The impact of wound-healing assay, phorbol myristate acetate (PMA) stimulation and siRNA-mediated FURIN gene silencing on endogenous retroviral ERVW-1 expression level in U87-MG astrocytoma cells

Abstract

Purpose

Human endogenous retroviruses (HERVs) are ubiquitous genomic sequences. Normally dormant HERVs, undergo reactivation by environmental factors. This deregulation of HERVs’ transcriptional equilibrium correlates with medical conditions such as multiple sclerosis (MS). Here we sought to explore whether exposing the U-87 MG astrocytoma cells to traumatic injury deregulates the expression of HERV-W family member ERVW-1 encoding syncytin-1. We also examined the expression of FURIN gene that is crucial in syncytin-1 synthesis.

Material and methods

Scratch assay was used as a model of cells injury in U-87 MG cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot (WB) and migration assay using Boyden chamber were used. Phorbol 12-myristate 13-acetate (PMA) and small interfering RNA (siRNA) were used for cell stimulation and gene expression inhibition, respectively.

Results

Results revealed reduced ERVW-1 expression in cells exposed to injury (p ​< ​0.05) while GFAP gene - a marker of active astrocytes, was upregulated (p ​< ​0.01). These findings were confirmed by both WB and RT-qPCR. Expression of FURIN gene was not altered after injury, but cell stimulation by PMA strongly increased FURIN expression, simultaneously downregulating ERVW-1 (p ​< ​0.01). SiRNA-mediated expression inhibition of ERVW-1 and FURIN influenced the mRNA level for SLC1A5 (ASCT2) - primary syncytin-1 receptor, that was significantly lower. FURIN inhibition by siRNA caused strong upregulation of ERVW-1 expression (p ​< ​0.01).

Conclusion

Results showed that mechanical impact affects the expression of endogenous retroviruses in U-87 MG astrocytoma cells by scratch assay. Regulation of FURIN, a crucial enzyme in ERVW-1 turnover may support the therapy of some neurological conditions.