Identification of microRNAs controlling human ovarian cell proliferation and apoptosis.
Previous studies have shown that microRNAs (miRNAs) can control steroidogenesis in cultured granulosa cells. In this study we wanted to determine if miRNAs can also affect proliferation and apoptosis in human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different constructs encoding human pre-miRNAs on the expression of the proliferation marker, PCNA, and the apoptosis marker, Bax was evaluated by immunocytochemistry. Eleven out of 80 tested miRNA constructs resulted in stimulation, and 53 miRNAs inhibited expression of PCNA. Furthermore, 11 of the 80 miRNAs tested promoted accumulation of Bax, while 46 miRNAs caused a reduction in Bax in human ovarian cells. In addition, two selected antisense constructs that block the corresponding miRNAs mir-15a and mir-188 were evaluated for their effects on expression of PCNA. An antisense construct inhibiting mir-15a (which precursor suppressed PCNA) increased PCNA, whereas an antisense construct for mir-188 (which precursor did not change PCNA) did not affect PCNA expression. Verification of effects of selected pre-mir-10a, mir-105, and mir-182 by using other markers of proliferation (cyclin B1) and apoptosis (TdT and caspase 3) confirmed specificity of miRNAs effects on these processes. This is the first direct demonstration of the involvement of miRNAs in controlling both proliferation and apoptosis by ovarian granulose cells, as well as the identification of miRNAs promoting and suppressing these processes utilizing a genome-wide miRNA screen.
Sirotkin
Alexander V
AV
Animal Production Research Centre, LuZianky, Slovakia.
Lauková
Marcela
M
Ovcharenko
Dmitriy
D
Brenaut
Pauline
P
Mlyncek
Milos
M
D000328:Adult; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D056744:Cyclin B1 / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D035683:MicroRNAs / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C494826:BAX protein, human; C534699:CCNB1 protein, human; D056744:Cyclin B1; D035683:MicroRNAs; D018809:Proliferating Cell Nuclear Antigen; D051028:bcl-2-Associated X Protein; C505407:CASP3 protein, human; D053148:Caspase 3