The effect of Tc-99m-labeled MDM2 antisense oligonucleotide on gene expression in human breast cancer MCF-7 cells.
To investigate the effect of radiolabed mouse double minute 2 (MDM2) antisense oligonucleotide on gene expression in human breast cancer MCF-7 cells, an antisense oligonucleotide (ASON) targeting MDM2 mRNA was synthesized and radiolabeled with 99Tcm. The labeling efficiency, radiochemical purity, and the ability of labeled ASON to hybridize to the sense oligonucleotides (SON) were investigated. To study whether the antisense probe hybridizes to respective sequence on MDM2 mRNA strand after radiolabeling, cells were incubated with radiolabeling oligonucleotides antisense oligonucleotide (0, 100, 500 nm/L) or mismatch oligonucleotide (ASONM) (500 nm/L) for 24 h, in the presence of Lipofectin 2000. RT-PCR and Western blotting was carried out to measure the MDM2 mRNA and protein levels. The antisense oligonucleotide was radiolabeled with the bifunctional chelator HYNIC at the labeling efficiency of 57.2 +/- 2.98% (n = 5) and the mismatch oligonucleotide was 56.3 +/- 3.01% (n = 5). The radiochemical purity was above 95% and labeled antisense oligonucleotide has the ability to hybridize to the sense oligonucleotide. The levels of mRNA and protein have significant differences in different concentration groups. The oligonucleotide can be successfully radiolabeled, and specially hybridized to the MDM2 mRNA and inhibit gene expression intensively as compared to mismatch oligonucleotide. This method will be very useful in the in vivo investigation of tumor targeting.
Zhao
Changjiu
C
Department of Nuclear Medicine, Union Hospital, Tongji Medicial College, Huazhong University of Science and Technology, Wuhan 430022, China.
Fu
Peng
P
Sheng
Yunxia
Y
Li
Zhongyuan
Z
D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D005260:Female; D015870:Gene Expression; D016158:Genes, p53; D006801:Humans; D006834:Hydrazines; D007553:Isotope Labeling; D009539:Nicotinic Acids; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D013667:Technetium; D016159:Tumor Suppressor Protein p53 / Q000032:analysis
C120392:6-hydrazinopyridine-3-carboxylic acid; D006834:Hydrazines; D009539:Nicotinic Acids; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D013667:Technetium; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2