Figure 2. TP53BP2 is a potential therapeutic target for the autophagy of trophoblasts in PE. (A) On gestational day 14, SD rats underwent surgical reduction of uterine perfusion pressure (RUPP), followed by a single injection of 10 μL of AAV-shTP53BP2 (1.5E+11) into the placenta. (B) The expression of TP53BP2 in placentas from preeclamptic rats was detected by western blot and qRT‒PCR. (C) Immunofluorescence staining showing TP53BP2 expression in placentas from preeclamptic rats. Scale bar=50 µm. (D) Immunohistochemical staining was used to detect the expression of TP53BP2 in placentas from preeclamptic rats. Scale bar=200 µm. (E) Systolic and diastolic blood pressure were measured via a noninvasive tail-cuff blood pressure measurement system in preeclamptic rats. (F) Total urine protein levels were measured via a protein assay in preeclamptic rats.(G) Gross appearance and birthweight of the fetus at embryonic day 18.5 (E18.5). (H) HE staining analysis of placental pathological changes in preeclamptic rats. Scale bar=500 μm.(I) The expression of LC3B-II and p62 in placentas from preeclamptic rats was detected by western blot. (J) Immunofluorescence staining was used to detect the expression of LC3B and p62 in placentas from preeclamptic rats. Scale bar=50 µm.(K) Immunohistochemical staining was used to detect the expression of LC3B and p62 in placentas from preeclamptic rats. Scale bar=200 µm. The data are presented as the means ± SDs.*P <0.05,**P <0.01,***P <0.001.
TP53BP2 enhances trophoblast autophagy by regulating Beclin-1 expression
Next, we performed RNA-seq on HTR8/SVneo cells with TP53BP2 knockdown to investigate the molecules involved in autophagy. The results revealed the upregulation of 1046 molecules and the downregulation of 1035 molecules (|fold change|≥2.0, P ≤0.05) (Figure 3A ). GO analysis revealed that these molecules are involved in autophagy, ATP binding, protein serine/threonine kinase activity, mTOR signaling, the HIF1 signaling pathway and AMPK signaling (Figure S2 ). A qRT‒PCR assay was subsequently employed to measure the levels of the top 10 downregulated genes. The results revealed a significant reduction in the level of Beclin-1, a key marker of autophagy, in the placentas of preeclamptic rats (Figure 3B, C ). To further confirm the role of Beclin-1 in TP53BP2-induced autophagy, sh-Beclin-1 and Ad-TP53BP2 were cotransfected into HTR8/SVneo or JEG-3 cells under hypoxia (Figure S3). As shown in Figure 3D , cotransfection with sh-Beclin-1 and Ad-TP53BP2 resulted in a decrease in LC3B-II levels and an increase in p62 expression in both HTR8/SVneo and JEG-3 cells. Bcl-2 is known to interact with Beclin-1, an initiator of autophagy. Previous results indicate that the release of Beclin-1 from the Bcl-2–Beclin-1 complex then initiates TP53BP2-induced autophagy. Here, we investigated the role of Bcl-2 binding to the BH3 domain of Beclin-1 in TP53BP2-induced autophagy in trophoblasts. Coimmunoprecipitation (co-IP) revealed an increase in the interaction between TP53BP2 and Bcl-2, whereas disruption of the interaction between Beclin-1 and Bcl-2 was observed in HTR8/SVneo and JEG-3 cells under hypoxia (Figure 3E ). Conversely, knockdown of TP53BP2 increased the interaction between Beclin-1 and Bcl-2 (Figure 3F ). Taken together, these results demonstrate that knockdown of TP53BP2 inhibits trophoblast autophagy by decreasing Beclin-1 expression and promoting the interaction between Beclin-1 and Bcl-2.