Regulation of heparanase on the expression of cytokines and the prediction of its molecular mechanism in trophoblast cells

Objective To explore the influence of heparanase (HPSE) on the expression of cytokines and predict its underlying molecular mechanism in trophoblast cells. Methods The human first-trimester extravillous trophoblast cell line HTR8/SVneo cells were selected as research subjects of this study. And a HPSE over-expression stably transfected HTR8/SVneo cell line and a HPSE normal-expression stably transfected HTR8/SVneo cell line were constructed. They were enrolled into test group and control group, respectively. And then 343 cytokines were semi-quantitatively detected using human cytokine antibody array kit and analyzed by Image J software to discover differential cytokine proteins in trophoblast cell lines of two groups. Western blotting was performed to validate the semi-quantitative results of cytokine antibody array by detecting AXL receptor tyrosine kinase, which had the most significant difference between trophoblast cell lines of two groups. The protein-protein interaction network was constructed through STRING protein online database, and the gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed by The Database for Annotation, Visualization and Integrated Discovery (DAVID) online bioinformatics resources. Results ①The semi-quantitative results of cytokine microarray showed that levels of 15 cytokines, including AXL, intercellular adhesion molecule (ICAM)1, TEK receptor tyrosine kinase, urokinase-type plasminogen activator receptor (PLAUR), tissue inhibitor of metalloproteinase (TIMP)2, matrix metalloprotein (MMP)9, lymphocyte-activation gene (LAG)3, tumor necrosis factor receptor superfamily (TNFRSF)1B, TNFRSF1A, cytotoxic and regulatory T cell molecule (CRTAM), tissue inhibitor of metalloproteinase (TIMP)4, human platelet reactive protein (THBS)1, interleukin 17 receptor B (IL17RB), junctional adhesion molecule-like protein (AMICA), and C-C chemotactic factor 16 (CCL16) were decreased in HPSE-overexpressed trophoblast cell line of test group, compared with the control trophoblast cell line of control group. And the ratio of levels of differential cytokines in trophoblast cell line between test group and control group was less than 0.666 7.②The result of Western blotting showed that the level of AXL in HPSE-overexpressed trophoblast cell line of test group was obviously lower than that in trophoblast cell line of control group, and the difference was statistically significant (t=-6.931, P=0.020), which was consistent with the semi-quantitative results of cytokines microarray. ③HPSE was associated with MMP9 by vascular endothelial growth factor (VEGF)A, tumor protein p53 (TP53) and CD44 by STRING protein online database. And MMP9 was directly or indirectly associated with other 12 types of differential cytokines except LAG3, CCL16 and IL17RB to establish an interaction network. ④GO enrichment analysis of cytokines showed that the differential cytokines were mainly involved in 41 biological processes, including negative regulation of apoptosis and extracellular matrix (ECM) disassembly. Results of KEGG signaling pathway analysis showed that the differential cytokines were mainly involved in 6 signaling pathways, including proteoglycan in cancer signaling pathway and tumor necrosis factor signaling pathway. Conclusions Overexpression of HPSE may regulate the expression of cytokines in trophoblast cells. And it may affect the biological functions of trophoblast cells through the proteoglycan in cancer signaling pathway, which MMP9 is involved in. This study can provide a research direction and basis for further study of the influences of HPSE in trophoblast cells associated diseases. Key words: Heparanase; Cytokines; Signaling pathway; Protein interaction; Trophoblast cell