Effect and mechanism of TNF-α and etanercept on the invasion ability of extravillous trophoblast cell in URSA patients

Abstract

Objective: To investigate the effect and mechanism of tumor necrosis factor α (TNF-α) and its inhibitor etanercept (ETA) on the invasion ability of extravillous trophoblast in patients with unexplained recurrent spontaneous abortion (URSA). Methods: (1) Patients were collected from March to June in 2019. They were divided into the URSA group (n=15) and the normal control group (n=15), according to whether diagnosed with URSA or not. The mRNA expression levels of TNF-α in villi tissue of patients in the two groups were detected by quantitative real-time PCR (qRT-PCR). (2) The mRNA and protein expression levels of matrix metalloproteinase-2 (MMP-2), Slug and CXC chemokine rceptor 4 (CXCR4) in HTR-8/SVneo cells were detected by qRT-PCR or western blot after being stimulated by exogenous TNF-α (0.2, 2, 20 ng/ml) alone or TNF-α along with ETA, or phosphate buffered saline (PBS) as control. (3) The invasion ability of HTR-8/SVneo cells was investigated by transwell test after stimulating by TNF-α alone or TNF-α along with ETA. (4) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells, which were stimulated by TNF-α (2 ng/ml) alone after nuclear factor-κB (NF-κB) inhibitor, BAY 11-7028, preconditioning, were detected by qRT-PCR or western blot. Results: (1) The mRNA expression level of TNF-α in villi tissue of URSA group (4.10±0.49) was 4.1 times as much as the normal control group (t=10.51, P<0.05). (2) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells of TNF-α group were significantly lower than those in PBS control group (P<0.05) and those in TNF-α along with ETA group (P<0.05). (3) The invasion ability of HTR-8/SVneo cells in TNF-α group was significantly decreased than PBS group and TNF-α along with ETA group (78±14 vs 373±26 vs 227±44, P<0.05). (4) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells with BAY 11-7028 preconditioning (mRNA: 1.03±0.10, 1.03±0.06, 1.09±0.08; protein: 1.09±0.03, 1.49±0.03, 1.12±0.03) were significantly higher than without preconditioning after being stimulated by TNF-α (all P<0.05). Conclusions: The expression of TNF-α in the villi of URSA patients is much higher than normal early pregnant women. TNF-α could decrease the capacity of invasion by suppressing the expression of MMP-2, Slug and CXCR4 through NF-κB signaling pathway in extravillous trophoblast cells. While ETA could improve the invasiveness capability of extravillous trophoblast cells through inhibiting the negative effect of TNF-α.