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

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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.

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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

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Acknowledgments

We sincerely thank Dr Li S for providing the FR-targeted liposomes for our experiments. This research work was fully supported by grant from Chinese Natural Science Foundation, Permit No. 30471802 and Grant from China Project 863, Permit No. 2007AA021004.

Conflict of interests

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Department of Pediatrics, Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, China

    Chen Feng, Jianwen Wang, Hui Long, Xiaoning Gao & Suoqin Tang

  2. Department of Hematology-Oncology, Capital Institute of Pediatrics, 2 Yabao Road, Beijing, 100020, China

    Tianyou Wang & Ruihong Tang

Authors
  1. Chen Feng
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  2. Tianyou Wang
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  3. Ruihong Tang
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  4. Jianwen Wang
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  5. Hui Long
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  6. Xiaoning Gao
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  7. Suoqin Tang
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Correspondence to Suoqin Tang.

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Feng, C., Wang, T., Tang, R. et al. Silencing of the MYCN gene by siRNA delivered by folate receptor-targeted liposomes in LA-N-5 cells. Pediatr Surg Int 26, 1185–1191 (2010). https://doi.org/10.1007/s00383-010-2703-5

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  • DOI: https://doi.org/10.1007/s00383-010-2703-5

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