The Impact Of Histone Deacetylase Inhibitor Trichostatin A On Proliferation And Apoptosis Of Porcine Granulosa Cells

Granulosa cells play important role in the different development stages of ovarian by synthesizing and secreting a variety of hormones and growth factors through autocrine/paracrine. Granulosa cells are crucial to oocyte maturation and follicular development. Related studies suggested that the apoptosis of granulosa cells is a key cause of follicular atresia. If apoptosis of more than 10% of follicular granulosa cell occurs, it indicates that the follicular have been or are being atresic. As the first found natural histone deacetylase inhibitor, recent studies have found that Trichostatin A (TSA) had inhibitory effect on many tumor cells by inducing tumor cell apoptosis and cell differentiation, blocking cell cycles and so on. As a promising anticancer drug, the effect of histone deacetylase inhibitors on normal cells remains unclear. Therefore, this study aims to investigate the effect of histone deacetylase inhibitors on porcine granulosa cells and its preliminary mechanism. All the results can provide a reference for clinical use. Specific aspects are involved in this study as follows.The effect of TSA on the viability of porcine granulosa cell was detected by MTT method. The results found no effect on porcine granulosa cells when TSA at low concentrations (10ng/mL or less), however, the growth of porcine granulosa cells was inhibited with the increase of drug concentration (P<0.05), and it is dose-dependent inhibition. No significant difference of OD550 value in different time was found between treated groups with 10ng/mL and 200ng/mL, it indicated the inhibition of TSA on porcine granulosa cells is not time-dependent.The effect of TSA on acetylation levels of porcine granulosa cells was studied by performing indirect immunofluorescence, and the mechanism was learned by analyzing molecular level with fluorescence quantitative PCR technology. Indirect immunofluorescence showed that the acetylation levels of histone H3 of granulosa cells treated with 200ng/mL TSA changed significantly, but no significant change at low concentration (10ng/mL). Quantitative RT-PCR results showed significant differences (P<0.05) of Hadc-1, Hadc-3 and p300 mRNA expression between treated group with 200 ng/mL TSA and the control group, but no significant difference (P>0.05) at low concentration (10ng/mL) compared to the control. It indicated that TSA has affect on the normal acetylation level of granulosa cells at higher concentrations, but no at low concentration.The apoptosis rate of granulosa cell treated with different concentrations TSA was detected by flow cytometry to determine whether apoptosis is the mechanism of growth inhibition of granulosa cells by treating with TSA. The results showed that the cell apoptosis rate in the 200ng/mL TSA treatment group was as high as 13.7%, which was significantly differenent than the rate of 9.1% in the control group (P<0.05), but no significance was found between thelOng/mL TSA treatment group and the control (P>0.05). This demonstrated that TSA with high concentration could accelerate cell apoptosis of granulosa, which imposes a key effect on the cell activity.The changes of granulosa cell cycle treated with different concentration of TSA were measured by flow cytometry and expressions of cell cycle-related genes were studied by quantitative RT-PCR technology. Flow cytometry results showed that in 200ng/mL TSA treatment group, the percentage of cells in G0/G1 phase was significantly increased (P<0.05), and cells in S phase was significantly lower (P<0.05), percentage of cells in G2/ M phase was not significantly changed compared with the control group. No significant changes of the percentage of cells were found at low concentration (10ng/mL) treatment. Similarly, quantitative PCR results showed that in 200ng/mL TSA treatment group, Cyclin D2 and CDK4 mRNA significant changed (P<0.05), while no significant difference (P>0.05) was displayed at low concentration (10ng/mL). It show that the cell cycle was blocked in the G0/G1 phase when the granulosa cells were treated with 200ng/mL TSA, and significantly reduction of Cyclin D2 and CDK4 mRNA expression might be an important reason for cell cycle blockage.