the most aggressive childhood tumors with highly invasive and metastatic potential. The increased expression of urokinase and its receptor is often associated with a negative prognosis in neuroblastoma patients.We have shown that targeting of the Plaur gene in mouse neuroblastoma Neuro 2A cells by CRISPR/Cas9n results in ~60% decrease in cell proliferation (p<0.05), reduction in the number of Ki-67 positive cells, caspase 3 activation and PARP-1 cleavage. Knockout of uPAR leads to downregulation of mRNA encoding full-length TrkC receptor, which is involved in p38MAPK and Akt signalling pathways. This finding provides a rationale to study a role of uPAR in neuroblastoma progression, since uPAR could be considered a potential therapeutic target in neuroblastoma treatment.

Highlights

Neuroblastoma is the most common pediatric extracranial solid tumour originating from the sympatoadrenal lineage residing in neural crest [1,2,3]
We showed that clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9n targeting of uPAR gene (Plaur) gene resulted in inhibition of neuroblastoma proliferation, significant reduction in the number of Ki-67 positive cells, caspase 3 activation and Poly (ADPribose) polymerase (PARP)-1 cleavage. uPA receptor (uPAR) downregulation correlated with the decrease in TrkC mRNA level and Akt phosphorylation
CRISPR/Cas9n application was predicted to result in a frameshift mutation close to the start codon of Plaur and to cause premature termination of uPAR translation

Summary

Introduction

Neuroblastoma is the most common pediatric extracranial solid tumour originating from the sympatoadrenal lineage residing in neural crest [1,2,3]. It accounts for 7–15% of childhood cancers [2, 4]. In older patients neuroblastoma can be metastatic with rapid progression and fatal outcome. Pharmacological agents, such as retinoic acid, NO, phenylacetate, can induce neuroblastoma cell differentiation or apoptosis in culture as well as tumor regression in clinics [5,6,7,8]. Even the most intensive multimodal therapy results in only modest improvement in the cure rate of aggressive neuroblastoma [9]

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Results

Conclusion