Gene profiling of cottontail rabbit papillomavirus-induced carcinomas identifies upregulated genes directly Involved in stroma invasion as shown by small interfering RNA-mediated gene silencing.
To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs was further confirmed by quantitative reverse transcription-PCR (RT-PCR) with additional biopsies. Eight papilloma-carcinoma pairs examined showed a constant upregulation of the transcripts for the multifunctional adaptor protein 14-3-3 zeta and the Y-box binding transcription factor YB-1, whereas transcripts for m-type calpain 2 and NB thymosin beta, which are involved in cell motility and tissue invasion, as well as casein kinase 1 alpha, chaperonin, and annexin I, were found to be upregulated in the majority of the cases. RNA-RNA in situ hybridization and laser capture microdissection in combination with quantitative RT-PCR analysis verified the deregulated expression of the transcripts in the tumor cells. In contrast, CRPV E7 transcript levels remained rather constant indicating no requirement for a further upregulation of E7 expression following tumor induction. Small interfering RNA-mediated interference with expression of genes encoding YB-1, m-type calpain 2, or NB thymosin beta in a CRPV-positive cell line established from New Zealand White rabbit keratinocytes resulted in decreased cell invasion in matrigel chamber assays.
Huber
Evamaria
E
Sektion Experimentelle Virologie, Universitaetsklinikum Tuebingen, 72076 Tuebingen, Germany.
Vlasny
Daniela
D
Jeckel
Sonja
S
Stubenrauch
Frank
F
Iftner
Thomas
T
D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D002478:Cells, Cultured; D012777:Cottontail rabbit papillomavirus / Q000472:pathogenicity*; D020869:Gene Expression Profiling; D020868:Gene Silencing; D015603:Keratinocytes; D009693:Nucleic Acid Hybridization; D010212:Papilloma / Q000378:metabolism / Q000473:pathology / Q000821:virology; D030361:Papillomavirus Infections / Q000378:metabolism / Q000473:pathology / Q000821:virology; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017154:Stromal Cells / Q000378:metabolism / Q000473:pathology / Q000821:virology; D015854:Up-Regulation
D011506:Proteins; D034741:RNA, Small Interfering