Knockdown of XBP1 by RNAi in Mouse Granulosa Cells Promotes Apoptosis, Inhibits Cell Cycle, and Decreases Estradiol Synthesis

Abstract

Granulosa cells are crucial for follicular growth, development, and follicular atresia. X-box binding protein 1 (XBP1), a basic region-leucine zipper protein, is widely involved in cell differentiation, proliferation, apoptosis, cellular stress response, and other signaling pathways. In this study, RNA interference, flow cytometry, western blot, real-time PCR, Cell Counting Kit (CCK8), and ELISA were used to investigate the effect of XBP1 on steroidogenesis, apoptosis, cell cycle, and proliferation of mouse granulosa cells. ELISA analysis showed that XBP1 depletion significantly decreased the concentrations of estradiol (E2). Additionally, the expression of estrogen synthesis enzyme Cyp19a1 was sharply downregulated. Moreover, flow cytometry showed that knockdown of XBP1 increased the apoptosis rate and arrests the cell cycle in S-phase in granulosa cells (GCs). Further study confirmed these results. The expression of CCAAT-enhancer-binding protein homologous protein (CHOP), cysteinyl aspartate specific proteases-3 (caspase-3), cleaved caspase-3, and Cyclin E was upregulated, while that of Bcl-2, Cyclin A1, and Cyclin B1 was downregulated. Simultaneously, CCK8 analysis indicated that XBP1 disruption inhibited cell proliferation. In addition, XBP1 knockdown also alters the expression of Has2 and Ptgs2, two essential genes for folliculogenesis. Collectively, these data reveal a novel critical role of XBP1 in folliculogenesis by regulating the cell cycle, apoptosis, and steroid synthesis of mouse granulosa cells.

miR-431 regulates granulosa cell function through the IRS2/PI3K/AKT signaling pathway

“…Cyclin B1 is also involved in the mitotic process as an activator of Cdk1 [24]. Together, the downregulation of Cyclin A1 and Cyclin B1 could lead to S phase arrest by stopping granulosa cells from entering the G2/M phase [25]. Moreover, the inhibition of IRS2/AKT resulted in a direct decrease in cyclin D2 in granulosa cells [26], which could further impair granulosa cell proliferation and follicular growth.…”

Endoplasmic reticulum stress is involved in lipopolysaccharide‐induced inflammatory response and apoptosis in goat endometrial stromal cells

“…To impose stress on the cells, we applied different concentrations of LPS at different time points and processed them for further experiments. The ESCs were plated into 96‐well plates as described previously (Wang et al, ) and then divided into three groups. The first group was treated with different doses of LPS (0, 10, 20, 30, and 40 μg/ml) at 6 hr, the second group was treated with different concentrations of LPS (0, 1, 5, 10, and 20 μg/ml) at 24 hr, and the third group was treated with 20 μg/ml LPS at 6, 12, and 24 hr, each group have four replications.…”

“…The sequences of the specific primers were used to amplify the related 16 genes that are listed in Table . Glyceraldehyde 3‐phosphate dehydrogenase was used as a control gene (Wang et al, ). The messenger RNA (mRNA) quantification was estimated using the method (Yi et al, ).…”

MicroRNA-205 affects mouse granulosa cell apoptosis and estradiol synthesis by targeting CREB1

“…Follicular atresia was closely associated with correlates with estrogen E2 5 concentrations in follicular fluid (14,20). To further investigate the effect of miR-205 on estrogen, the concentration of E2 in the medium of mGCs transfected with miR-205 mimics was measured using a RIA kit.…”