ABSTRACT
Inadequate invasion and excessive apoptosis of trophoblast cells are associated with the development of preeclampsia. Vitamin D deficiency in pregnant women may lead to an increased risk of preeclampsia. However, the underlying mechanisms by which vitamin D is effective in preventing preeclampsia are not fully understood. The objectives of this study were to investigate the role of lysosome-associated membrane glycoprotein 3 (LAMP3) in the pathogenesis of preeclampsia and to evaluate whether vitamin D supplementation would protect against the development of preeclampsia by regulating LAMP3 expression. Firstly, the mRNA and protein levels of LAMP3 were significantly upregulated in the placentas of preeclampsia patients compared to normal placentas, especially in trophoblast cells (a key component of the human placenta). In the hypoxia/reoxygenation (H/R)-exposed HTR-8/Svneo trophoblast cells, LAMP3 expression was also upregulated. H/R exposure repressed cell viability and invasion and increased apoptosis of trophoblast cells. siRNA-mediated knockdown of LAMP3 increased cell viability and invasion and suppressed apoptosis of H/R-exposed trophoblast cells. We further found that 1,25(OH)2D3 (the hormonally active form of vitamin D) treatment reduced LAMP3 expression in H/R exposed trophoblast cells. In addition, 1,25(OH)2D3 treatment promoted cell viability and invasion and inhibited apoptosis of H/R-exposed trophoblast cells. Notably, overexpression of LAMP3 abrogated the protective effect of 1,25(OH)2D3 on H/R-exposed trophoblast cells. Collectively, we demonstrated trophoblast cytoprotection by vitamin D, a process mediated via LAMP3.
ABSTRACT
@#[Abstract] Objective: To investigate the effect of Beclin1 knockdown on cisplatin resistance in ovarian cancer A2780 cells and its related mechanisms. Methods: The mRNA and protein expressions of Beclin1 in A2780 cells and drug resistant A2780/DDP cells were determined by qPCR and Western blotting. After transfection with Beclin1 siRNA, the sensitivity of A2780/DDP cells to cisplatin was detected by MTT assay; Cell clone formation and apoptosis were detected by the Colony formation assay and Flow cytometry assay, respectively; cell autophagy was monitored by monodansylcadaverin (MDC) staining. Furthermore, the protein levels of cell autophagy related proteins, lysosomal associated membrane protein Lamp-2 and Cathepsin B were detected by Western blotting. Results: The mRNA and protein expression levels of Beclin1 in cisplatin-resistant A2780/DDP cells were significantly higher than those in A2780 cells (all P<0.05). The expression of Beclin1 was significantly increased in A2780 cells after treated with cisplatin (P<0.05). Beclin1 knockdown promoted cisplatin induced apoptosis of A2780/DDP cells (P<0.05), inhibited autophagy and cell colony formation (all P<0.05), and increased cell sensitivity to cisplatin (P<0.05). Meanwhile, Western blotting showed that Beclin1 knockdown increased the protein levels of cleaved-caspase 3 and Cathepsin B in A2780/DDP cells, while down-regulated the protein expressions of Atg3, Atg7, LC3Ⅱ/Ⅰand Lamp-2 (all P<0.05). Conclusion: Beclin1 knockdown can improve the sensitivity of A2780/DDP cells to cisplatin, and the mechanism may be related to the inhibition of protective autophagy of cells by regulating the expressions of autophagy related proteins, and the regulation of lysosomes, thus further promoting cisplatin-induced apoptosis of drug-resistant cells.