RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

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 CYP19A1 and 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.

Fig. 1.

Transfection and knockdown efficiency of INHBB RNAi vectors. Three (3) groups of INHBB RNAi recombinant plasmids were transfected in mouse GCs named as pshRNA-B1, pshRNA-B2, pshRNA-B3 and pshRNA-negative. After 48 h, the expression of GFP in the recombinant plasmids were shown, which implied that INHBB recombinant plasmids could be high efficiently expressed in mouse GCs. The best efficient RNAi vector (pshRNA-B3) was selected for further investigation.

Fig. 2.

Expression of INHBB in transfected GCs. The mRNA level of INHBB in mouse GCs was detected after transfection with INHBB knockdown vectors, respectively. The ratio is relative to the pshRNA-negative control group. All silencing vectors could efficiently knockdown the expression of INHBB with pshRNA-B3 having the best silencing efficiency, which was chosen for further study. Values are presented as the mean ± SEM (n = 3 in each group). Bars with different letters indicate significantly different at P < 0.05.

Fig. 3.

INHBB protein levels in transfected GCs. INHBB protein levels were detected by western blot in mouse GCs 48 h after transfection. Lanes 1 to 4 represent the pshRNA-B1, pshRNA-B2, pshRNA-B3, and pshRNA-negative, respectively. Normalized ratios of INHBB band intensities were calculated by dividing the mean signal intensity for 3 biological replicates by the mean signal intensity with GAPDH. Values are presented as the mean ± SEM. Different letters (a, b) indicate significantly different at P < 0.05.

Fig. 4.

DNA-content of transfected mouse GCs by flow cytometry. Following a 48-h transfection with pshRNA-B3 and pshRNA-negative respectively, GCs were treated for DNA content by propidium iodide. ( A ) Histogram showing the proportions of cells that are in the three phases of the cell cycle using flow cytometry to measure their relative DNA content. G0/G1-phase cells are diploid (2N) and express half the DNA content of tetraploid G2/M phase cells (4N). S phase cells contain varying amounts of DNA between the G1 and G2 phases. (B) A graph presenting the % of GCs in each of the cell cycle phases. Values are presented as means ± SEM (n = 3 in each group). An asterisk (*) indicates significantly different at P < 0.05.

Fig. 5.

Protein levels of related genes in transfected GCs. The protein levels of Cyclin D1, Cyclin E, BCL-2 and BAX were detected by western blot 48 h after transfection with the pshRNA-B3 and pshRNA-negative plasmids, respectively. The normalized ratio for each protein was calculated by dividing the mean signal intensity from 3 biological replicates by the mean signal intensity with GAPDH. Values are presented as the mean ± SEM (n = 3 in each group). Different letters (a, b) indicate significantly different at P < 0.05.

Fig. 6.

Expression of CYP19A1 and CYP11A1 in transfected GCs. The mRNA levels of CYP19A1 and CYP11A1 genes in GCs transfected with pshRNA-B3 and pshRNA-negative respectively were determined 48 h after transfection by q-PCR. The results showed that the mRNA level of CYP19A1 (P < 0.05) (A) and CYP11A1 (P < 0.05) (B) were significantly downregulated in pshRNA-B3 group compared with pshRNA-negative group. Values are presented as the mean ± SEM, n = 3 in each group. Bars with different mark indicate significantly different at P < 0.05.