Pediatric Surgery International

, Volume 26, Issue 12, pp 1185–1191

Silencing of the MYCN gene by siRNA delivered by folate receptor-targeted liposomes in LA-N-5 cells

  • Chen Feng
  • Tianyou Wang
  • Ruihong Tang
  • Jianwen Wang
  • Hui Long
  • Xiaoning Gao
  • Suoqin Tang
Original Article

DOI: 10.1007/s00383-010-2703-5

Cite this article as:
Feng, C., Wang, T., Tang, R. et al. Pediatr Surg Int (2010) 26: 1185. doi:10.1007/s00383-010-2703-5

Abstract

Introduction

MYCN amplification is highly associated with malignancy and correlates with poor prognosis in patients with neuroblastoma.

Materials and methods

We developed a novel liposome-MYCN siRNA-folic acid complex, and the transfection efficacy was measured in LA-N-5 cells by cy-3 fluorescence density in each microgram of protein from the transfected cell lysate. MYCN expression and cell growth were studied with quantitative RT-PCR and MTT assays, and the expression of MYCN protein was studied with Western blot, respectively. An SCID mouse model with subcutaneous LA-N-5 xenografted tumor was established. The animals were divided into four groups (n = 5) and they were peritoneally injected with liposome-encapsulated MYCN siRNA (siRNA 125 μg/kg/day), lipid-encapsulated control siRNA, MYCN siRNA, or liposome only, respectively, for 5 consecutive days. The animals were killed 24 h after the last injection, and the expression of MYCN mRNA in tumor tissue was detected by RT-PCR.

Results

Our results are as follows: the transfect efficacy reached 1808.5 ± 140.2 pg siRNA/μg protein in LA-N-5 lysates after treatment with 100 nmol/L MYCN siRNA encapsulated with lipid, and fluorescence could be visualized in 92% of LA-N-5 cells after transfection. At 72 h post-transfection, MYCN mRNA expression in LA-N-5 cells was downregulated by 79.2%, MYCN protein was downregulated by 71.3% and cell growth was inhibited by 66.2%, as measured by MTT assay. In the in vivo study, MYCN mRNA expression was knocked down 53.1% in tumor tissues with injection of liposome-encapsulated MYCN siRNA as compared to control siRNA.

Conclusion

These results suggest that targeted delivery of MYCN siRNA by folate receptor-targeted lipid vesicles into LA-N-5 cells is efficacious and capable of suppressing MYCN mRNA expression both in vitro and in vivo.

Keywords

Neuroblastoma Target therapy MYCN gene Folate receptor Liposome 

Abbreviations

NB

Neuroblastoma

siRNA

Small interference RNA

RNAi

RNA interference

ODN

Oligonucleotides

DSPC

1,2-Distearoyl-sn-glycero-phosphocholine

DODAC

N-N-dioleyl-N-N-dimethylammonium chloride

PEG-CerC16

N-palmitoylsphingosine-1-[succinyl-(methoxypoly (ethylene glycol) 2000]

FR

Folate receptor

dTd

Thymidine residues

PBS

Phosphate buffered saline

RT-PCR

Transcription polymerase chain reaction

hGAPDH

Human glyceraldehyde-3-phosphate dehydrogenase

DMSO

Dimethyl sulfoxide

SCID

Severe combined immune deficient

MTT

3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Chen Feng
    • 1
  • Tianyou Wang
    • 2
  • Ruihong Tang
    • 2
  • Jianwen Wang
    • 1
  • Hui Long
    • 1
  • Xiaoning Gao
    • 1
  • Suoqin Tang
    • 1
  1. 1.Department of PediatricsChinese PLA General HospitalBeijingChina
  2. 2.Department of Hematology-OncologyCapital Institute of PediatricsBeijingChina

Personalised recommendations