RNAi-mediated knockdown of INHBB
increases apoptosis and inhibits steroidogenesis in mouse granulosa
cells |
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Authors: | Mohamed M’BAYE Guohua HUA Hamid Ali KHAN Liguo YANG |
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Institution: | 1)Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science & Technology, Huazhong Agricultural University, Wuhan 430070, PR China |
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Abstract: | Inhibins are members of the TGFβ superfamily and act as suppressors
of follicle stimulating hormone (FSH) secretion from pituitary glands
via a negative feedback mechanism to regulate folliculogenesis. In
this study, the INHBB gene was knocked down by three
RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids
to explore the effects of INHBB silencing on
granulosa cell (GC) cell cycle, apoptosis and steroid production
in vitro. Quantitative real-time polymerase chain
reaction, Western blot, flow cytometry and ELISA were performed to
evaluate the role of INHBB in the mouse GC cell
cycle, apoptosis and steroid production in vitro. The
results showed that the relative mRNA and protein expression of
INHBB in mouse GCs can be significantly reduced by
RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3
having the best knockdown efficiency. Downregulation of the expression
of INHBB significantly arrests cells in the G1 phase
of the cell cycle and increases the apoptosis rate in GCs. This was
further confirmed by downregulation of the protein expressions of
Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was
upregulated. In addition, specific downregulation of
INHBB markedly decreased the concentration of
estradiol and progesterone, which was further validated by the
decrease in the mRNA levels of CYP19A1and
CYP11A1. These findings suggest that inhibin βB is
important in the regulation of apoptosis and cell cycle progression in
granulosa cells. Furthermore, the inhibin βB subunit has a role in the
regulation of steroid hormone biosynthesis. Evidence is accumulating
to support the concept that inhibin βB is physiologically essential
for early folliculogenesis in the mouse. |
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Keywords: | Apoptosis Granulosa cells INHBB Knockdown Steroidogenesis |
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