Effect of 99Tcm-labeled mouse double minute 2 antisense oligonucleotide on target gene expression of prostatic cancer cells / 中华核医学与分子影像杂志

ABSTRACT

Objective To investigate the effect of mouse double minute 2 (MDM2) mRNA ASON and mismatched oligonucleotide (ASONM) radiolabeled with 99Tcm on target gene expression in LNCaP cells.Methods The ASON and ASONM targeted to MDM2 mRNA were synthesized and radiolabeled by 99Tcm with the bifunctional chelator of HYNIC.The labeling efficiency,radiochemical purity,stability and molecular hybridization activity were investigated.The different concentrations of 99Tcm-HYNIC-ASON (0,100,500 nmol/L) and 99Tcm-HYNIC-ASONM (500 nmol/L) coated with lipofectamin 2000 were incubated with prostate cancer cells for 24 h,then RT-PCR and Western blot were carried out to assay the MDM2,p53 mRNA and the corresponding protein level.The variables of RT-PCR and Western blot were analyzed using one-way analysis of variance and q test.Results The labeling efficiency of ASON and ASONM were (65.15± 2.05)％ (n=5) and (64.93±2.18)％ (n=5),respectively.The radiochemical purity were both more than 90％.99Tcm-HYNIC-ASON had a good stability and could hybridize to the sense oligonucleotide (SON).The contents of MDM2 mRNA in 0,100,500 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM groups were 0.458±0.035,0.250±0.026,0.174±0.032,0.463±0.033,respectively,and there were significant differences between each 2 groups except between 0 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM groups (F=33.69,q =24.32-91.45,all P＜0.01).The average density of MDM2 protein in the 4 groups were 90.712±3.042,71.218±2.915,32.775±3.062,88.121±2.710,respectively (F=235.93,q=6.43-19.14,all P＜0.01; except 0 nmol/L99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM).The contents of p53 mRNA in the 4 groups were 0.185±0.046,0.203±0.040,0.213±0.027,0.163±0.049,respectively(F =2.18,P＞ 0.05).The average density of p53 protein was 33.865 ± 2.213,70.445±2.180,99.025±3.012,38.351±3.271,respectively (F=53.98,q =3.32-6.74,all P＜0.01 ; except 0 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM).Conclusions The MDM2 antisense probe can accumulate in the prostate cancer cells,and specially hybridize to the MDM2 mRNA and inhibit target gene expression.This novel molecular probe has a promising potential for the diagnosis of prostate cancer at gene level.