Suppression of Mitf by small interfering RNA induces dedifferentiation of chick embryonic retinal pigment epithelium.
Recent studies indicate a key role of Mitf (microphthalmia-associated transcription factor) in the differentiation of the retinal pigment epithelium (RPE). To explore transdifferentiation processes, we used small interfering RNA (siRNA) targeting Mitf. Transfection of embryonic chick RPE cells with a non-silencing fluorescein-labelled control siRNA demonstrated a high (84%) transfection efficiency. Transfection of Mitf siRNA reduced Mitf synthesis at the mRNA and protein levels as analysed by real-time RT-PCR and Western blot. Mitf siRNA suppressed the expression of Mitf RNA to 25.0% of the level in control cells (P<0.005). The expression of melanosomal matrix glycoprotein 115 (MMP115), a marker of differentiated pigment cells, was also markedly suppressed (to 52.2+/-6.6%) (P<0.0005). Moreover, the expression of Pax6 was increased by Mitf siRNA (to 143.5+/-18.0%) (P<0.0005), and induced dedifferentiation of the RPE cells. These data suggest siRNA can be an effective gene silencing approach in RPE, and reduction of Mitf expression is essential for the dedifferentiation of RPE cells.
Iwakiri
Ryo
R
Department of Ophthalmology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan.
Kobayashi
Kaori
K
Okinami
Satoshi
S
Kobayashi
Hiroshi
H
D000818:Animals; D030161:Avian Proteins / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D005786:Gene Expression Regulation; D020868:Gene Silencing; D051739:Microphthalmia-Associated Transcription Factor; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D030161:Avian Proteins; D004268:DNA-Binding Proteins; D051739:Microphthalmia-Associated Transcription Factor; D034741:RNA, Small Interfering; D014157:Transcription Factors; C071193:MMP115 protein, Gallus gallus