Original Article
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Otorhinolaryngol. Feb 28, 2012; 2(1): 1-7
Published online Feb 28, 2012. doi: 10.5319/wjo.v2.i1.1
Effects of p27Kip1- and p53- shRNAs on kanamycin damaged mouse cochlea
Laura Pietola, Jussi Jero, Reetta Jalkanen, Teemu J Kinnari, Olga Jero, Mikko Frilander, Katri Pajusola, Marjo Salminen, Antti A Aarnisalo
Laura Pietola, Jussi Jero, Teemu J Kinnari, Olga Jero, Antti A Aarnisalo, Department of Otorhinolaryngology, University of Helsinki, 00014 Helsinki, Finland
Reetta Jalkanen, Folkhälsan Institute of Genetics, Biomedicum Helsinki, 00014 Helsinki, Finland
Mikko Frilander, Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland
Katri Pajusola, Molecular Cancer Biology Program, Biomedicum Helsinki, 00014 Helsinki, Finland
Marjo Salminen, Department of Basic Veterinary Science, Division of Biochemistry, University of Helsinki, 00014 Helsinki, Finland
Author contributions: Pietola L, Jero J, Jalkanen R, Kinnari TJ, Jero O and Aarnisalo AA performed the research and analyzed the data; Jero J, Salminen M and Aarnisalo AA designed the research; Frilander M and Pajusola K contributed shRNAs and virus vectors; Pietola L, Jero J, Jalkanen R, Salminen M and Aarnisalo AA wrote the paper.
Supported by grants from a Helsinki University Central Hospital Research Funds; the Sigrid Jusélius Foundation; the Instrumentarium Research Foundation; and the Finnish Medical Foundation.
Correspondence to: Antti A Aarnisalo, MD, PhD, Department of Otorhinolaryngology, University of Helsinki, 00014 Helsinki, Finland. antti.aarnisalo@hus.fi
Telephone: +358-9-4711 Fax: +358-9-47175010
Received: March 22, 2011
Revised: November 2, 2011
Accepted: February 27, 2012
Published online: February 28, 2012
Abstract

AIM: To study the effects of adeno-associated virus (AAV) delivered short hairpin RNAs (shRNAs) on adult CD-1 mouse cochlea damaged by aminoglycoside antibiotic kanamycin.

METHODS: Three different shRNAs were designed (p27 Kip1, p53 and p27 Kip1+p53) and tested in COS cells. A total of 20 adult CD-1 mice were used in the experiment. Mice were divided into five different groups (four animals/group) depending on the AAV-shRNA construct they received and whether they received kanamycin or not. Saline and AAV-EGFP injected animals were used as controls. All constructs were injected through the round window membrane (RWM) into the cochlea. Cochleae were harvested after 1 mo. Apoptosis was detected with Tunel labeling from paraffin-embedded cochlear tissue sections.

RESULTS: AAV2/2-p27 Kip1-shRNA and AAV2/2-p53-shRNA were tested in COS cells. Western blotting analysis confirmed that both constructs silenced their target genes effectively in the cell culture. AAV2/2-shRNA constructs were injected into the cochlea of CD-1 mice through the intact RWM. Cotransduction of individual AAV2/2-shRNAs with AAV2/2-EGFP resulted in EGFP expression in the organ of Corti. Kanamycin treatment had no effect on the expression pattern of the EGFP. AAV2/2-shRNA treated mice (either with p53 or p27Kip1and p53 together) showed fewer apoptotic hair cells in the cochlea than the control group (P < 0.05; AAV2/2-p53-shRNA vs saline P = 0.00014; AAV2/2-p27+p53-shRNA vs saline P = 0.0011). AAV2/2-p27-shRNA injected cochleae showed no significant difference in the number of apoptotic cells when compared to the saline injected cochleae.

CONCLUSION: Silencing of p53 protein in the kanamycin treated ears may decrease cell death in the organ of Corti.

Keywords: Inner ear, Adeno-associated virus, Short hairpin RNA, p27, p53, Kanamycin, Apoptosis