A Study On Suppresion Of MYCN Expression In Neruoblastoma Tissue By Folic Acid Receptor Targeted Lipisome Mediated MYCN SiRNA In Vivo

Backgroud: Neuroblastoma(NB) is the most common extracranial malignant solid tumor in infants and children. MYCN oncogene activation through amplification is the hallmark of advanced disease and is associated with very poor prognosis. MYCN is a potential target for gene therapy of neuroblastoma, thus it is of value to establish a neuroblastoma animal model for further study.Objective: To establish a neuroblastoma mouse subcutaneous metastasis model; to study MYCN suppression in neruoblastoma tissue by folic acid receptor targeted lipisome mediated MYCN RNAi in vivo.Methods: LA-N-5 cells were cultured and 0.5ml cell suspension with concentration of 1×10~7 / ml were inoculated in 10 SPF severe combined immune deficient (SCID) mice to establish a human neuroblastoma model. Another 28 SCID mice were selected, establish neuroblastoma animal model, randomized divided them into two groups(n=14 each).When the diameter of tumor mass reached 0.5cm, four mice in each group had been sacrificed for tumor tissue MYCN mRNA expression detection before the experiment. Then folic acid targeted lipisome mediated MYCN siRNA(3.5mg/kg/day) was injected daily into tumor mass,continuously for 3 days, while same volume of normal saline, with the same protocol was injected in mice with control. Three hours after last day injection, all the mice were sacrificed, tumor mass was collected for MYCN and Glyceraldehyde phosphate dehydrogenase(GAPDH) mRNA expression detection by real time RT-PCR. The MYCN mRNA expression was calculated based on MYCN copies/GAPDH copies.Results: 1. All the mice had developed mass around inoculated area about 5-6 days after injection, with the average volume of 4.6±2.9mm~3 at day 8. Undermicroscopic examination, the tumor cells are aggregation and the cells are anomalism, rosette like arrangement, NSE immunostaining (+); 2. Before the experiment, MYCN mRNA expression was 17.8±2.3 and 17.5±0.9, in each group, while after treatment the MYCN mRNA expression was 18.1±4.6 and 8.8±3.5, respectively in control and experiment group. Statistical analysis revealed that there is no difference in MYCN expression between two group(t = 1.6, p=0.1357, p>0.05), while after treatment, MYCNexpression is significant lower than that in control group(t =4.8, p=0.001, p<0.05).Conclusion: Mouse neuroblastoma subcutaneous metastasis model can be successfully established by inoculation of 0.5ml cell suspension with concentration of 1×10~7/ml LA-N-5 cells. Folic acid receptor targeted lipisome mediated MYCN siRNA can significantly down-regulate MYCN mRNA expression in vivo.