Activation of the P2X7 receptor leads to a rapid, bidirectional flux of cations, causing broad range of biological responses including cytotoxicity. However, the mechanism of P2X7-mediated cytotoxicity remains largely unexplored. In our previous study, the lack of P2X7-mediated calcium response under normal conditions was found in P2X7(+) hematopoietic cell lines. In this study, the P2X7-mediated cytotoxicity in different type of cells (P2X7(-), P2X7(+) with calcium response, and P2X7(+) without calcium response) was investigated. Our results showed that P2X7 agonists, adenosine 5'-triphosphate (ATP) or 2',3'-O-(4 benzoylbenzoyl)-ATP, dose-dependently reduced the cell viability in all P2X7(+) cells tested, including J6-1, LCL, and Namalva cells which are negative for P2X7-mediated calcium response, although these effects were lower than those observed in KG1a cells which has normal P2X7 functions. The cytotoxic effect could be blocked by P2X7 antagonists, oxidized ATP and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine. In addition, externalization of phosphatidylserine could be detected in a time-dependent manner and apoptotic morphological changes could be observed after the activation of P2X7 receptor in J6-1 cells. Furthermore, P2X7-mediated pore formation could be detected in KG1a and J6-1 cells under low-ionic conditions, but not under low-divalent conditions. These effects could not be observed in P2X7(-) Ramos cells. These results suggested that P2X7 receptor-mediated cytotoxic effects may occur independent of calcium response.

Background: Preeclampsia (PE) is a serious obstetric complication. Recent studies point out that the functions of long intergenic noncoding RNA 00473 (linc00473), miR-424-5p, and Wnt/β-catenin signaling pathway were involved in the invasion and migration of extravillous trophoblast. Here, we investigated the role and mechanism of linc00473 in HTR-8/SVneo trophoblastic cell line and its role in PE. Method: The expression levels of linc00473 and miR-424-5p in placental tissues and the transfection efficiency of miR-424-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). HTR-8/SVneo cell invasion and proliferation were determined by transwell and Cell Counting Kit-8 (CCK-8) assays. The protein expressions of wnt3a, p-GSK3β, GSK3β, active β-catenin, and total β-catenin were detected by Western blot. The apoptosis and migration of HTR-8/SVneo cells were detected by flow cytometry and wound healing assays. The targeting relationships between linc00473, miR-424-5p, and wnt3a were predicted by ENCORI database and TargetScan V7.2 and were determined using dual-luciferase reporter assay.Results: The expression level of linc00473 was low and miR-424-5p was high in placenta of PE patients. Linc00473 can target miR-424-5p, while miR-424-5p target wnt3a. High expression of linc00473 and wnt3a promoted cell proliferation, migration, invasion, and inhibited cell apoptosis. However, miR-424-5p mimic inhibited HTR-8/SVneo cells proliferation, migration, invasion, while promoted cell apoptosis, partially reversed the effect of linc00473, while wnt3a overexpression partially counteracted the effect of miR-424-5p mimic. Conclusion: Linc00473 mediates the regulation of Wnt/β-catenin signaling pathway by miR-424-5p to affect the invasion and migration ability of trophoblastic cell line HTR-8/SVneo. It indicated that linc00473 is involved in PE and could be a therapeutic target.