Cancer inhibition in nude mice after systemic application of U6 promoter-driven short hairpin RNAs against PLK1.
BACKGROUND
RNA interference initiated by small interfering RNAs effectively suppresses gene expression, but the suppression is transient, which limits the therapeutic use of this technique. Polo-like kinase 1 (PLK1) is a key cell cycle regulator that is overexpressed in various human tumors. We used a xenograft mouse model to determine whether an RNA interference-based strategy that used short hairpin RNAs (shRNAs) to suppress PLK1 expression could inhibit tumor growth in vivo.


METHODS
HeLa S3 cervical and A549 lung cancer cell lines were transfected with plasmids containing U6 promoter-driven shRNAs against human PLK1 or control (parental or scrambled) plasmids. Plasmids were treated with the nuclease inhibitor aurintricarboxylic acid (ATA) as protection against nucleases in murine blood. Nude mice carrying xenograft tumors were injected with shRNA plasmids, and their xenograft tumor growth was assessed. Northern and western blot analyses were used to measure PLK1 mRNA and protein expression, respectively, in transfected cultured cells and in xenograft tumors. All statistical tests were two-sided.


RESULTS
Levels of PLK1 mRNA and protein were lower in HeLa S3 and A549 cancer cells transfected with PLK1 shRNA plasmids than in corresponding cells transfected with control parental or scrambled PLK1S shRNA plasmids. Proliferation of cells transfected with PLK1 shRNA was lower than that of cells transfected with either control plasmid, and proliferation of cells transfected with ATA-treated PLK1 shRNA plasmids was even lower. In mice with human xenograft tumors, PLK1 shRNA expression from ATA-treated plasmids reduced tumor growth to 18% (95% confidence interval [CI] = 12% to 26%; P =.03) and from untreated plasmids reduced tumor growth to 45% (95% CI = 26% to 64%; P =.1) of that of tumors in mice treated with scrambled control PLK1S shRNA plasmids.


CONCLUSIONS
The combination of shRNA-mediated gene silencing with effective in vivo gene delivery strategies appears to generate a long-lasting silencing signal.
Spänkuch
Birgit
B
Department of Obstetrics and Gynecology, School of Medicine, J. W. Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany.
Matthess
Yves
Y
Knecht
Rainald
R
Zimmer
Brigitte
B
Kaufmann
Manfred
M
Strebhardt
Klaus
K
D000199:Actins / Q000037:antagonists & inhibitors; D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage* / Q000494:pharmacology*; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D004273:DNA, Neoplasm / Q000302:isolation & purification; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000188:drug therapy*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000008:administration & dosage* / Q000494:pharmacology*; D014162:Transfection; D014183:Transplantation, Heterologous; D000097869:Polo-Like Kinase 1
D000199:Actins; D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D004273:DNA, Neoplasm; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D000097869:Polo-Like Kinase 1; C405486:U6 small nuclear RNA