Expression of interleukin-6 (IL-6), signal transducer and activator of transcription-3 (STAT-3) and telomerase in choriocarcinomas

Blastocyst implantation and neoplastic invasion have some common properties related to tissue invasion, mediated by various cytokines. Aim To compare the expression of IL-6, STAT-3 and telomerase in material of abortions in the first trimester of pregnancy, at term placentas and in choriocarcinomas. Methods Immunohistochemical reactions were performed on formalin fixed and included in paraffin samples from 3 groups: abortions, normal at term placentas and choriocarcinomas. Western Blot and Real-Time PCR assays were performed on fresh material from BeWo cell line and in primary culture cells of normal placenta. Results Immunohistochemical reactions: IL-6 expression was moderate in the first trimester abortion samples and high in at term placentas and choriocarcinomas. STAT-3 was strongly positive in all groups. Telomerase expression was absent in normal at term placentas but was increased in BeWo cells. Conclusion IL-6 and STAT-3 are present in the invasion process of the normal placental development and they are maintained during the malignant transformation to choriocarcinoma. The intense telomerase expression observed in BeWo cells was strongly associated with the malignant phenotype, confirming it as a good marker for cell transformation and tumor progression.

Transcriptomic profiling of trophoblast fusion using BeWo and JEG-3 cell lines

In human placenta, alteration in trophoblast differentiation has a major impact on placental maintenance and integrity. However, little is known about the mechanisms that control cytotrophoblast fusion. The BeWo cell line is used to study placental function, since it forms syncytium and secretes hormones after treatment with cAMP or forskolin. In contrast, the JEG-3 cell line fails to undergo substantial fusion. Therefore, BeWo and JEG-3 cells were used to identify a set of genes responsible for trophoblast fusion. Cells were treated with forskolin for 48 h to induce fusion. RNA was extracted, hybridised to Affymetrix HuGene ST1.0 arrays and analysed using system biology. Trophoblast differentiation was evaluated by real-time PCR and immunocytochemistry analysis. Moreover, some of the identified genes were validated by real-time PCR and their functional capacity was demonstrated by western blot using phospho-specific antibodies and CRISPR/cas9 knockdown experiments. Our results identified a list of 32 altered genes in fused BeWo cells compared to JEG-3 cells after forskolin treatment. Among these genes, four were validated by RT-PCR, including salt-inducible kinase 1 (SIK1) gene which is specifically upregulated in BeWo cells upon fusion and activated after 2 min with forskolin. Moreover, silencing of SIK1 completely abolished the fusion. Finally, SIK1 was shown to be at the center of many biological and functional processes, suggesting that it might play a role in trophoblast differentiation. In conclusion, this study identified new target genes implicated in trophoblast fusion. More studies are required to investigate the role of these genes in some placental pathology.

442. Functional perspectives of caspase-14 in the human placenta

The functional barrier for exchange between the mother and fetus in the placenta is created by the fusion of cytotrophoblasts with one another to form a continuous, multinuclear syncytiotrophoblast, which is maintained by the incorporation of underlying proliferative cytotrophoblasts. Disruption to this process has been hypothesised to be involved in the aetiology of preeclampsia. Recently we investigated caspase-14 in the context of trophoblast differentiation as it is crucial to epidermal differentiation and keratin stabilisation, revealing disparate expression of caspase-14 in the differentiating BeWo cell line. Consequently, further examination as to its functional role in trophoblast differentiation was conducted using RNA Interference (RNAi), with the hypothesis that differentiation would be suppressed following caspase-14 silencing. 100nM siRNA were delivered into the BeWo cell line for 16 h before the addition of 20µM Forskolin. Cultures were incubated for a further 24, 48 or 72 h before the extraction of RNA and protein. Transcription of the trophoblast hormone β-hCG, the endothelial mediator of eNOS, and cytokeratin 18 were found to be increased after both 24 and 48 h of differentiation following silencing, implicating caspase-14 in the regulation of these pathways. As both β-hCG and eNOS are significantly increased with trophoblast differentiation, this indicates that caspase-14 functionally suppresses BeWo differentiation. Furthermore, the differential expression of cytokeratin 18 indicates a conserved role for caspase-14 in keratin homeostasis in barrier formation. In conclusion, the suppression of caspase-14 in the BeWo cell line resulted in augmented differentiation, a trait often observed in preeclampsia. Further investigation of caspase-14 activity would provide important insight into mechanisms of trophoblast differentiation, particularly in relation to preeclampsia.