Protein kinases controlling PCNA and p53 expression in human ovarian cells.
The aim of this study was to identify protein kinases (PKs) involved in the expression of proliferating cell nuclear antigen (PCNA) and p53, markers of proliferation and apoptosis in human ovarian cells. Cultured ovarian granulosa cells were subjected to transfection with 264 small-interfering RNA (siRNA) constructs from a siRNA library, which selectively blocked the expression of 88 known PKs. The efficiency of transfection and siRNA knockdown were validated by fluorescent microscopy, real-time reverse transcription polymerase chain reaction, and immunocytochemical analysis. The expression of PCNA and p53, before and after transfection with siRNA constructs, was evaluated by immunocytochemistry. The siRNA constructs suppressed the expression of their targets molecules by up to 84%. Knockdown of 32 of the 88 PKs inhibited the expression of PCNA, while the knockdown of seven of the PKs stimulated PCNA expression. Knockdown of 30 of the 88 PKs reduced the expression of p53, while knockdown of five PKs enhanced p53 expression. Our results illustrate that siRNA constructs are useful tools for understanding the role of PKs in the control of ovarian cell functions, such as proliferation and apoptosis. The specific knockdown of individual PKs has enabled the identification of a number of new PKs that control the expression of PCNA and p53 in human ovarian cells.
Sirotkin
Alexander V
AV
Research Institute of Animal Production, Slovak Centre of Agricultural Studies, Nitra, Slovakia. sirotkin@scpv.sk
Ovcharenko
Dmitriy
D
Benco
Andrej
A
Mlyncek
Milos
M
D000328:Adult; D017209:Apoptosis; D049109:Cell Proliferation; D002478:Cells, Cultured; D005260:Female; D006107:Granulosa Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D018809:Proliferating Cell Nuclear Antigen; D034741:RNA, Small Interfering; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D011494:Protein Kinases