Abnormal apoptosis of trophoblastic cells is related to the up-regulation of CYP11A gene in placenta of preeclampsia patients

Abstract

Abnormal placenta trophoblast proliferation and apoptosis is related to the pathogenesis of preeclampsia. Emerging evidence has also indicated that key pregnancy-associated hormones, such as hCG, progesterone, are found in high concentration at the maternal-fetal interface. The purpose of this study was to investigate the expression of CYP11A, a key enzyme in steroid hormone synthesis and metabolism, in normal pregnancy and severe preeclampsia placenta and to explore the underlying mechanism of the relationship between the altered CYP11A expression and onset of preeclampsia. Immunohistochemistry method was used to study the localization of CYP11A-encoded protein P450scc in the placenta; reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to examine CYP11A expression at mRNA and protein levels in patients with severe preeclampsia and normal placental tissue. CYP11A overexpression in trophoblastic cells was used to evaluate the effect on viability. TUNEL staining was used to determine whether overexpression of CYP11A could affect trophoblastic cell apoptosis. The results showed that CYP11A was selectively expressed in the cytoplasm of the placental trophoblastic cells. CYP11A expression were significantly increased in severe preeclampsia compared with normal pregnancy in both mRNA and protein levels. Multiple regression analysis indicated that CYP11A gene expression was positively correlated to ALT level and Plt, while negatively correlated to INR. Overexpression of CYP11A reduced trophoblastic cell proliferation and induced HTR8/SVneo cells apoptosis through activation of activated caspase-3 expression. These results suggest that abnormally high expression of CYP11A inhibits trophoblastic proliferation and increases apoptosis and therefore could be involved in the pathogenesis of preeclampsia.

Figure 1. Immunohistochemistry staining of P450scc in placenta tissue.

(A) Upper panel: Visible P450scc positive expression located in the cytoplasm of syncytiotrophoblast of placenta (arrow). Left: negative control without primary antibody. Right: P450scc (CYP11A) staining; Manification 600x; scale bar = 40 uM. Down panel: the higher manification pictures of the insert part of the above figures (B) Western blot shows that there is no significant different of CYP11A expression between first and third trimester placentas.

Figure 2. P450scc protein expression in preeclampsia group was significantly higher than normal control.

Left panel: representative western blot result of the placenta samples, Right panel: statistic of western blot result from 53 normal samples and 59 preeclampsia samples (p<0.05).

Figure 3. Linear multiple regression result of P450scc protein expression and clinical parameters.

(A) CYP11A expression negatively correlated to INR (P = 0.095). (B) CYP11A expression positively correlated to Plt (P = 0.018). (C) CYP11A expression positively correlated to ALT (P = 0.02).

Figure 4. Overexpression of CYP11A reduced HTR-8/SVneo cell viability.

(A) Determining the retention of Geimsa stain by cells after 72 h of culture in hypoxia condition. The average optical density (means±SEM) of the Geimsa stain is shown in the bar chart (p<0.05). (B) Cell counts of the CYP11A gene overexpressed and vector control HTR-8/SVneo cell (p<0.05). (C) Representative image of the CYP11A overexpressed (right) and vector control (left) HTR-8/SVneo cells.

Figure 5. Enhanced apoptosis in CYP11A overexpressed HTR-8/SVneo cells.

(A) TUNEL staining shows increased positive signals in CYP11A overexpressed HTR-8/SVneo cells compared to vector control when cultured in hypoxia condition. (B) Western blot result showed increased cleaved caspase-3 expression in CYP11A overexpressed HTR-8/SVneo compared to vector control cells. The image shown is representative result of two independant experiments.