Ephrin type-A receptor 2-antisense RNA1/2 promote proliferation and migration of MDA-MB-231 cells through EPHA2-dependent Ras signaling pathway mediated by MAPK8/JNK1, MAPK9/JNK2-NFATC2/NFAT1 and JUND

Abstract

Ephrin type-A receptor 2 (EPHA2) is a receptor tyrosine kinase that is overexpressed in a variety of cancers, including breast cancer. EPHA2 expression may be causally related to tumorigenesis; therefore, it is important to understand how EPHA2 expression is regulated. We previously reported that EPHA2 antisense RNA (EPHA2-AS), a natural antisense transcript, is an important modulator of EPHA2 mRNA levels and hence production of EPHA2 protein. EPHA2-AS encodes two splice variants, EPHA2-AS1 and EPHA2-AS2. The two variants are constitutively expressed in a concordant manner with EPHA2 mRNA in human breast adenocarcinoma cell lines and in patient samples, with the highest levels detected in the basal-like/triple-negative molecular subtype of breast cancer cells. In this study, we investigated the mechanism of EPHA2-AS1/2 in triple-negative breast cancer using MDA-MB-231 cells. We performed RNA-seq transcriptome analyses of MDA-MB-231 cells treated with AHCC^®, which suppressed expression of EPHA2-AS1/2 and EPHA2 mRNA, and EPHA2-AS1/2-silenced MDA-MB-231 cells. Bioinformatics analyses identified 545 overlapping differentially expressed genes that were significantly up- or down-regulated by these treatments. Subsequent functional enrichment analyses of the overlapping genes in combination with in vitro assays indicated that EPHA2-AS1/2 may promote the proliferation and migration of MDA-MB-231 cells through the EPHA2-dependent Ras signaling pathways mediated by MAPK8/JNK1, MAPK9/JNK2-NFATC2/NFAT1 (proliferation and migration) and JUND (migration). These results thus suggest that EPHA2-AS1/2 may represent a potential molecular target for triple-negative breast cancer treatment.

Keywords: EPHA2; EPHA2-AS1/2; JUND; MAPK8/JNK1; MAPK9/JNK2; NFATC2/NFAT1; migration; proliferation.

FIGURE 1

Effects of AHCC^® treatment on EPHA2 -AS1/2, EPHA2 mRNA and protein expressions and the proliferation and migration of MDA-MB-231 cells. (A) Levels of EPHA2-AS1/2 (top) and EPHA2 mRNA (bottom) in the AHCC®-treated MDA-MB-231 cells were quantified by strand-specific RT-qPCR. Copy numbers of EPHA2-AS1/2 and EPHA2 mRNA were determined as described in the Materials and Methods section. The results are presented as the mean ± s.e.m. of three samples. (B) AHCC^® (1.25 mg/mL)-treated MDA-MB-231 cells were subjected to western blot analysis after 24 h. Densitometric scanning analysis of immunoblots was performed; the results presented beneath the panel indicates the normalization of EPHA2 to β-actin. MM: molecular size marker (kDa). (C) MDA-MB-231 cells were cultured in the presence of various concentrations of AHCC^® and the number of live cells were counted after trypan blue staining. (D) (Left) MDA-MB-231 cells were treated with AHCC^® at either 0 or 1.25 mg/mL, scratched with a p200 pipette tip and imaged for 24 h using a microscope equipped with point visiting and a live-cell apparatus. Note that the cells remain in contact during their directed and coordinated movement into the gap. Scale bar = 100 µm. (right) The gap areas were imaged at four different sites at each time point and the number of cells that migrated into the areas were counted twice. Values of a representative experiment of three independent experiments are presented as the mean ± s.e.m. of a total eight counts each at the indicated time points. Error bars cannot be seen as they are smaller than the graph symbols. (E) (Left) MDA-MB-231 cells were transfected with ncseODN or seODN3 and subjected to wound-healing assays. Images are shown from a representative experiment of three independent experiments. Scale bar = 100 µm. (right) The number of cells from each transfection group that migrated into the wounded areas were counted. Values are presented as described in the legend (D). *p < 0.05 and **p < 0.01.

FIGURE 2

Identification of SDE genes from MDA-MB-231 cells after AHCC^® treatment and EPHA2-AS1/2 silencing identified by RNA-seq and functional enrichment analysis of overlapping genes. (A) Identification of the SDE genes between AHCC®-treated and EPHA2-AS1/2-silenced MDA-MB-231 cells using a Venn diagram. (B) Identification of hub genes from the SDE genes by DAVID. The Venn diagram showed that 33 genes of the 38 genes were enriched in all three GO categories.

FIGURE 3

The levels of downstream signaling proteins were reduced upon EPHA2-AS1/2 silencing. (A) seODN3 and ncseODN-transfected MDA-MB-231 cells were subjected to western blot analysis. Cells were analyzed 26 h after transfection (except for analysis for JNK2, which was performed at 36 h). MM: molecular size marker (kDa). (B) Densitometric scanning analysis was performed and the results were normalized to the densities of β-actin. Relative protein expression levels in EPHA2-AS1/2-silenced cells were calculated from levels in ncseODN-transfected cells. A representative western blot result of three independent experiments is shown. **p < 0.01, and ***p < 0.001.

FIGURE 4

The effects of NFATC2/NFAT1 silencing on MDA-MB-231 cell proliferation. (A) Cells were transfected with control siRNA (siCNT) or siRNA against NFATC2/NFAT1 mRNA (siNFAT1) and subjected to 5-EdU cell proliferation assay and flow cytometry. Histograms and percentages of EdU + cells in the total population of MDA-MB-231 cells at the indicated times are shown; a representative experiment of three independent experiments is shown. (B) Quantification of the flow cytometric analysis from (A). The number of EdU + cells in siNFAT1-transfected MDA-MB-231 cells were normalized to those in the control siRNA-transfected cells at each time point; data are presented as the ‘relative EdU-labeled MDA-MB-231 cells (%)’ ± s.e.m. of triplicate samples. *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 5

Effects of silencing of JUND, NFATC2/NFAT1 on the migration of MDA-MB-231 cells. (A) MDA-MB-231 cells were transfected with control siRNA (siCNT), siRNAs against JUND mRNA (siJUND) or NFATC2/NFAT1 mRNA (siNFAT1) and subjected to wound-healing assay. Images were obtained after 12 h and 24 h. Images are shown from a single representative experiment of three independent experiments. Scale bar = 100 µm. (B) The number of cells from each transfection group that migrated into the wounded areas were counted. Values of a representative experiment of three independent experiments are presented as the mean ± s.e.m. of a total eight counts each at the indicated time points.