trophoblasts in PE
To elucidate the molecular mechanisms underlying the autophagy of
placental trophoblasts in early-onset PE, we employed RNA-seq to
examine autophagy-related differentially expressed genes (DEGs) in
placentas derived from early-onset PE pregnancies compared with those
from non-PE pregnancies. The functionally related genes were enriched
predominantly in autophagy-related signaling pathways, such as the
PI3K-Akt signaling, AMPK signaling, and mTOR signaling pathways
(Figure 1A ). The cluster heatmaps show the top 20 DEGs involved
in autophagy-related signaling pathways in placentas from PE pregnancies
(Figure 1B ). Among them, TP53BP2 was the most robust inducer
(Figure 1C ). Moreover, TP53BP2 expression was markedly greater
in placentas from PE pregnancies than in those from non-PE pregnancies(Figure 1D ). Immunohistochemical and immunofluorescence
staining further revealed the upregulation of TP53BP2 in trophoblasts
from PE pregnancies (Figure 1E, F) . To investigate the
functional role of TP53BP2, we conducted TP53BP2 overexpression and
knockdown in HTR8/Svneo and JEG-3 cells by transfecting Ad-TP53BP2 or
three independent sh-TP53BP2 vectors, respectively (Figure S1 ). TEM revealed increased autophagosome and autolysosome
formation in trophoblasts transfected with Ad-TP53BP2, whereas the
opposite effect was observed in those transfected with sh-TP53BP2
(Figure 1G ). Additionally, an autophagic flux assay using
tandem fluorescent GFP-RFP-LC3 revealed a decrease in total
autophagosome and autolysosome formation in trophoblasts transfected
with sh-TP53BP2 (Figure 1H ). LC3B-II and p62 serve as
complementary markers of autophagy, with LC3B-II reflecting
autophagosome formation and p62 indicating the process of substrate
recognition and degradation. Therefore, the protein expression of
LC3B-II and p62 was assayed to determine the extent of autophagy. The
results of the western blot analysis confirmed the effect of TP53BP2 on
the LC3B-II and p62 levels in HTR8/SVneo and JEG-3 cells under hypoxia
(Figure 1I ). These results demonstrated that the upregulation
of TP53BP2 could increase trophoblast autophagy in PE placentas.