LncRNA FOXD1-AS1 acts as a potential oncogenic biomarker in glioma

Abstract

Aims: Altered activities of long noncoding RNAs (lncRNAs) have been associated with cancer development, and lncRNA FOXD1-AS1 (FOXD1-AS1) is the antisense transcript of the gene encoding for FOXD1, known for its role as an oncogene in several tumor types including glioma. However, the role of FOXD1-AS1 in the differentiation and progression of glioma is not well known.

Methods: Expression profile chip and qPCR were used to screen and identify FOXD1-AS1. Glioma cells were transfected with siRNA or eukaryotic expression vector to observe FOXD1-AS1 function in vitro and in vivo. Dual luciferase reporter gene analysis, Western blot, and ChIRP-MS were used to detect microRNAs and protein that combine with FOXD1-AS1.

Results: FOXD1-AS1 was upregulated and directly correlated with the glioma grade, and it was localized in both the nucleus and the cytoplasm of the glioma cell. FOXD1-AS1 silencing caused tumor suppressive effects via inhibiting cell proliferation, migration, and apoptosis, while FOXD1-AS1 overexpression resulted in opposite effects. Additionally, in vivo experiments showed that FOXD1-AS1 knockdown reduced tumor volume and weight. More importantly, mechanical studies revealed that FOXD1-AS1 targeted both miR339-5p and miR342-3p (miR339/342). Furthermore, protein eukaryotic translation initiation factor 5 subunit A (eIF5a) resulted a direct target of FOXD1-AS1.

Conclusions: These data indicated that FOXD1-AS1, a miR339/342 target, affected biological processes via protein eIF5a; thus, it might be considered as a new therapeutic target for glioblastoma.

Keywords: eIF5a; glioma; lncRNA FOXD1-AS1; lncRNAs; miR-339/342.

Figure 1

FOXD1‐AS1 is upregulated in glioma tissue. A, Microarray analysis showing 3334 lncRNAs with more than 2‐fold expression in glioma tissue compared with normal brain tissue. B, CPAT (Coding Potential Assessment Tool) indicated that FOXD1‐AS1 did not have any coding ability. C, FOXD1‐AS1 expression was significantly higher in the high‐grade glioma tissues compared with that in the normal brain tissues and low‐grade glioma tissues. D, FOXD1‐AS1 was higher in U87 and U251, while it was the lowest in Hs683 by qPCR. E, FOXD1‐AS1 was present in both the nucleus and the cytoplasm, although highly expressed in the cytoplasm. Data are presented as mean ± SEM *P < 0.05

Figure 2

FOXD1‐AS1 promotes glioma cell proliferation and tumor growth in vitro and in vivo. A, siRNA and plasmid regulate FOXD1‐AS1 expression efficiently in human glioma cell lines. B, C, MTS and colony formation assay. FOXD1‐AS1 silencing dramatically reduced the proliferative ability of glioma cells. In contrast, FOXD1‐AS1 overexpression increased the proliferative ability of Hs683 cells. D, EdU assay. U251 and Hs683 glioma cell lines were treated with siRNA or plasmid alone, and EdU assay was performed. E, Wound healing assay. Wound healing assay indicates that FOXD1‐AS1 expression contributed to glioma cell migration. F‐H, Xenograft tumors obtained using FOXD1‐AS1 silenced cells resulted in a smaller mean volume and weight than controls. Tumors obtained using cells overexpressing FOXD1‐AS1 resulted in a larger mean volume and weight, with a more rapid development than tumors obtained from control cells. Data are presented as mean ± SEM *P < 0.05, **P < 0.01, and ***P < 0.001

Figure 3

FOXD1‐AS1 is a direct target of miR339/342. A, B, Dual luciferase assay showed a significant decrease in luciferase activity after cotransfection of miR339/342 and wild‐type FOXD1‐AS1 expression vector, but not when a mutant FOXD1‐AS1 was used. C, miR339/342 overexpression significantly downregulated FOXD1‐AS1. D, FOXD1‐AS1 overexpression did not affect miR339/342 expression. Data are presented as mean ± SEM **P < 0.01, and ***P < 0.001

Figure 4

miR339/342 inhibit cell proliferation by regulating FOXD1‐AS1. A, miRNA mimics enhanced miR339/342 expression efficiently in human glioma cell lines. B, MTS assay. The result showed that miR339/342 overexpression dramatically reduced proliferation of glioma cells, compared with the mock group. The mixture group also prevented glioma cell growth. Interestingly, no change in proliferation was observed between miR339/342 group and mixture group. C, Flow cytometric analysis also showed that the miR339/342 mimics and mixture group prevented glioma cell growth compared to the mock‐treated group. D, Colony formation assay. Long‐term colony formation assay revealed that the mixture group dramatically reduced colony formation ability of glioma cells compared to the miR339/342 mimics group. Data are presented as mean ± SEM **P < 0.01, and ***P < 0.001

Figure 5

FOXD1‐AS1 affects biological processes via protein eIF5a. A, ChIRP‐MS diagram. B, Intersection of the identified DEGs in GSE4290 and GSE7696, revealing 962 DEGs in GBM. C, Western blot results showing that eIF5a could bind to FOXD1‐AS1, whereas the corresponding protein CBL could not. D, eIF5a mRNA was not changed when the expression of FOXD1‐AS1 was adjusted in glioma cells. E, No correlation was observed between eIF5a mRNA and FOXD1‐AS1 in glioma tissue. F, Western blot was used to investigate the relationship between FOXD1‐AS1 and eIF5a at a protein level. The result showed that eIF5a protein was positively proportional to FOXD1‐AS1 protein

Figure 6

A summary of this study. FOXD1‐AS1, a miR339/342 target, might function as an oncogene, facilitating tumor cell proliferation and inhibiting apoptosis via targeting eIF5a in glioma