Korean J Physiol Pharmacol.  2019 Nov;23(6):475-482. 10.4196/kjpp.2019.23.6.475.

FoxD2-AS1 is a prognostic factor in glioma and promotes temozolomide resistance in a O⁶-methylguanine-DNA methyltransferase-dependent manner

• Shangguan
• Lv X
• Tian N

• KMID: 2461040
• DOI: http://doi.org/10.4196/kjpp.2019.23.6.475

Abstract

Glioma is the most common brain tumor with a dismal prognosis. While temozolomide (TMZ) based chemotherapy significantly improves survival in glioma patients, resistance against this compound commonly leads to glioma treatment failure. Overexpression of long-noncoding RNA (LncRNA) FoxD2 adjacent opposite strand RNA 1 (FoxD2-AS1) was identified to promote glioma development, but the role in TMZ resistance remains unclear. In this paper, we found that FoxD2-AS1 was overexpressed in recurrent glioma, high FoxD2-AS1 expression was significantly correlated with poor patient outcome. Methylation of O⁶-methylguanine-DNA methyltransferase (MGMT) is significantly less frequent in high FoxD2-AS1 expression patients. Knockdown of FoxD2-AS1 decreased the proliferation, metastatic ability of glioma cells and promote the sensitivity to TMZ in glioma cells. Furthermore, knockdown of FoxD2-AS1 induced hypermethylation of the promoter region of MGMT. Our data suggested that FoxD2-AS1 is a clinical relevance LncRNA and mediates TMZ resistance by regulating the methylation status of the MGMT promoter region.

Keyword

Drug Resistance; Glioma; Long-noncoding RNA; Methylation; Temozolomide

MeSH Terms

Brain Neoplasms
Drug Resistance
Drug Therapy
Glioma*
Humans
Methylation
Prognosis
Promoter Regions, Genetic
RNA
RNA, Long Noncoding
Treatment Failure
RNA
RNA, Long Noncoding

Figure

• 

  Fig. 1 FoxD2-AS1 is associated with clinical outcome of patients with glioma. (A) FoxD2-AS1 expression in recurrent glioma tissues and non-recurrent tissues obtained from the Cancer Genome Atlas (TCGA). (B–D) Kaplan–Meier plots of patient outcome based on FoxD2-AS1 expression. (E–H) Kaplan–Meier plots of patient outcome based on FoxD2-AS1 expression in glioma, stratified according to the annotated IDH mutation status and glioma subtype. Data, statistical evaluation, and visualization were obtained using the R2 website “R2: Genomics Analysis and Visualization Platform” (http://r2.amc.nl) and http://www.betastasis.com/. REMBRANDT, Repository for Molecular Brain Neoplasia Data.

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  Fig. 2 FoxD2-AS1 knockdown sensitizes glioma cells to temozolomide (TMZ) treatment. (A) Cell viabilities of siFoxD2-AS1 or siNC transducted glioma cells under treatment of indicated doses of TMZ. (B) Colony formation of siFoxD2-AS1 or siNC transducted glioma cells after TMZ treatment. (C) Flow cytometry analysis shown the cell apoptosis of siFoxD2-AS1 or siNC transducted glioma cells after TMZ treatment. (D) Western blot analysis of cleaved caspase-3 and Bax in glioma cells transfected with siFoxD2-AS1 or siNC after TMZ treatment. siFox, siFoxD2-AS1. *p < 0.05, **p < 0.01.

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  Fig. 3 FoxD2-AS1 knockdown inhibits the metastatic ability of glioma cells. (A) Effect of FoxD2-AS1 knockdown on the migration of glioma cells. (B) Effect of FoxD2-AS1 knockdown on the invasion of glioma cells. Cells were counted after staining with crystal violet (magnification, ×200). Data represent means ± standard deviation. of at least three independent experiments. siFox, siFoxD2-AS1. **p < 0.01.

• 

  Fig. 4 FoxD2-AS1 is associated with the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter in glioma cells. (A) MS-PCR analysis depicts the methylation status of the MGMT promoter in siFoxD2-AS1 or siNC transducted glioma cells. The density of each band was quantified using imaging analysis and the relative band density values were calculated as the ratio of methylated MGMT to that of methylated plus un-methylated MGMT. MGMT unmethylated: 92 bp; MGMT methylated: 8l bp. (B) Western blot analysis of MGMT expression in siFoxD2-AS1 or siNC transducted glioma cells. U, unmethylated; M, methylated; MM, molecular marker; siFox, siFoxD2-AS1. **p < 0.01.

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  Fig. 5 A diagram summarizing the main findings of this study. FoxD2-AS1 contributes to temozolomide (TMZ) resistance of glioma cells by regulating the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter region.

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