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

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.